CN111514133A

CN111514133A - Application of costunolide and/or dehydrocostuslactone in preparing medicine for treating melanoma

Info

Publication number: CN111514133A
Application number: CN202010419304.7A
Authority: CN
Inventors: 刘蕊; 王丹丹; 张景照; 唐旭东
Original assignee: Shenzhen Songle Biotechnology Co ltd
Current assignee: Shenzhen Songle Biotechnology Co ltd
Priority date: 2020-05-18
Filing date: 2020-05-18
Publication date: 2020-08-11

Abstract

The invention provides an application of costunolide and/or dehydrocostuslactone in preparing a medicine for preventing and/or treating melanoma, belonging to the technical field of biological medicine. In the invention, the costunolide and/or dehydrocostuslactone can inhibit the proliferation of melanoma cells B16 and induce apoptosis, and has the effect of resisting melanoma.

Description

Application of costunolide and/or dehydrocostuslactone in preparing medicine for treating melanoma

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of costunolide and/or dehydrocostuslactone in preparing a medicine for treating melanoma.

Background

Melanoma, also known as Malignant Melanoma (MM), is the most dangerous one of skin cancers, has very strong proliferative capacity, and is the skin cancer that causes the most number of deaths. The detection rate of melanoma in China is increased, and new diseases are continuously increased every year. At present, the main conventional methods for treating melanoma are surgery, radiotherapy and chemotherapy, but have the technical problems of large wound and obvious adverse reaction.

Disclosure of Invention

The invention aims to provide application of costunolide and/or dehydrocostuslactone in preparing a medicine for treating melanoma.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides application of costunolide and/or dehydrocostuslactone in preparing a medicine for preventing and/or treating melanoma.

Preferably, the costunolide and/or dehydrocostuslactone treat melanoma by inhibiting melanoma cell proliferation and/or inducing melanoma cell apoptosis.

Preferably, when the medicine comprises costunolide and dehydrocostuslactone, the mass ratio of the costunolide to the dehydrocostuslactone is (1-10): (1-10).

Preferably, the medicine also comprises ginsenoside Rh2 or astragalus polysaccharide.

Preferably, when the medicine comprises costunolide and ginsenoside Rh2, the mass ratio of the costunolide to the ginsenoside Rh2 is (1-10): (1-10).

Preferably, when the medicine comprises costunolide and astragalus polysaccharide, the mass ratio of the costunolide to the astragalus polysaccharide is (1-10): (1-10).

Preferably, the dosage forms of the medicine comprise an oral preparation and an external preparation; the oral preparation comprises granules, tablets, decoctions or capsules; the external preparation comprises ointment or powder.

Preferably, the medicine also comprises auxiliary materials; when the dosage form of the medicine is granules, the auxiliary materials comprise beta-cyclodextrin and magnesium stearate; when the dosage form of the medicine is a tablet, the auxiliary materials comprise hypromellose, magnesium stearate and maltodextrin; when the medicament is in the form of an ointment, the auxiliary material comprises vaseline.

The invention has the beneficial effects that: the invention provides application of costunolide and/or dehydrocostuslactone in preparing a medicine for preventing and/or treating melanoma. In the invention, the costunolide and/or dehydrocostuslactone can inhibit the proliferation of melanoma cells B16 and induce apoptosis, and has the effect of resisting melanoma. Costunolide treatment of B16 cells for 24h, IC of 48h₅₀Values were 28.34. mu. mol/L and 19.21. mu. mol/L, respectively; IC of dehydrocostuslactone treated B16 cells for 24h and 48h₅₀The values were 29.54. mu. mol/L and 21.22. mu. mol/L, respectively; after costunolide is used for treating B16 cells for 48h, the cell morphology is obviously changed, mainly the change of apoptosis related morphology, including cell contraction, high chromatin condensation, visible formation of apoptotic bodies and nuclear degradation; after the dehydrocostuslactone is used for treating B16 cells for 48h, the cell morphology is obviously changed, mainly the change of apoptosis-related morphology, including cell contraction, high chromatin condensation, visible formation of apoptotic bodies and nuclear degradation; after costunolide is used for treating cells for 48 hours, 35.1 percent of B16 cells are induced to die when the concentration is 25 mu mol/L; costunolide at a concentration of 50 μmol/L induced apoptosis of 50.2% of B16 cells; induces 57.6% of B16 apoptosis at a concentration of 100. mu. mol/L; after dehydrocostuslactone treatment of cells for 48h, 36% of B16 cells were induced to die at a concentration of 25. mu. mol/L; dehydroalcoholic at a concentration of 50. mu. mol/L induced 47.9% of B16 cell apoptosis; at a concentration of 100. mu. mol/L, 57.5% of B16 cells were induced to undergo apoptosis.

Drawings

FIG. 1 is a graph of the effect of costunolide on B16 cell proliferation;

FIG. 2 is a graph of the effect of dehydrocostuslactone on B16 cell proliferation;

FIG. 3-1 is a blank control B16 cell morphology;

FIG. 3-2 shows the morphology of B16 cells after 25. mu. mol/L costunolide treatment;

FIG. 4-1 is a blank control B16 cell morphology;

FIG. 4-2 shows the morphology of B16 cells after 25. mu. mol/L dehydrocostuslactone treatment;

FIG. 5-1 shows the results of flow cytometry analysis of apoptosis rate of blank control B16 cells;

FIG. 5-2 is a flow cytometer analyzing the apoptosis rate of B16 cells by 25. mu. mol/L costunolide;

FIG. 5-3 shows the apoptosis rate of B16 cells by flow cytometry analysis of 50. mu. mol/L costunolide;

FIG. 5-4 shows the apoptosis rate of B16 cells analyzed by flow cytometry at 100. mu. mol/L costunolide;

FIG. 6-1 shows the results of flow cytometry analysis of apoptosis rate of blank control B16 cells;

FIG. 6-2 is a flow cytometer analyzing the apoptosis rate of B16 cells by 25. mu. mol/L dehydrocostuslactone;

FIG. 6-3 shows the flow cytometry analysis of the apoptosis rate of B16 cells by 50. mu. mol/L dehydrocostuslactone;

FIG. 6-4 shows the flow cytometry analysis of the apoptosis rate of B16 cells by 100. mu. mol/L dehydrocostuslactone;

FIG. 7 shows Western Blot analysis of B16 apoptosis-related protein expression by costunolide.

Detailed Description

The invention provides an application of costunolide and/or dehydrocostuslactone in preparing a medicament for preventing and/or treating melanoma; the costunolide and/or dehydrocostus lactone can be used for treating melanoma by inhibiting proliferation of melanoma cells and/or inducing apoptosis of melanoma cells; the costunolide releases Cyto c through mitochondria so as to activate caspase apoptosis pathway, finally cause B16 cell apoptosis, and play a role in resisting melanoma.

In the invention, the chemical structural formula of the Costunolide (Costunolide) is shown as a formula I, the Costunolide is from conventional markets, the purity is more than 98 percent, and the Costunolide has a CAS number: 553-21-9.

In the invention, the chemical structural formula of the Dehydrocostus Lactone (Dehydrocostus Lactone) is shown as a formula II, the Dehydrocostus Lactone is from conventional markets, the purity is more than 98 percent, and the CAS number is as follows: 477-43-0.

In the invention, when the medicine comprises costunolide and dehydrocostuslactone, the mass ratio of the costunolide to the dehydrocostuslactone is preferably (1-10): (1-10), more preferably (1-4): (1-4).

In the present invention, the medicament preferably further comprises ginsenoside Rh2 or astragalus polysaccharide; when the medicine comprises costunolide and ginsenoside Rh2, the mass ratio of the costunolide to the ginsenoside Rh2 is preferably (1-10): (1-10), more preferably (1-4): (1-4); when the medicine comprises costunolide and astragalus polysaccharide, the mass ratio of the costunolide to the astragalus polysaccharide is preferably (1-10): (1-10), more preferably (1-4): (1-4).

In the invention, the ginsenoside Rh2 has an anticancer effect, and can also enhance the immunity of the organism and quickly recover the physique; the Astragalus polysaccharides have antitumor, antioxidant, and antiaging effects. In the invention, the ginsenoside Rh2 and the astragalus polysaccharide are from conventional markets.

In the present invention, the dosage forms of the drug product include oral preparations and external preparations; the oral preparation preferably comprises granules, tablets, decoction or capsules; the external preparation preferably comprises an ointment or powder; the medicine preferably also comprises auxiliary materials;

when the dosage form of the medicine is granules, the auxiliary materials preferably comprise beta-cyclodextrin and magnesium stearate, and the mass ratio of the active ingredients, the beta-cyclodextrin and the magnesium stearate in the medicine is preferably (105-110): (5-7): (5-7), more preferably 108: 6: 6; when the dosage form of the medicine is a tablet, the auxiliary materials preferably comprise hypromellose, maltodextrin and magnesium stearate, and the mass ratio of the active ingredients, the hypromellose, the maltodextrin and the magnesium stearate in the medicine is preferably (85-95): (5-7): (3-5): (0.3 to 0.8), more preferably 89.5: 6: 4: 0.5; when the dosage form of the medicine is decoction, the medicine is decocted, dregs are removed, and the medicine is obtained without auxiliary materials; when the medicament is in a capsule form, the auxiliary materials comprise starch, magnesium stearate and hydroxypropyl methylcellulose in a dextrorotation mode; when the preparation form of the medicine is an ointment, the auxiliary material preferably comprises vaseline, and the mass ratio of the active ingredients in the medicine to the vaseline is preferably 1-3: 7-9, more preferably 1: 4; when the medicament is in the form of powder, the auxiliary materials preferably comprise starch, talcum powder and dextrin.

In the invention, the dosage standard of the medicine is as follows: granules: the packaging specification is 2 g/bag. The administration method comprises 1 time 1 bag and 1 day 2 times; and (3) tablet preparation: the packaging specification is 0.5 g/tablet. The administration method comprises taking two tablets 1 time, and taking 3 times per day; cream preparation: applied to affected part for 3 times a day.

In the invention, the oral preparation is orally taken, and the effective components are absorbed by gastrointestinal tracts in vivo, act on tumor positions through blood circulation and resist tumors; the external preparation is directly acted on the skin without oral administration or injection administration, and has local or systemic treatment effect; the process of the external preparation for transdermal exertion: the skin surface is horny layer, and the external preparation penetrates the horny layer of the skin and enters the horny layer-dermis junction layer to play an anti-tumor role in melanoma.

The medicine of the present invention can prevent melanoma, and because melanotic nevus located at the interface between epidermis and dermis is easily malignant, it is considered as the prodromal stage of melanoma.

The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The materials used in the examples of the present invention:

costunolide (commercially available) (purity > 98%) (CAS No.: 553-21-9).

Dehydrocostuslactone (Dehydrocostus Lactone) (commercially available) (purity > 98%) (CAS number: 477-43-0).

Ginsenoside Rh2 (commercially available) (CAS number 78214-33-2).

Astragalus polysaccharides, commercially available.

Mouse B16 melanoma cells.

Example 1MTT assay to determine the Effect on melanoma cell B16 cell proliferation

Taking mouse B16 melanoma cell in logarithmic growth phase, microscopically counting the cell, and adjusting the cell concentration to 1 × 10⁵one/mL, inoculated into a 96-well plate at 100. mu.L per well, i.e., 1 × 10⁴And each well, after the cells grow in an adherent manner overnight, respectively adding 100 mu L of fresh culture medium containing the aromatic hydrocarbon lactone or the dehydrocostuslactone, setting the drug concentration to be 3.125 mu M, 6.25 mu M, 12.5 mu M, 25 mu M and 50 mu M, simultaneously setting blank control groups without adding drugs, setting 6 multiple wells for each group, respectively culturing for 24, 48 and 72 hours, adding 20 mu LMTT solution into each well, continuously culturing for 4 hours in a cell culture box, discarding the old culture medium, adding 200 mu LDMSO into each well, and measuring the absorbance value by an enzyme reader A570 nm.

The cell proliferation inhibition ratio (%) (a blank group-a administration group)/a blank group × 100%.

The results are as follows:

after costunolide treatment of mouse B16 melanoma cells for 24h and 48h, compared with the blank control group without administration, the costunolide can obviously inhibit the proliferation of mouse B16 melanoma cells (figure 1, note that P < 0.05 has significant difference,denoted by "; p < 0.01 with very significant differences, indicated by x). When B16 cells are treated by costunolide at 0-100 μmol/L, the inhibition rate increases with the increase of concentration; and the inhibition rate is also increasing with time. For example, the inhibition rate of costunolide with the concentration of 25 mu mol/L on B16 cells for 24h is 42.01%, and the inhibition rate at 48h is 62.23% (FIG. 1). Costunolide treatment of B16 cells for 24h, IC of 48h₅₀The values were 28.34. mu. mol/L and 19.21. mu. mol/L, respectively.

After the dehydrocostuslactone treatment of mouse B16 melanoma cells for 24h and 48h, the proliferation of mouse B16 melanoma cells was significantly inhibited compared to the control group without the drug (FIG. 2, P < 0.05 is significantly different and is indicated by x; P < 0.01 is significantly different and is indicated by x). When B16 cells are treated by 0-100 μmol/L dehydrocostuslactone, the inhibition rate increases with the increase of concentration; and with the increase of time, the inhibition rate is also 40.21% at 24h and 60.06% at 48h when the B16 cells are inhibited by increasing the concentration of the dehydrocostus hydrocarbon ester of 25 mu mol/L (figure 2). IC of dehydrocostuslactone treated B16 cells for 24h and 48h₅₀The values were 29.54. mu. mol/L and 21.22. mu. mol/L, respectively.

Therefore, costunolide and dehydrocostuslactone have strong capability of resisting B16 melanoma cell proliferation.

Example 2 fluorescent microscopy of apoptosis after DAPI staining of nuclei

Take 3mL1 × 10⁵Inoculating mLB16 cell fluid into a 60mm culture dish with a slide, after the cell fluid grows overnight by adhering to the wall, discarding the old culture medium, respectively adding 3mL of fresh culture medium containing costunolide or dehydrocostunolide with the concentration of 25 MuM, setting a blank control group, placing the culture medium in a cell culture box for continuous culture for 48h, washing twice by PBS, adding precooled 75% ethanol, fixing the mixture in a refrigerator at 4 ℃ overnight, discarding the fixing fluid, adding PBS for washing 3 times, adding DAPI staining fluid, keeping out of the sun at 37 ℃ for 30min, washing by PBS, observing the cell morphology under a fluorescence inversion microscope, and photographing and analyzing.

The results are as follows:

after B16 cells are treated by costunolide, the number of the cells is obviously reduced under the observation of a microscope, and the phenomena of shrinkage and digestion appear. The influence of costunolide on the morphology of B16 cells was observed under an inverted microscope after DAPI staining (fig. 3-1, fig. 3-2), wherein fig. 3-1 is blank control B16 cell morphology, fig. 3-2 is 25 μmol/L costunolide treated B16 cell morphology, and after costunolide treated B16 cells for 48h, the cell morphology changed significantly, mainly apoptosis-related morphological changes including cell contraction, hyperchromatin condensation, visible formation of apoptotic bodies and nuclear degradation.

After B16 cells are treated by dehydrocostuslactone, the number of the cells is obviously reduced, and the phenomena of shrinkage and digestion appear under the microscope. The effect of dehydrocostuslactone on the morphology of B16 cells was observed under an inverted microscope after DAPI staining (fig. 4-1, fig. 4-2), wherein fig. 4-1 is blank control B16 cell morphology, fig. 4-2 is B16 cell morphology after 25 μmol/L dehydrocostuslactone treatment, and significant changes in cell morphology, mainly apoptosis-related morphological changes, including cell contraction, hyperchromatic aggregation, visible formation of apoptotic bodies and nuclear degradation, occurred after 48h dehydrocostuslactone treatment of B16 cells.

Indicating that costunolide and dehydrocostuslactone cause apoptosis of B16 cells.

Example 3 flow cytometry FCM assay to analyze apoptosis

Take 3mL1 × 10⁵Inoculating mLB16 cell liquid into a 60mm culture dish, after the cell liquid grows in an adherent manner overnight, discarding old culture medium, adding fresh culture medium containing medicines with different concentrations, setting a medicine-free group as a control group, placing the cell liquid in a cell culture box for continuous culture for 48 hours, digesting cells by pancreatin without EDTA, centrifuging the cell liquid at 1000rpm for 5min, abandoning supernatant, washing the cells twice by PBS, re-suspending the cells by PBS, taking 1 × 104 re-suspended cells, centrifuging the cells, adding 195 mu L of annexin V-FITC binding solution, re-suspending the cells, adding 5 mu L of annexin V-FITC staining solution, mixing the cells gently, adding 10 mu L of PI, mixing the mixture, incubating the mixture at room temperature in a dark place for 20min, then placing the mixture on ice, detecting by a flow cytometer, and analyzing the apoptosis rate.

The results are as follows:

a flow cytometry detects that costunolide induces B16 cell apoptosis, and after B16 cells are treated by medicines with different concentrations for 48 hours, flow detection results show that costunolide can significantly induce B16 cell apoptosis and increase the apoptosis number, and the apoptosis rate is increased along with the increase of the concentration. After costunolide is used for treating cells for 48 hours, 35.1 percent of B16 cells are induced to die when the concentration is 25 mu mol/L; costunolide at a concentration of 50 μmol/L induced apoptosis of 50.2% of B16 cells; 57.6% of B16 cells were induced at a concentration of 100. mu. mol/L (FIG. 5-1, FIG. 5-2, FIG. 5-3, FIG. 5-4, where FIG. 5-1 is the results of flow cytometry analysis of apoptosis rate of blank control B16 cells; FIG. 5-2 is the results of flow cytometry analysis of apoptosis rate of 25. mu. mol/L costunolide on B16 cells; FIG. 5-3 is the results of flow cytometry analysis of apoptosis rate of 50. mu. mol/L costunolide on B16 cells; FIG. 5-4 is the results of flow cytometry analysis of apoptosis rate of 100. mu. mol/L costunolide on B16 cells).

A flow cytometry detects that dehydrocostuslactone induces B16 cell apoptosis, and after B16 cells are treated by medicines with different concentrations for 48 hours, flow detection results show that the dehydrocostuslactone can significantly induce B16 cell apoptosis and increase the cell apoptosis number, and the cell apoptosis rate is increased along with the increase of the concentration. After dehydrocostuslactone treatment of cells for 48h, 36% of B16 cells were induced to die at a concentration of 25. mu. mol/L; dehydrocostuslactone at a concentration of 50 μmol/L induced apoptosis of 47.9% of B16 cells; 57.5% of B16 cells were induced at a concentration of 100. mu. mol/L (FIG. 6-1, FIG. 6-2, FIG. 6-3, FIG. 6-4, where FIG. 6-1 is the result of flow cytometry analysis of apoptosis rate of blank control B16 cells; FIG. 6-2 is the result of flow cytometry analysis of apoptosis rate of 25. mu. mol/L dehydrocostuslactone on B16 cells; FIG. 6-3 is the result of flow cytometry analysis of apoptosis rate of 50. mu. mol/L dehydrocostuslactone on B16 cells; FIG. 6-4 is the result of flow cytometry analysis of apoptosis rate of 100. mu. mol/L dehydrocostuslactone on B16 cells).

Flow results and microscopic observation results show that costunolide and dehydrocostuslactone can induce the apoptosis activity of B16 melanoma cells.

Example 4 detection of apoptotic proteins by Western blot

Collecting control group cells and B16 cells treated by costunolide treatment groups with different concentrations (25, 50, 100 μmol/L) for 48h, adding prepared RIPA cell lysate containing protease and phosphatase inhibitor, and sufficiently cracking on ice to extract protein. The BCA protein quantification kit measures the protein concentration. According to the volume ratio of the protein sample to 5 × loading buffer of 4: 1 preparation, denaturation at 95 ℃ for 8 min. Taking 40 mu g of protein to perform polyacrylamide gel electrophoresis, electrically transferring the protein to a PVDF membrane, sealing with 5% skimmed milk powder for 1h, adding diluted primary antibody (Cyto C; Caspase 3; Caspase 9; beta-Actin), incubating overnight at 4 ℃, washing the membrane for 3 times (5 min for each time) with TBST, incubating for 1h at room temperature with secondary antibody, washing the membrane for 3 times (5 min for each time) with TBST, adding a developing solution, using a Tanon chemiluminescence image analysis system, collecting images, and performing strip analysis.

The results are as follows:

the results of the WesternBlot experiment show that after costunolide treatment group B16 cells were treated for 48h, the expression of cytoc, clear caspase 3 and clear caspase 9 in costunolide group cells was significantly increased at different concentrations (25, 50, 100. mu. mol/L) and concentration-dependently promoted apoptosis of B16 cells, and the difference was statistically significant (P < 0.05) compared with the blank control group (FIG. 7). The costunolide-induced B16 apoptosis is characterized in that Cyto c is released by mitochondria so as to activate caspase apoptosis pathway (one of the pathways for resisting tumor and inducing tumor apoptosis), and finally B16 apoptosis is caused, thus playing a role in resisting melanoma.

Example 5

1) Costunolide (commercially available);

2) dehydrocostuslactone (commercially available);

3) costunolide (commercially available): dehydrocostuslactone (commercially available) (mass ratio 1: 1);

4) costunolide (commercially available): ginsenoside Rh2 (commercially available) (mass ratio 1: 1);

5) costunolide (commercially available): astragalus polysaccharides (commercially available) (mass ratio 1:1)

Vacuum drying any one of 1) -5), pulverizing, adding sucrose and beta-cyclodextrin, mixing, granulating by dry method, and packaging to obtain granule. See table 1 for the formulation.

Vacuum drying conditions: 100 ℃ for 2h or 80 ℃ for 3 h;

dry granulation parameters: the pressure of a rolling wheel of the GK-70 dry type granulator is 6.0MPa, and the rotating speed of the rolling wheel is 500rpm

Particle size of the granules: irregular particles of 60-80 meshes.

TABLE 1 granule formulation

Example 6

Preparation of tablets: dissolving any one of 1) -5) in hypromellose solution, dissolving with ultrasonic aid, dispersing the medicine in water with high speed disperser, adding appropriate amount of maltodextrin, preheating for 5min, pumping the medicine into fluidized bed spray gun, granulating, mixing with 0.5% magnesium stearate, and tabletting. See table 2 for the formulation. The concentration of the hypromellose solution is 3%, that is, 3g of hypromellose is dissolved in 100mL of water, and the amplification factor can be increased by the same method, for example, 6g of hypromellose is dissolved in 200mL of water.

TABLE 2 tablet formulation

Example 7

Anti-melanoma ointment:

the components: costunolide (commercially available): dehydrocostuslactone (commercially available) (10 g: 10g)

The preparation method comprises the following steps: adding vaseline 80g to obtain ointment 20%.

Example 9 in vivo anti-melanoma animal experiments

B16 cells were cultured to exponential growth phase, trypsinized, cells were collected and formulated to be in finger 1 × 10⁷A cell suspension; selecting nude mice with 6 weeks old and about 20g weight, subcutaneously injecting 0.2ml of cell suspension at the traagula of each nude mouse right side, and grouping the nude mice into 6 mice each when the cell tumor grows to 2-3 mm in diameter.

The experiments were divided into 9 groups: no administration control, positive control (2mg/kg), costunolide low dose (15mg/kg), costunolide high dose (30mg/kg), dehydrocostunolide low dose (15mg/kg), dehydrocostunolide high dose (30mg/kg), costunolide: dehydrocostuslactone (1:1) group (30mg/kg), costunolide: ginsenoside Rh2(1:1) group (30mg/kg), costunolide: astragalus polysaccharides (1:1) (30 mg/kg).

The administration mode comprises the following steps: and (5) performing intragastric administration.

The administration time is as follows: after 3 weeks, the nude mice were sacrificed and the tumors were taken and weighed.

The experimental results are as follows:

in the experimental process, the survival state of the nude mice is normal, no adverse reaction is found, and no anaphylactic reaction is found. No dead mice appeared.

The experimental results show that the positive control group (2mg/kg), the costunolide low dose group (15mg/kg), the costunolide high dose group (30mg/kg), the dehydrocostunolide low dose group (15mg/kg), the dehydrocostunolide high dose group (30mg/kg), the costunolide: dehydrocostuslactone (1:1) group (30mg/kg), costunolide: ginsenoside Rh2(1:1) group (30mg/kg), costunolide: the tumor volume and weight of the nude mice of the group (30mg/kg) of astragalus polysaccharides (1:1) were significantly reduced, indicating that both could inhibit tumor cell growth.

Tumor inhibition rate is (average volume of control group-average volume of experimental group)/average volume of control group x 100%.

TABLE 3 tumor inhibition rate of the drugs

Numbering	Experiment grouping	Tumor weight (g)	Tumor inhibition Rate (%)
				1	Blank control group	3.89	0.00
2	Positive control group	3.03	22.11
				3	Costunolide low dose group	2.56	34.19
4	Costunolide high dose compositions	1.78	54.24
				5	Dehydrocostuslactone low dose group	2.61	32.90
6	Dehydrocostuslactone high dose group	1.54	60.41
				7	Costunolide: dehydrocostuslactone (1:1) group	2.76	29.05
8	Costunolide: ginsenoside Rh2(1:1) group	2.87	26.22
				9	Costunolide: astragalus polysaccharides (1:1) group	2.64	32.13

Example 10

Animal experiment external use anti-ulcer melanoma

Experimental animals: and (3) inoculating melanoma cells subcutaneously to the nude mice to establish an ulcerative melanoma model.

B16 cells were cultured to exponential growth phase, trypsinized, cells were collected and formulated to be in finger 1 × 10⁷A cell suspension; nude mice of 6 weeks old and about 20g in body weight were selected, 0.2ml of cell suspension was subcutaneously injected at the traagula of each right nude mouse, and when cell tumors grew to ulcerative extravasation, the experiment was divided into a blank control group, a treatment group and a prevention group.

The prevention group process comprises the following steps: before subcutaneous inoculation of melanoma cells, the ointment of example 7 was applied to gauze approximately 3mm thick, applied externally to the nude mice at the position of the "right murine traagulants" to be inoculated with the tumor, fixed with medical tape, 1 time per day for 2 weeks. After 2 weeks, tumor cells were inoculated and the administration was continued as above.

Treatment group administration: the ointment of example 7 was applied to gauze about 3mm thick, and then applied to the area of ulcerative melanoma in nude mice, fixed with medical tape, 1 time per day, and used for 2 weeks.

The effect is as follows: the treatment group, ulcerative melanoma, had controlled ulcer surface, significantly reduced exudate, and significantly slowed melanoma growth compared to the control group. In the prevention group, the melanoma cells grow slowly, form small tumor nodules and hardly form tumor masses.

TABLE 4 tumor inhibition rate of the cream

Numbering	Experiment grouping	Tumor weight (g)	Tumor inhibition Rate (%)
				1	Blank control group	3.89	0.00
2	Cream treatment group	2.21	43.19
				3	Cream preventive group	0.71	81.74

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. Application of costunolide and/or dehydrocostuslactone in preparing medicine for preventing and/or treating melanoma is provided.

2. Use according to claim 1, wherein costunolide and/or dehydrocostunolide treats melanoma by inhibiting proliferation and/or inducing apoptosis of melanoma cells.

3. Use according to claim 1, wherein when the drug product comprises costunolide and dehydrocostuslactone, the mass ratio of costunolide to dehydrocostuslactone is (1-10): (1-10).

4. The use of claim 1, wherein the medicament further comprises ginsenoside Rh2 or astragalus polysaccharide.

5. The use according to claim 4, wherein when the medicament comprises costunolide and ginsenoside Rh2, the mass ratio of costunolide to ginsenoside Rh2 is (1-10): (1-10).

6. The use according to claim 4, wherein when the medicament comprises costunolide and astragalus polysaccharide, the mass ratio of costunolide to astragalus polysaccharide is (1-10): (1-10).

7. The use of any one of claims 1 to 6, wherein the pharmaceutical product is in a dosage form comprising an oral preparation and an external preparation; the oral preparation comprises granules, tablets, decoctions or capsules; the external preparation comprises ointment or powder.

8. The use of claim 7, wherein the medicament further comprises an adjuvant; when the dosage form of the medicine is granules, the auxiliary materials comprise beta-cyclodextrin and magnesium stearate; when the dosage form of the medicine is a tablet, the auxiliary materials comprise hypromellose, magnesium stearate and maltodextrin; when the medicament is in the form of an ointment, the auxiliary material comprises vaseline.