CN110974826B - Application of ouabain or 12-epi-ouabain in preparation of medicine for treating leukemia - Google Patents
Application of ouabain or 12-epi-ouabain in preparation of medicine for treating leukemia Download PDFInfo
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Abstract
The invention provides application of ouabain or 12-epi-ouabain in preparation of a medicament for treating leukemia, and belongs to the field of medicines. The inventor researches find that both the euaconitine and the 12-epi-euaconitine can effectively inhibit the proliferation of leukemia cells, and simultaneously can promote the apoptosis of the leukemia cells, and particularly has more remarkable effect on myelogenous leukemia cells. Therefore, the euaconitine and the 12-epi-euaconitine have the activities of resisting leukemia and hematopoietic tissue tumors, can be singly or jointly used as medicines for treating leukemia and hematopoietic tissue tumors, and have wide application prospect.
Description
Technical Field
The invention relates to the field of medicines, in particular to application of ouabain or 12-epi-ouabain in preparation of medicines for treating leukemia.
Background
At present, the development and utilization of the plant medicine firstly extracts and separates the effective chemical components with relevant curative effects, and purifies the effective chemical components to obtain single components with certain curative effects. Wherein, the alkaloid is a nitrogenous organic compound with obvious physiological activity, and a plurality of medicinal plants are rich in the alkaloid, thus being an important component of traditional Chinese medicines. The separation and purification of the alkaloid effective components are the difficulty and key of the development of traditional Chinese medicine.
The plurality of aconite (Aconitum karakolicum rap) are plants of Aconitum (Aconitum L.) belonging to Ranunculaceae, are distributed on hillside grasslands with the altitude of 2000-3000 m in the Yi-plow area of Xinjiang in China, are concentrated in distribution, and are mainly used for treating neuralgia, angina, nephritis and other diseases. Wherein the plurality of aconite roots contains abundant alkaloids, and the content of the ouabain (napelline) and the 12-epi-ouabain (12-epi-napelline) is higher.
Leukemia (blood cancer) is a group of diseases caused by abnormal proliferation of hematopoietic stem cells. Leukemia is a malignant tumor with the highest morbidity and mortality rate under 35 years old and rarely occurring in young people. Leukemia is classified as one of ten major high-grade tumors in China. Currently, the number of leukemia patients in China reaches more than 400 tens of thousands, and 4 tens of thousands of patients are newly added each year. The impoverishment of therapeutic strategies results in high mortality in leukemia patients. Therefore, development of new drugs and therapeutic strategies for leukemia treatment is an urgent problem to be solved.
Disclosure of Invention
The invention aims to provide an application of ouabain or 12-epi-ouabain in preparation of medicines for treating leukemia, and provides a new strategy for treating leukemia.
In order to achieve the above object of the present invention, the following technical solutions are specifically adopted:
application of eugeniin or 12-epi-eugeniin in preparing medicine for treating leukemia is provided.
Further, in a preferred embodiment of the present invention, the leukemia is a myeloid leukemia.
Further, in a preferred embodiment of the present invention, the myelogenous leukemia comprises chronic myelogenous leukemia.
Further, in a preferred embodiment of the present invention, the above-mentioned myelogenous leukemia is a disease caused by K-562 chronic myelogenous leukemia cells or by HL-60 myelogenous leukemia cells.
Application of ouabain or 12-epi-ouabain in preparing cell proliferation inhibitor for myeloid leukemia is provided.
Application of ouabain or 12-epi-ouabain in preparation of apoptosis promoter for myeloid leukemia is provided.
A pharmaceutical composition comprising an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is at least one of ouabain and 12-epi-ouabain.
Compared with the prior art, the invention has the beneficial effects that:
the inventor researches find that both the euaconitine and the 12-epi-euaconitine can effectively inhibit the proliferation of leukemia cells, and simultaneously can promote the apoptosis of the leukemia cells, and particularly has more remarkable effect on myelogenous leukemia cells. Therefore, the euaconitine and the 12-epi-euaconitine have the activities of resisting leukemia and hematopoietic tissue tumors, can be singly or jointly used as medicines for treating leukemia and hematopoietic tissue tumors, and have wide application prospect.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 shows the inhibition of K-562 cell proliferation by ouabain (μg/ml) in example 1, wherein: p <0.05, P <0.01 compared to control;
FIG. 2 shows the inhibition of HL-60 cell proliferation by ouabain (μg/ml) of example 1, wherein: p <0.05, P <0.01 compared to control;
FIG. 3 shows the inhibition of K-562 cell proliferation by 12-epi-Ouwu alkali (μg/ml) in example 1, wherein: p <0.05, P <0.01 compared to control;
FIG. 4 shows the inhibitory effect of 12-epi-Ouwu alkali (μg/ml) on HL-60 cell proliferation in example 1, wherein: p <0.05, P <0.01 compared to control;
FIG. 5 is a flow chart showing apoptosis of K-562 cells by Ouwusu (μg/ml) in example 2;
FIG. 6 is a graph showing apoptosis of K-562 cells by Ouwusu (μg/ml) in example 2;
FIG. 7 is a flow chart showing apoptosis of the HL-60 cells by Ouwu base (. Mu.g/ml) in example 2;
FIG. 8 is a graph showing apoptosis of the HL-60 cells by Ouwusu (μg/ml) in example 2;
FIG. 9 is a flow cytometry distribution of apoptosis of K-562 cells by 12-epi-Ouwu' ine (μg/ml) in example 2;
FIG. 10 is a graph showing apoptosis of K-562 cells by 12-epi-Ouwusu (μg/ml) in example 2;
FIG. 11 is a flow cytometry distribution of apoptosis of 12-epi-Ouwu alkali (μg/ml) to HL-60 cells of example 2;
FIG. 12 is a graph showing apoptosis of HL-60 cells by 12-epi-ouabain (μg/ml) in example 2;
FIG. 13 is a graph showing the results of morphological detection of apoptosis of K-562 cells by Ouwusu (μg/ml) in example 3;
FIG. 14 is a graph showing the results of morphological detection of apoptosis of K-562 cells by 12-epi-Ouwusu (μg/ml) in example 3;
FIG. 15 is a graph showing the results of morphological detection of apoptosis of HL-60 with ouabain (μg/ml) in example 3;
FIG. 16 is a graph showing the results of morphological detection of apoptosis of HL-60 cells by 12-epi-Ouwu-ine (. Mu.g/ml) in example 3.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instrumentation used were not manufacturer-specific and were conventional products commercially available.
Ouwusu and 12-epi-Ouwusu are isomers with molecular formula of C 22 H 33 NO 3 All belong to C-20 diterpenoid alkaloids. The difference between the two is that the configuration of the hydroxyl at the 12-position of the ouabain is alpha-type, while the configuration of the hydroxyl at the 12-position of the 12-epi-ouabain is beta-type, and the chemical structures of the two are shown in the following formulas.
In the invention, the euaconitine and the 12-epi-euaconitine can be used as active ingredients independently or can be combined together to play roles in inhibiting the proliferation of leukemia cells and promoting the apoptosis of leukemia cells.
In order to allow the pharmaceutical composition to release the active ingredient rapidly, continuously and over a long period of time, the pharmaceutical composition of the present invention may be manufactured according to conventional methods disclosed in those skilled in the art. The pharmaceutical compositions of the present invention are administered orally, nasally, or parenterally. The preparation of the pharmaceutical composition can be powder, granule, tablet, emulsion, syrup, aerosol, soft capsule, hard capsule, sterilized injection, sterilized powder, etc.
The term "pharmaceutically acceptable" as used herein means that the compounds are physiologically acceptable when ouabain and 12-epi-ouabain are administered to humans and do not undergo gastrointestinal disturbances, dizziness and other allergic reactions or systemic allergic reactions like these.
In the present invention, "pharmaceutically acceptable carrier" includes but is not limited to: binders (e.g., microcrystalline cellulose, alginate, gelatin, and polyvinylpyrrolidone), fillers (e.g., starch, sucrose, dextrose, and anhydrous lactic acid), disintegrants (e.g., crosslinked PVP, crosslinked sodium carboxymethyl starch, crosslinked sodium carboxymethyl cellulose, and low-substituted hydroxypropyl cellulose), lubricants (magnesium stearate, aluminum stearate, talc, polyethylene glycol, sodium benzoate), wetting agents (e.g., glycerin), surfactants (e.g., cetyl alcohol), and absorption enhancers, flavoring agents, sweeteners, diluents, coating agents, and the like.
In the present invention, the active ingredient in the pharmaceutical composition also includes a "pharmaceutically acceptable salt" formed by ouabain or 12-epi-ouabain. Herein, "pharmaceutically acceptable salts" include pharmaceutically acceptable non-toxic salts of ouabain or 12-epi-ouabain formed by standard reaction with inorganic or organic acids. Wherein the inorganic acid comprises, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and the like; the organic acid includes, for example, formic acid, acetic acid, citric acid, cis-butenedioic acid, tartaric acid, ascorbic acid, salicylic acid, sorbic acid, benzoic acid, methanesulfonic acid, p-toluenesulfonic acid, and the like.
The content of the eugenoline or the 12-epi-eugenoline in the pharmaceutical composition is a pharmaceutical effective dose. The term "pharmaceutically effective dose" as used herein means an amount of the active ingredient sufficient to bring about the desired physiological or pharmacological activity when administered to an animal or human. However, the pharmaceutically effective dose may be adjusted according to the age, weight, health condition, sex, and administration route and treatment period of the patient, etc.
The features and capabilities of the present invention are described in further detail below in connection with the following examples:
example 1
Anti-leukemia cell proliferation activity assay:
the activity of the anti-K-562 and HL-60 cells of the euaconitine and the 12-epi-euaconitine is detected by adopting a CCK-8 method.
(1) Pharmaceutical formulation
The eugeniin and the 12-epi-eugeniin are weighed by an electronic balance, and the medicament is completely dissolved in DMSO.
(2) Solvent preparation
Preparing a culture medium: prepared according to the high-sugar DMEM culture medium and serum, wherein the diabody=100:10:1
Preparation of PBS: the electronic balance weighed 8g of NaCl, 0.2g of KCl, 1.42g of Na2HPO4 and 0.27g of KH2PO 4; approximately 800ml of deionized water was added and dissolved by stirring thoroughly on a magnetic stirrer, and then the volume was set to 1L. Autoclaving at 120deg.C for 30min, and cooling to 4deg.C.
Preparing frozen stock solution: prepared in serum, dmso=9:1, ready to use.
(3) Cell culture
In various experiments of the invention, K-562 human chronic myelogenous leukemia cells and HL-60 human myelogenous leukemia cells are purchased from Shanghai cell bank of the Chinese sciences. K-562 and HL-60 are suspension cells, and both cell types are at 37deg.C and 5% CO 2 Growing in the environment, and performing experiments in a high-sugar DMEM complete medium containing 10% of fetal calf serum.
Cell exchange liquid: cells from the cell bank were scraped with 75% alcohol from the flask and observed for cell morphology and status under an inverted microscope. Cells were collected in 50ml centrifuge tubes, centrifuged at 1000r for 5min, the supernatant discarded, the cells resuspended in fresh medium containing serum, and the cells inoculated into fresh flasks and washed 1-2 times with PBS if there were more cell debris.
Cell passage: when the cells grow to 70% -80% of the culture flask, the cells need to be passaged. Collecting cells in a centrifuge tube 1000r for 5min, centrifuging, and discarding the supernatant; the solution was washed 1 time with PBS, centrifuged at 1000r for 5min, and the supernatant was discarded. Cells were resuspended in serum-containing medium and plated on average in new flasks, 1 to 2 or 3 depending on cell density.
Cell cryopreservation: in order to prevent the cells from being bad in cell state due to contamination or passage of algebra during the culture, the cells need to be frozen. Taking log to growCells at stage 1000r were collected, centrifuged for 5min and the supernatant was discarded; resuspension of cells with frozen stock, adjusting cell density to 5×10 6 -1×10 7 And (3) packaging the cell suspension in about 1mL of frozen storage tubes per mL, and storing in a refrigerator at-80 ℃.
Cell resuscitation: taking out the frozen tube from-80deg.C, soaking in 37 deg.C water bath, centrifuging at 1000r for 5min after the liquid in the frozen tube is completely dissolved, discarding supernatant, resuspension with serum-containing culture medium, inoculating to culture flask, and adding into 37 deg.C CO 2 Culturing in an incubator.
(4) CCK-8 assay for detecting cell proliferation
(1) K-562 cells
Cells grown in log phase were taken at 2X 10 4 Wells were seeded in 96 well cell culture plates at 100. Mu.l per well at 37℃with 5% CO 2 Culturing in incubator for several hours, and adding medicine after cell is stable. Blank, control, ouwu base (20, 40, 60, 80. Mu.g/ml), 12-epi-Ouwu base (12.5, 25, 50, 100. Mu.g/ml) and 3 duplicate wells per group were set. The blank group contains no cells, the vehicle control group incubates cells with fresh complete DMEM medium, the Ouwu alkali group incubates cells with fresh complete DMEM medium containing the drug at final concentration of 20, 40, 60, 80 μg/ml, and the 12-epi-Ouwu alkali group incubates cells with fresh complete DMEM medium containing the drug at final concentration of 12.5, 25, 50, 100 μg/ml. After the treatment in the above manner, at 37℃5% CO 2 After conventional culture in incubator for 24h and 48h, CCK-8 μl was added in each well in the absence of light, and absorbance of each well was measured at 450nm using an enzyme-labeled instrument after further culture for 1-3 h. The number of living cells is indirectly reflected by absorbance, the survival rate of the cells acted by the medicines with different concentrations is calculated, and the experiment is repeated for 3 times to record the result.
(2) HL-60 cells
Taking cells growing in logarithmic phase, inoculating HL-60 cells into 96-well cell culture plate at 4×104/well, 100 μl per well, and culturing at 37deg.C under 5% CO 2 Culturing in incubator for several hours, and adding medicine after cell is stable; blank, control, ouwu base (15, 30, 50, 90. Mu.g/ml), 12-epi-Ouwu base (7.5, 15, 30, 60. Mu.g/ml)g/ml) 3 duplicate wells per group. The blank group contained no cells, the vehicle control group incubated cells with fresh complete DMEM medium, the euwu base group incubated cells with fresh complete DMEM medium containing drug at final concentrations of 15, 30, 50, 90 μg/ml, respectively, and the 12-epi-euwu base group incubated cells with fresh complete DMEM medium containing drug at final concentrations of 7.5, 15, 30, 60 μg/ml, respectively. After the treatment in the above manner, at 37℃5% CO 2 After conventional culture in incubator for 24h and 48h, CCK-8 μl was added in each well in the absence of light, and absorbance of each well was measured at 450nm using an enzyme-labeled instrument after further culture for 1-3 h. The number of living cells is indirectly reflected by absorbance, the survival rate of the cells acted by the medicines with different concentrations is calculated, and the experiment is repeated for 3 times to record the result.
The calculation formula is as follows: survival = ((dosing group-blank)/(control group-blank)). 100
(5) Data processing
The SPSS 22.0 statistical software is used for processing data, the metering data are expressed in terms of x+/-s, the mean value of two samples is compared by using a Student T-Test, the mean value between multiple sample groups is compared by using One-way ANOVA, P <0.05 is statistically significant, and P <0.01 is very significant in the difference tested.
(6) Experimental results
(1) Inhibition of K-562 and HL-60 cell proliferation by ouabain
Ouwusu (20-80 μg/ml) has significant inhibition of K-562 cell proliferation after acting on K-562 tumor cells, and is time-dose dependent, see FIG. 1.
Ouwusu (15-90 μg/ml) has significant inhibition of HL-60 cell proliferation after acting on HL-60 tumor cells, and is time-dose dependent, see FIG. 2.
(2) Inhibition of K-562, HL-60 cell proliferation by 12-epi-Ouwusu
After 12-epi-ouabain (12.5-100 μg/ml) acted on K-562 tumor cells, it had obvious inhibition effect on proliferation of K-562 cells, and was time-dose dependent, see FIG. 3.
12-epi-Ouwusu (7.5-60 μg/ml) has inhibitory effect on proliferation of HL-60 cells after acting on HL-60 tumor cells, and is time-dose dependent, see FIG. 4.
Example 2
Pro-leukemia apoptosis experiments:
apoptosis of K-562 and HL-60 cells by using flow cytometry to detect ouabain and 12-epi-ouabain
(1) Pharmaceutical formulation
The euler alkali and a proper amount of 12-epi-euler alkali are weighed by an electronic balance, and DMSO completely dissolves the medicine.
(2) Seed plate
(1) K-562 cells: collecting K-562 cells in logarithmic growth phase, inoculating into 6-well plate at a density of 6×105/well, and inoculating into 5% CO at 37deg.C 2 Culturing in incubator for several hours, and stabilizing the cells.
(2) HL-60 cells: taking HL-60 cells in logarithmic growth phase, collecting cells, inoculating into 6-well plate at 1×106/well, inoculating into 5% CO at 37deg.C 2 Culturing in incubator for several hours, and stabilizing the cells.
(3) Dosing
(1) K-562 cells: after cell stabilization, ouabain (40, 60. Mu.g/ml) and 12-epi-ouabain (25, 50. Mu.g/ml) were added. At 37 ℃,5% CO 2 Culturing in incubator for 24 hr.
(2) HL-60 cells: after cell stabilization, ouabain (30, 50. Mu.g/ml) and 12-epi-ouabain (15, 30. Mu.g/ml) were added. At 37 ℃,5% CO 2 Culturing in incubator for 24 hr.
(4) Flow cytometry to detect apoptosis of tumor cells
(1) Apoptosis: after 24h incubation, the cells were collected in a centrifuge tube, centrifuged at 1000r for 5min, the supernatant was discarded, the cells were resuspended in 1ml ice-bath pre-chilled PBS and transferred to a 1.5ml centrifuge tube, centrifuged at 1000r for 5min again, and the supernatant was discarded. Under the condition of avoiding light, 195 mu l of Annexin V-FITC binding solution is added into each sample to resuspend cells, 5 mu l of Annexin V-FITC is added and mixed evenly, 10 mu l of propidium iodide staining solution is added and mixed evenly, incubation is carried out for 10-20min at room temperature and avoiding light, and flow cytometry detection is repeated for 3 times.
(2) Cycle: after 24h, the cells were collected in a centrifuge tube, centrifuged at 1000r 5min, the supernatant was discarded, the cells were resuspended in 1ml ice-bath pre-chilled PBS and transferred to a 1.5ml centrifuge tube, centrifuged at 1000r 5min again, and the supernatant was discarded. 1ml of ice-bath pre-chilled 70% ethanol was added, after 2h fixation at 4℃1000r 5min centrifugation, the supernatant was discarded, 1ml of ice-bath pre-chilled PBS was added to re-suspend the cells, 1000r 5min re-centrifugation, and the supernatant was discarded. Preparation of propidium iodide staining solution for 1 sample: 0.5ml of staining buffer, 25. Mu.l of propidium iodide staining solution (20X), 10. Mu.l of RNaseA (50X); after preparation of propidium iodide staining solution, 0.5ml of propidium iodide staining solution was added to each sample, and the cells were resuspended and incubated at 37℃for 30min in the absence of light. Flow cytometry detection was repeated 3 times.
(5) Experimental results
(1) Apoptosis of K-562 cells and HL-60 cells by ouabain
After 24h of the euaconitine (40, 60. Mu.g/ml) was applied to K-562 cells, the apoptosis rate increased with increasing concentration, as shown in FIGS. 5 and 6.
After 24h of the application of ouabain (30, 50. Mu.g/ml) to HL-60 cells, the apoptosis rate increased with increasing concentration, as shown in FIGS. 7 and 8.
(2) Apoptosis of K-562 cells and HL-60 cells by 12-epi-Ouwusu
After 12-epi-euwu alkali (25. Mu.g/ml, 50. Mu.g/ml) was applied to K-562 cells for 24 hours, the apoptosis rate was significantly different and statistically significant (P0.01) compared with the control group, and as the concentration increased, the apoptosis rate increased as shown in FIGS. 9 and 10.
After 12-epi-euwu alkali (15. Mu.g/ml, 30. Mu.g/ml) was applied to HL-60 cells for 24 hours, the apoptosis rate was significantly different and statistically significant (P0.01) compared with the control group, and as the concentration increased, the apoptosis rate increased as shown in FIGS. 11 and 12.
Example 3
Morphological detection of leukemia cell apoptosis:
apoptosis of K-562 and HL-60 cells by detecting ouabain and 12-epi-ouabain with Hoechst staining
(1) Seed plate
(1) K-562 cells: taking K-562 cells in logarithmic growth phaseThe cells were collected at 6X 10 5 Density of wells/density of wells was seeded into 6-well plates at 37 ℃,5% co 2 Culturing in incubator for several hours, and stabilizing the cells. A blank group, a low concentration group and a high concentration group are set.
(2) HL-60 cells: taking HL-60 cells in logarithmic growth phase, collecting cells, and culturing at 1×10 6 Density of wells/density of wells was seeded into 6-well plates at 37 ℃,5% co 2 Culturing in incubator for several hours, and stabilizing the cells. A blank group, a low concentration group and a high concentration group are set.
(2) Dosing
(1) K-562 cells: after cell stabilization, ouabain (40, 60. Mu.g/ml) and 12-epi-ouabain (25, 50. Mu.g/ml) were added. At 37 ℃,5% CO 2 Culturing in incubator for 24 hr and 48 hr.
(2) HL-60 cells: after cell stabilization, ouabain (30, 50. Mu.g/ml) and 12-epi-ouabain (15, 30. Mu.g/ml) were added. At 37 ℃,5% CO 2 Culturing in incubator for 24 hr and 48 hr.
(3) Hoechst staining
(1) After 24h and 48h, the cells were collected in a 1.5ml centrifuge tube, 0.5ml of fixative was added, the cells were slowly suspended and fixed for 15min.
(2) The fixative was removed by centrifugation and washed twice with PBS for 3min each.
(3) After centrifugation, the bulk of the liquid was aspirated to leave approximately 50 μl of liquid, the cells were slowly suspended and added dropwise to the slide to distribute the cells as evenly as possible.
(4) And the cells are adhered to the glass slide by a little air drying, so that the cells are not easy to flow along with the liquid.
(5) Uniformly dripping 0.5ml Hoechst 33258 staining solution, staining for 5min, sucking the solution from the edge with absorbent paper, and slightly air-drying.
(6) The staining solution was removed, washed twice with PBS for 3min each time, and the solution was removed by pipetting, and shaking.
(7) Dropping a drop of anti-fluorescence quenching sealing liquid on the glass slide, and covering a clean cover glass to avoid bubbles as much as possible.
(8) Blue nuclei can be detected by a fluorescence microscope, the excitation wavelength is about 350nm, and the emission wavelength is about 460 nm.
(4) Experimental results
(1) Detection of K-562 apoptosis by Hoechst staining
Results of treatment of K-562 cells with different concentrations of Ouwu's alkali for 24h and 48h are shown in FIG. 13; the results of treatment of K-562 cells with varying concentrations of Ouwu's base for 24h and 48h are shown in FIG. 14.
As can be seen from fig. 13 and 14, the blank control group K-562 cells have clear nucleus limitation, are round or oval, and have normal blue fluorescence; the euaconitine and 12-epi-euaconitine treated groups can show that the cell nuclei are densely concentrated, or are densely concentrated in a block shape, and the color is somewhat whitish. The degree of apoptosis of K-562 cells was found to be dose-time dependent by fluorescence microscopy.
(2) Hoechst staining for detecting HL-60 apoptosis
Results of treatment of HL-60 cells with different concentrations of Ouwusu for 24h and 48h are shown in FIG. 15; results of treatment of HL-60 cells with varying concentrations of Ouwusu for 24h and 48h are shown in FIG. 16.
As can be seen from fig. 15 and 16, the blank HL-60 cells had clear nucleus limitation, and had a circular or oval shape and a normal blue fluorescence; the euaconitine and 12-epi-euaconitine treated groups can show that the cell nuclei are densely concentrated, or are densely concentrated in a block shape, and the color is somewhat whitish. The degree of apoptosis of HL-60 was observed to be dose-time dependent by fluorescence microscopy.
Formulation example 1
Taking 10g of euaconitine, adding proper auxiliary materials of tablets (including sustained release tablets, skeleton tablets, dispersible tablets and the like), and preparing the anti-leukemia tablets according to a preparation process of the tablets.
Formulation example 2
Taking 10g of 12-epi-Ouwusu, adding proper auxiliary materials of injection, and preparing the anti-leukemia injection according to the preparation process of the injection.
Formulation example 3
Taking 5g of Ouwusu and 5g of 12-epi-Ouwusu, adding capsule proper auxiliary materials, and preparing into anti-leukemia capsules according to the capsule preparation process.
While particular embodiments of the present invention have been illustrated and described, it will be appreciated that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.
Claims (1)
1.12-epi-euwu alkali as the only active ingredient in the preparation of the medicine for treating leukemia; the leukemia is myelogenous leukemia; the myelogenous leukemia is a disease caused by K-562 chronic myelogenous leukemia cells or HL-60 myelogenous leukemia cells.
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