CN110974826A

CN110974826A - Use of ouwu alkali or 12-epi-ouwu alkali in preparing medicine for treating leukemia

Info

Publication number: CN110974826A
Application number: CN201910595654.6A
Authority: CN
Inventors: 张帆; 韩佳; 陈泳洁
Original assignee: North Sichuan Medical College
Current assignee: North Sichuan Medical College
Priority date: 2019-07-03
Filing date: 2019-07-03
Publication date: 2020-04-10
Anticipated expiration: 2039-07-03
Also published as: CN110974826B

Abstract

The invention provides an application of ouwu alkali or 12-epi-ouwu alkali in preparing a medicament for treating leukemia, belonging to the field of medicaments. The inventor researches and discovers that both the orubine and the 12-epi-orubine can effectively inhibit the proliferation of leukemia cells, can promote the apoptosis of the leukemia cells, and particularly has more remarkable effect on myelogenous leukemia cells. Therefore, the eudragine and the 12-epi-eudragine both have the activity of resisting leukemia and hematopoietic tissue tumor, can be used as the medicine for treating leukemia and hematopoietic tissue tumor singly or in combination, and have wide application prospect.

Description

Use of ouwu alkali or 12-epi-ouwu alkali in preparing medicine for treating leukemia

Technical Field

The invention relates to the field of medicines, and in particular relates to application of ouwu alkali or 12-epi-ouwuwu alkali in preparation of a medicine for treating leukemia.

Background

At present, the development and utilization of the plant medicine are firstly to extract and separate the effective chemical components with related curative effects and purify the effective chemical components to obtain a single component with a certain curative effect. Wherein, the alkaloid is a nitrogenous organic compound with remarkable physiological activity, and a plurality of medicinal plants are rich in alkaloid and are an important group part of traditional Chinese medicines. The separation and purification of alkaloid effective components are the difficult and key points of traditional Chinese medicine development.

Aconitum carmichaeli (Aconitum karakolicum Rap.) is a plant of Aconitum L of Ranunculaceae, is distributed on hilly grassland with altitude of 2000-3000 m in Yili area of Xinjiang in China, is concentrated in distribution, and is mainly used for treating diseases such as neuralgia, angina pectoris, nephritis, etc. Wherein the radix Aconiti Kusnezoffii contains abundant alkaloids, and has high content of ouwuline (napelline) and 12-epi-ouwuline (12-epi-napelline).

Leukemia (leukemia) is a group of diseases caused by abnormal proliferation of hematopoietic stem cells. Leukemia usually occurs in young people, and is a malignant tumor with the highest morbidity and mortality rate below 35 years old. Leukemia is listed as one of ten high-incidence tumors in China. At present, more than 400 million leukemia patients are treated in the whole country, and 4 million leukemia patients are newly added every year. The poor therapeutic strategy results in a high mortality rate for patients with leukemia. Therefore, the development of new leukemia therapeutic drugs and therapeutic strategies is an urgent problem to be solved.

Disclosure of Invention

The invention aims to provide application of the eudragine or 12-epi-eudragine in preparing a medicament for treating leukemia, and provides a new strategy for treating leukemia.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

use of ouwu-alkaloid or 12-epi-ouwu-alkaloid 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 above-mentioned myeloid leukemia includes chronic myelogenous leukemia.

Further, in a preferred embodiment of the present invention, the above-mentioned myelocytic leukemia is a disease caused by K-562 chronic myelogenous leukemia cells or by HL-60 myelocytic leukemia cells.

Use of ouwu alkaloid or 12-epi-ouwu alkaloid in preparation of cell proliferation inhibitor for myelocytic leukemia is provided.

Use of ouwu alkaloid or 12-epi-ouwu alkaloid in preparing 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 ouwuline and 12-epi-ouwuline.

Compared with the prior art, the invention has the beneficial effects that:

the inventor researches and discovers that both the orubine and the 12-epi-orubine can effectively inhibit the proliferation of leukemia cells and promote the apoptosis of the leukemia cells, and particularly have more remarkable effect on myelogenous leukemia cells. Therefore, the eudragine and the 12-epi-eudragine both have the activity of resisting leukemia and hematopoietic tissue tumor, can be used as the medicine for treating leukemia and hematopoietic tissue tumor singly or in combination, 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 is a graph showing the inhibitory effect of ouabain (. mu.g/ml) on the proliferation of K-562 cells in example 1, wherein: p <0.05, P <0.01 compared to control;

FIG. 2 is a graph showing the inhibitory effect of ouabain (. mu.g/ml) on HL-60 cell proliferation in example 1, wherein: p <0.05, P <0.01 compared to control;

FIG. 3 is a graph of the inhibition of K-562 cell proliferation by 12-epi-ouwuweidine (. mu.g/ml) in example 1, wherein: p <0.05, P <0.01 compared to control;

FIG. 4 is a graph of the inhibitory effect of 12-epi-ouwuweidine (. mu.g/ml) on HL-60 cell proliferation in example 1, wherein: p <0.05, P <0.01 compared to control;

FIG. 5 is a flow cytometric distribution chart of the apoptotic effect of ouabain (. mu.g/ml) on K-562 cells in example 2;

FIG. 6 is a graph showing the apoptotic effect of ouabain (. mu.g/ml) on K-562 cells in example 2;

FIG. 7 is a flow cytometric distribution chart of the apoptotic effect of ouabain (. mu.g/ml) on HL-60 cells in example 2;

FIG. 8 is a graph showing the apoptotic effect of ouabain (. mu.g/ml) on HL-60 cells in example 2;

FIG. 9 is a flow cell distribution diagram of the apoptotic effect of 12-epi-ouwuweidine (. mu.g/ml) on K-562 cells in example 2;

FIG. 10 is a graph of the apoptotic effect of 12-epi-ouwuweidine (. mu.g/ml) on K-562 cells in example 2;

FIG. 11 is a flow cell distribution diagram of the apoptotic effect of 12-epi-ouwuweidine (. mu.g/ml) on HL-60 cells in example 2;

FIG. 12 is a graph of the apoptotic effect of 12-epi-ouwuweidine (. mu.g/ml) on HL-60 cells in example 2;

FIG. 13 is a graph showing the results of the morphological measurement of apoptosis of K-562 cells by eudrine (. mu.g/ml) in example 3;

FIG. 14 is a graph showing the results of the morphological measurement of apoptosis of K-562 cells by 12-epi-ouwuweine (. mu.g/ml) in example 3;

FIG. 15 is a graph showing the results of morphological examination of apoptosis of HL-60 cells by eudrine (. mu.g/ml) in example 3;

FIG. 16 is a graph showing the results of morphological examination of apoptosis of HL-60 cells by 12-epi-ouwuweine (. 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 illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which the specific conditions are not specified, were conducted under the conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are conventional products available commercially.

The ouwuweizi and 12-epi-ouwuweizi are isomers, and the molecular formula is C₂₂H₃₃NO₃Both belong to C-20 type diterpenoid alkaloids, the difference between the two types is that the configuration of 12-position hydroxyl of the eudragit is α -type,and the configuration of 12-epi-ouwuweizi at 12-position hydroxyl is β -type, and the chemical structures of the two are shown in the following formulas.

In the invention, the eugonine and the 12-epi-eugonine can be used as active ingredients independently or can be used together in a combined way to play the effects of inhibiting the proliferation of leukemia cells and promoting the apoptosis of the leukemia cells together.

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 technical fields. The route of administration of the pharmaceutical composition of the present invention is oral, nasal inhalation, or parenteral administration. The pharmaceutical composition can be prepared into powder, granule, tablet, emulsion, syrup, aerosol, soft capsule, hard capsule, sterile injection, sterile powder, etc.

Herein, the term "pharmaceutically acceptable" refers to a compound which is physiologically acceptable when both ouabain and 12-epi-ouabain are administered to humans, and does not cause allergic reactions such as gastrointestinal disorders, dizziness or systemic allergic reactions like these.

In the present invention, "pharmaceutically acceptable carrier" includes but is not limited to: binders (such as microcrystalline cellulose, alginates, gelatin, and polyvinylpyrrolidone), fillers (such as starch, sucrose, glucose, and anhydrous lactic acid), disintegrants (such as 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 (such as glycerin), surfactants (such as cetyl alcohol), and absorption enhancers, taste modifiers, sweeteners, diluents, coating agents, and the like.

In the present invention, the active ingredient of the pharmaceutical composition also includes "pharmaceutically acceptable salts" formed from ouwuline or 12-epi-ouwuline. Herein, "pharmaceutically acceptable salts" include pharmaceutically acceptable non-toxic salts of ouwubase or 12-epi-ouwubase formed by standard reactions 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 comprises, for example, formic acid, acetic acid, citric acid, maleic acid, tartaric acid, ascorbic acid, salicylic acid, sorbic acid, benzoic acid, methanesulfonic acid, p-toluenesulfonic acid and the like.

The content of the ouwuweizi or 12-epi-ouwuweizi in the pharmaceutical composition of the present invention is a pharmaceutically effective dose. As used herein, the term "pharmaceutically effective dose" 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, body weight, health condition, sex, and administration route and treatment period of the patient.

The features and properties of the present invention are further described in detail below with reference to examples:

example 1

Anti-leukemia cell proliferation activity assay:

the CCK-8 method is adopted to detect the activity of the eusines in resisting K-562 and HL-60 cells of the eusines in combination with 12-epi-eusines.

(1) Pharmaceutical formulation

Weighing appropriate amount of eudrine and 12-epi-eudrine with electronic balance, and dissolving the medicine completely with DMSO.

(2) Solvent formulation

Preparation of a culture medium: prepared according to the proportion of high-sugar DMEM medium to serum and double antibody 100:10:1

Preparation of PBS: weighing 8g of NaCl, 0.2g of KC l0, 41.42g of Na2HPO41 and 40.27g of KH2PO40 by using an electronic balance; adding about 800ml of deionized water, fully stirring and dissolving on a magnetic stirrer, and then making the volume constant to 1L. Autoclaving at 120 deg.C for 30min, and placing in a refrigerator at 4 deg.C.

Preparing a frozen stock solution: prepared according to the ratio of DMSO to 9:1, and is used as the preparation.

(3) Cell culture

In the 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 Chinese academy of sciences.K-562 and HL-60 are suspension cells, and both types of cells are at 37 ℃ and 5% CO₂Growth was performed in ambient conditions and experiments were performed in high glucose DMEM complete medium containing 10% fetal bovine serum.

Cell liquid change: cells from the cell bank were wiped with 75% alcohol and the morphology and state of the cells were observed under an inverted microscope. The cells were collected in a 50ml centrifuge tube, centrifuged at 1000r for 5min, the supernatant was discarded, the cells were resuspended in a fresh serum-containing medium, the cells were inoculated into a fresh flask and, if the cell debris was large, washed 1-2 times with PBS.

Cell passage: when the cells grow to 70% -80% of the culture flask, the cells need to be passaged. Collecting cells, centrifuging the cells in a centrifuge tube for 5min at 1000r, and removing supernatant; PBS wash 1 time, 1000r, 5min centrifugation, discard supernatant. The cells were resuspended in serum-containing medium and the cells were inoculated on average into new flasks, either 1 pass 2 or 3 depending on cell density.

Freezing and storing cells: in order to prevent the cells from having too many cell states due to contamination or passage during the culture process, the cells need to be frozen. Collecting cells in logarithmic phase, centrifuging for 5min at 1000r, and removing supernatant; resuspending the cells in a freezing medium and adjusting the cell density to 5X 10⁶-1×10⁷And (4) subpackaging the cell suspension into about 1mL of cryopreservation tubes, and storing the tubes in a refrigerator at the temperature of-80 ℃.

Cell recovery: taking out the freezing tube from-80 deg.C, soaking in 37 deg.C water bath, centrifuging at 1000r for 5min after the liquid in the freezing tube is completely dissolved, discarding supernatant, resuspending with serum-containing culture medium, inoculating into culture bottle, and adding 37 deg.C CO₂Culturing in an incubator.

(4) CCK-8 method for detecting cell proliferation test

① K-562 cells

Cells grown in log phase at 2X 10 were taken⁴ Perwell 100. mu.l/well of 96-well cell culture plates at 37 ℃ with 5% CO₂Culturing for several hours in incubator, and adding medicine after cell is stable. Setting blank group, control group, ouwu alkali group (20, 40, 60, 80 μ g/ml), 12-epi-ouwu alkali group (12.5, 25, 50, 1)00. mu.g/ml) of 3 replicates per group. The blank group contained no cells, the vehicle control group incubated cells with fresh complete DMEM medium, the ouwuweizi group incubated cells with fresh complete DMEM medium containing drugs at final concentrations of 20, 40, 60, 80 μ g/ml, respectively, and the 12-epi-ouwuweizi group incubated cells with fresh complete DMEM medium containing drugs at final concentrations of 12.5, 25, 50, 100 μ g/ml, respectively. After the above treatment, 5% CO was added at 37 deg.C₂After the conventional culture in an incubator for 24h and 48h, CCK-810 mu l is added into each hole in a dark place, and after the continuous culture for 1-3h, the absorbance of each hole is measured at 450nm by using a microplate reader. The number of living cells is indirectly reflected by the absorbance, the survival rate of the cells acted by the drugs with different concentrations is calculated, and the experiment is repeated for 3 times to record the result.

② HL-60 cells

Taking log-phase grown cells, HL-60 cells were seeded at 4X 104/well in 96-well cell culture plates at 100. mu.l/well at 37 ℃ in 5% CO₂Culturing for several hours in an incubator by a conventional method, and adding the medicine after the cells are stabilized; 3 duplicate wells of blank, control, ouabain (15, 30, 50, 90. mu.g/ml), 12-epi-ouabain (7.5, 15, 30, 60. mu.g/ml) groups were set. The blank group contained no cells, the vehicle control group incubated cells with fresh complete DMEM medium, the ouniu alkali group incubated cells with fresh complete DMEM medium containing drugs at final concentrations of 15, 30, 50, 90 μ g/ml, respectively, and the 12-epi-ouniu alkali group incubated cells with fresh complete DMEM medium containing drugs at final concentrations of 7.5, 15, 30, 60 μ g/ml, respectively. After the above treatment, 5% CO was added at 37 deg.C₂After the conventional culture in an incubator for 24h and 48h, CCK-810 mu l is added into each hole in a dark place, and after the continuous culture for 1-3h, the absorbance of each hole is measured at 450nm by using a microplate reader. The number of living cells is indirectly reflected by the absorbance, the survival rate of the cells acted by the drugs with different concentrations is calculated, and the experiment is repeated for 3 times to record the result.

Calculating the formula: survival rate ═(dosing group-blank group)/(control group-blank group)). 100

(5) Data processing

SPSS 22.0 statistical software is adopted to process data, metering data is expressed by x +/-s, Student T-Test is adopted to compare the mean values of two samples, One-way ANOVA is adopted to compare the mean values among multiple sample groups, P <0.05 shows that the statistical significance is achieved, and P <0.01 shows that the tested difference has very significant significance.

(6) Results of the experiment

① Eudragit-L inhibiting effect on K-562 and HL-60 cell proliferation

The ouwuweidine (20-80 μ g/ml) has significant inhibitory effect on K-562 cell proliferation after acting on K-562 tumor cells, and is time-dose dependent, see FIG. 1.

Eudragine (15-90 μ g/ml) has remarkable inhibitory effect on HL-60 cell proliferation after acting on HL-60 tumor cells, and is time-dose dependent, see FIG. 2.

② 12 inhibition of K-562, HL-60 cell proliferation by epieudrine

After 12-epi-ouwu alkaloid (12.5-100 mug/ml) acts on K-562 tumor cells, the proliferation of the K-562 cells is obviously inhibited, and the time-dose dependence is shown in figure 3.

After 12-epi-ouwu alkali (7.5-60 μ g/ml) acts on HL-60 tumor cells, the proliferation of HL-60 cells is inhibited, and the time-dose dependence is shown in figure 4.

Example 2

Leukemia cell apoptosis promotion experiment:

flow cytometry is adopted to detect apoptosis effect of ouwu alkali and 12-epi-ouwu alkali on K-562 and HL-60 cells

(1) Pharmaceutical formulation

Weighing appropriate amount of eudrine and 12-epi-eudrine by electronic balance, and dissolving the medicine completely with DMSO.

(2) Blank plate

① K-562 cells in logarithmic growth phase K-562 cells were harvested, plated at 6X 105/well in 6-well plates at 37 ℃ with 5% CO₂Culturing in incubator for several hours until the cells are stable.

② HL-60 cells, collecting HL-60 cells in logarithmic growth phase, inoculating to 6-well plate at 1 × 106/well density, and culturing at 37 deg.C under 5% CO₂Culturing for several hours in incubatorAnd (4) stabilizing.

(3) Dosing

① K-562 cells, adding eugonine (40, 60 μ g/ml) and 12-epi-eugonine (25, 50 μ g/ml) after the cells are stabilized, and adding 5% CO at 37 deg.C₂Culturing in incubator for 24 hr.

② HL-60 cells, adding ouwu alkali (30, 50 μ g/ml) and 12-epi-ouwu alkali (15, 30 μ g/ml) after the cells are stabilized, and adding 5% CO at 37 deg.C₂Culturing in incubator for 24 hr.

(4) Flow cytometry detection of apoptosis of tumor cells

① apoptosis, namely culturing for 24 hours, collecting cells in a centrifuge tube, centrifuging for 5min at 1000r, discarding the supernatant, adding 1ml of PBS precooled by ice bath to resuspend the cells, transferring the cells into a 1.5ml centrifuge tube, centrifuging for 5min at 1000r again, discarding the supernatant, adding 195 mu l of Annexin V-FITC binding solution to each sample under the condition of keeping out of the sun to resuspend the cells, adding 5 mu l of Annexin V-FITC to mix uniformly, adding 10 mu l of propidium iodide staining solution to mix uniformly, incubating for 10-20min at room temperature in the shade, and repeating the detection for 3 times by a flow cytometer.

② period, 24h later, collecting cells in a centrifuge tube, centrifuging for 1000r 5min, discarding the supernatant, adding 1ml PBS precooled by ice bath, resuspending the cells and transferring the cells into a 1.5ml centrifuge tube, centrifuging again for 1000r 5min, discarding the supernatant, adding 1ml PBS precooled by ice bath, 70% ethanol, fixing for 2h at 4 ℃, centrifuging for 1000r 5min, discarding the supernatant, adding 1ml PBS precooled by ice bath, re-centrifuging again for 1000r 5min, discarding the supernatant, preparing 1 sample propidium iodide staining solution, wherein the preparation of the staining buffer solution is 0.5ml, the propidium iodide staining solution (20x)25 μ l, the RNaseA (50x)10 μ l, after the preparation of the propidium iodide staining solution, 0.5ml propidium iodide staining solution is added to each sample, resuspending the cells, and carrying out warm bath at 37 ℃ for 30min in a dark place, and repeating the detection by a flow cytometer for 3 times.

(5) Results of the experiment

① apoptosis of Eudrine on K-562 cell and HL-60 cell

The apoptosis rate of eudrine (40, 60 μ g/ml) was increased with increasing concentration after 24h on K-562 cells, as shown in FIGS. 5 and 6.

After the eudrine (30, 50 mu g/ml) acts on HL-60 cells for 24h, the apoptosis rate of the cells is increased along with the increase of the concentration, as shown in figure 7 and figure 8.

② 12 apoptosis of K-562 cells and HL-60 cells by using epi-ouwu alkali

After the 12-epi-ouwuweidine (25. mu.g/ml, 50. mu.g/ml) was applied to K-562 cells for 24h, the apoptosis rate was significantly different and statistically significant (P0.01) compared to the control group, and the apoptosis rate increased with increasing concentration, as shown in FIGS. 9 and 10.

After 12-epi-ouwuweidine (15 μ g/ml, 30 μ g/ml) was applied to HL-60 cells for 24h, the apoptosis rate was significantly different and statistically significant (P0.01) compared to the control group, and the apoptosis rate increased with increasing concentration, as shown in fig. 11 and 12.

Example 3

Morphological detection of leukemia cell apoptosis:

detecting apoptosis of K-562 and HL-60 cells by Hoechst staining with eudragine and 12-epi-eudragine

(1) Blank plate

① K-562 cells in logarithmic growth phase, collecting cells at 6X 10⁵Density of one well was inoculated in 6-well plates at 37 ℃ with 5% CO₂Culturing in incubator for several hours until the cells are stable. A blank control group, a low concentration group and a high concentration group were set.

② HL-60 cells, collecting HL-60 cells in logarithmic growth phase, and culturing at 1 × 10⁶Density of one well was inoculated in 6-well plates at 37 ℃ with 5% CO₂Culturing in incubator for several hours until the cells are stable. A blank control group, a low concentration group and a high concentration group were set.

(2) Dosing

① K-562 cells, adding eugonine (40, 60 μ g/ml) and 12-epi-eugonine (25, 50 μ g/ml) after the cells are stabilized, and adding 5% CO at 37 deg.C₂Culturing in incubator for 24h and 48 h.

② HL-60 cells, adding ouwu alkali (30, 50 μ g/ml) and 12-epi-ouwu alkali (15, 30 μ g/ml) after the cells are stabilized, and adding 5% CO at 37 deg.C₂Culturing in incubator for 24h and 48 h.

(3) Hoechst staining

① 24h and 48h later, collecting cells in a 1.5ml centrifuge tube, adding 0.5ml fixing solution, slowly suspending the cells, and fixing for 15 min.

② the fixative was centrifuged off and washed twice with PBS for 3min each.

③ after centrifugation, most of the liquid was aspirated and approximately 50. mu.l of the liquid was retained, the cells were suspended slowly and dropped onto the slide to distribute the cells as evenly as possible.

④ air dried slightly to make the cells not easy to flow with the liquid when attached to the slide.

⑤ and 0.5ml of Hoechst 33258 staining solution is dropped on the cloth evenly, and the cloth is stained for 5min, and the liquid is sucked from the edge by absorbent paper and dried slightly.

⑥ removing staining solution, washing twice with PBS for 3min, sucking off the solution, and washing with shaker.

⑦ A drop of the anti-fluorescence quenching mounting solution was placed on the slide and covered with a clean coverslip to avoid air bubbles as much as possible.

⑧ the fluorescence microscope can detect blue cell nucleus, the excitation wavelength is about 350nm, and the emission wavelength is about 460 nm.

(4) Results of the experiment

① Hoechst staining for detecting K-562 apoptosis

The results of treating K-562 cells with different concentrations of ouwuweine for 24h and 48h are shown in FIG. 13; the results of treating K-562 cells with different concentrations of ouwuweine for 24h and 48h are shown in FIG. 14.

As can be seen from FIGS. 13 and 14, the blank control group K-562 cells had clear nuclear boundaries, were round or oval, and exhibited normal blue fluorescence; the eudrine and 12-epi-eudrine treated group showed compact and thick staining of cell nucleus, or massive compact and thick staining, and some whitish color. The apoptosis degree of K-562 cells is observed in a fluorescence microscope and is in a dose-time dependence.

② Hoechst staining for detecting HL-60 cell apoptosis

The results of different concentrations of ouwu alkali treatment of HL-60 cells for 24h and 48h are shown in FIG. 15; the results of different concentrations of ouwuweizine after 24h and 48h treatment of HL-60 cells are shown in FIG. 16.

As can be seen from FIGS. 15 and 16, the blank group HL-60 cells had clear nuclear boundaries, were circular or oval, and exhibited normal blue fluorescence; the eudrine and 12-epi-eudrine treated group showed compact and thick staining of cell nucleus, or massive compact and thick staining, and some whitish color. The observation of a fluorescence microscope shows that the apoptosis degree of HL-60 cells is in dose-time dependence.

Formulation example 1

Taking 10g of the eudragine, adding proper auxiliary materials into tablets (including sustained release tablets, matrix tablets, dispersible tablets and the like), and preparing the anti-leukemia tablets according to the preparation process of the tablets.

Formulation example 2

Taking 10g of 12-epi-ouwu alkali, adding proper auxiliary materials of the injection, and preparing the anti-leukemia injection according to the preparation process of the injection.

Formulation example 3

Taking 5g of ouwu alkali and 5g of 12-epi-ouwu alkali, adding appropriate capsule adjuvants, and making into capsule for treating leukemia according to capsule preparation process.

While particular embodiments of the present invention have been illustrated and described, it would be obvious 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. Use of ouwu-alkaloid or 12-epi-ouwu-alkaloid in preparing medicine for treating leukemia is provided.

2. The use according to claim 1, wherein the leukemia is a myeloid leukemia.

3. The use according to claim 2, wherein the myeloid leukemia comprises chronic myelogenous leukemia.

4. The use according to claim 2, wherein the myeloid leukemia is a disease caused by K-562 chronic myelogenous leukemia cells or by HL-60 myeloid leukemia cells.

5. Use of ouwu alkaloid or 12-epi-ouwu alkaloid in preparation of cell proliferation inhibitor for myelocytic leukemia is provided.

6. Use of ouwu alkaloid or 12-epi-ouwu alkaloid in preparing apoptosis promoter for myeloid leukemia is provided.

7. A pharmaceutical composition comprising an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is at least one of ouwuline and 12-epi-ouwuline.