Composition capable of resisting doxorubicin cardiotoxicity as well as preparation method, application and preparation thereof

Abstract

The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to a composition for resisting doxorubicin cardiotoxicity and a preparation method, application and a preparation thereof. The invention provides a composition capable of resisting doxorubicin cardiotoxicity, the composition comprises diosmetin and loureirin B, the diosmetin and the loureirin B can be extracted through natural plants, the raw materials are low in cost and easy to obtain, and clinical popularization and application are facilitated. The cell experiment research finds that the combined administration of diosmetin and loureirin B can significantly improve the survival rate of adriamycin-damaged myocardial cells and significantly reduce the active oxygen level and positive cell rate, and verifies the synergistic interaction of diosmetin and loureirin B in resisting adriamycin cardiotoxicity.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparations, and particularly relates to a composition having anti-doxorubicin myocardial toxicity, a preparation method and application thereof, and a preparation. Background Art

[0002] Doxorubicin is an antibiotic drug with the chemical formula C 27 H 29 NO 11 , and is applicable to acute leukemia (lymphocytic and granulocytic), malignant lymphoma, breast cancer, bronchogenic carcinoma (undifferentiated small cell and non-small cell), ovarian cancer, soft tissue sarcoma, osteosarcoma, rhabdomyosarcoma, Ewing sarcoma, blastoma, neuroblastoma, bladder cancer, thyroid cancer, prostate cancer, head and neck squamous cell carcinoma, testicular cancer, gastric cancer, liver cancer, etc. However, doxorubicin can produce a wide range of biochemical effects on the body and has a strong cytotoxic effect, and its side effects can cause myocardial injury, ultimately leading to arrhythmia, systolic and diastolic dysfunction, dilated cardiomyopathy, and congestive heart failure.

[0003] Therefore, on the basis of the anti-tumor effect of doxorubicin, the development of drugs to protect the heart from damage is still a research hotspot at present. For example, Chinese Patent Publication No. CN119499263A discloses the application of pentagalloyl glucose in the preparation of drugs for anti-doxorubicin cardiotoxicity; another Chinese Patent Publication No. CN119499237A discloses the application of hydroxydaphnetin (HGK) in the preparation of drugs for preventing and treating anti-tumor drug-induced cardiotoxicity.

[0004] Diosmetin is a natural flavonoid compound widely present in citrus fruits, chrysanthemums and other plants, and has biological activities such as antioxidant, anti-inflammatory and anti-tumor.

[0005] Dracaenoside B is a stilbene compound extracted from plants of the genus Dracaena, and has antibacterial, anti-inflammatory, antithrombotic and anti-tumor activities. Its mechanism of action includes inducing apoptosis of tumor cells, inhibiting angiogenesis and regulating immune function.

[0006] Although it has been reported in the prior art that diosmetin and dracaenoside B have various biological activities, so far, it has not been reported that the combination of diosmetin and dracaenoside B can reduce doxorubicin-induced myocardial toxicity. Summary of the Invention

[0007] Aiming at the problems existing in the prior art, the present invention provides a composition having anti-doxorubicin myocardial toxicity, a preparation method and application thereof, and a preparation.

[0008] To achieve the above object, the technical scheme adopted by the present invention is as follows: A composition with anti-doxorubicin myocardial toxicity, said composition comprising diosmetin and dracorhodin B.

[0009] Preferably, the composition comprises 5 μM diosmetin and 8 - 12 μM dracorhodin B.

[0010] More preferably, the composition comprises 5 μM diosmetin and 10 μM dracorhodin B.

[0011] The present invention also provides a preparation method of the above composition, comprising the following steps: (1) First, dissolve diosmetin and dracorhodin B in solvents respectively, and perform ultrasonic treatment to obtain a diosmetin solution and a dracorhodin B solution; (2) Then, dilute the diosmetin solution and the dracorhodin B solution respectively, and mix the two to obtain the product.

[0012] Preferably, in step (1), the temperature of the ultrasonic treatment is 20 - 30 °C, the ultrasonic power is 80 - 120 W, and the ultrasonic time is 4 - 6 min.

[0013] Preferably, stirring is performed before the ultrasonic treatment, the rotation speed of the stirring is 1400 - 1600 rpm, and the stirring time is 20 - 40 s.

[0014] Preferably, the solvent in step (1) is dimethyl sulfoxide; in step (2), the dilution is performed using a basal medium.

[0015] The present invention also provides a preparation with anti-doxorubicin myocardial toxicity, comprising the above composition and pharmaceutically acceptable excipients.

[0016] Preferably, the excipients include fillers, surfactants, binders, antioxidants, preservatives, flavoring agents, lubricants, coating agents or coloring agents.

[0017] Preferably, the type of the preparation is capsule, tablet, granule, pellet, lyophilized powder or injection.

[0018] The present invention also provides the use of the above composition or the above preparation in the preparation of a drug with anti-doxorubicin myocardial toxicity.

[0019] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention provides a new idea for the method of anti-doxorubicin myocardial toxicity, and provides a composition with anti-doxorubicin myocardial toxicity. This composition comprises diosmetin and dracorhodin B, and diosmetin and dracorhodin B can be extracted from natural plants, with low raw material cost and easy availability, which is conducive to clinical promotion and application.

[0020] (2)Through cell experiment research, it is found in the present invention that the combined administration of diosmetin and dracorhodin B can significantly improve the survival rate of cardiomyocytes damaged by doxorubicin, significantly reduce the levels of reactive oxygen species and the positive cell rate, verifying the synergistic effect of diosmetin and dracorhodin B in anti-doxorubicin myocardial toxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a graph of the percentage of cell viability.

[0022] Figure 2 It is a graph of the synergy score of diosmetin and dracorhodin B.

[0023] Figure 3 It is a representative fluorescence image of mitochondrial reactive oxygen species.

[0024] Figure 4 It is a graph of the average fluorescence value of mitochondrial reactive oxygen species and the positive cell rate.

[0025] Figure 1 and Figure 4 In, compared with the normal group, ## indicates p <0.01; compared with the model group, ** indicates p <0.01, * indicates p <0.05. DETAILED DESCRIPTION OF THE INVENTION

[0026] It should be noted that the raw materials used in the present invention are all ordinary commercially available products.

[0027] Diosmetin, with a content of 98%, was purchased from Shanghai Yuanye Co., Ltd.; Dracorhodin B, with a content of 98%, was purchased from Shanghai Yuanye Co., Ltd.; Basic medium was purchased from WISENT INC; High-glucose DMEM basic medium was purchased from WISENT INC; H9c2 cells were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences; CCK8 solution was purchased from Biosharp; Doxorubicin, with a content of 99.82%, was purchased from Selleck; Cyanidin chloride, with a content of 98%, was purchased from Shanghai Yuanye Co., Ltd.; Mitochondrial superoxide indicator was purchased from ThermoFisher Scientific; Hoechst 33342 was purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0028] Among them, the high-glucose DMEM complete medium is composed of high-glucose DMEM basic medium, 10% fetal bovine serum and 1% penicillin-streptomycin.

[0029] Take 2.01 mg of diosmetin and 334.7 μL of dimethyl sulfoxide (DMSO). First, shake it at a speed of 1500 rpm in a vortex oscillator for 30 s, and then ultrasonicate it at a temperature of 25°C for 5 min with a power of 100 W to completely dissolve it, and prepare a 20 mM diosmetin stock mother solution, which is stored at -20°C for later use.

[0030] Take 2.21 mg of loureirin B and 350 μL of dimethyl sulfoxide (DMSO). Similarly, first shake it at a speed of 1500 rpm in a vortex oscillator for 30 s, and then ultrasonicate it at a temperature of 25°C for 5 min with a power of 100 W to completely dissolve it, and prepare a 20 mM loureirin B stock mother solution, which is stored at -20°C for later use.

[0031] Example 1 A method for preparing a composition with anti-doxorubicin cardiotoxicity, the steps are as follows: Before use, dilute the 20 mM diosmetin stock mother solution 2000x with the basal medium to obtain a 10 μM diosmetin solution; dilute the 20 mM loureirin B stock solution 1000x with the basal medium to obtain a 20 μM loureirin B solution. Then mix the 10 μM diosmetin solution and the 20 μM loureirin B solution in equal volume, and that's it. At this time, the concentration of diosmetin in the composition is 5 μM, and the concentration of loureirin B is 10 μM.

[0032] Comparative Example 1 Dilute the 20 mM diosmetin stock mother solution 4000x with the basal medium to obtain a 5 μM diosmetin solution.

[0033] Comparative Example 2 Dilute the 20 mM diosmetin stock mother solution 2000x with the basal medium to obtain a 10 μM diosmetin solution.

[0034] Comparative Example 3 Dilute the 20 mM diosmetin stock mother solution 1000x with the basal medium to obtain a 20 μM diosmetin solution.

[0035] Comparative Example 4 Dilute the 20 mM loureirin B stock solution 4000x with the basal medium to obtain a 5 μM loureirin B solution.

[0036] Comparative Example 5 Dilute the 20 mM loureirin B stock solution 2000x with the basal medium to obtain a 10 μM loureirin B solution.

[0037] Comparative Example 6 Dilute the 20 mM dracoresene B stock solution 1000x with the basal medium to obtain a 20 μM dracoresene B solution.

[0038] Comparative Example 7 A method for preparing a composition with anti-doxorubicin cardiotoxicity, the steps are as follows: Dilute the 20 mM daphnetin stock mother solution 2000x with the basal medium to obtain a 10 μM daphnetin solution; dilute the 20 mM dracoresene B stock solution 2000x with the basal medium to obtain a 10 μM dracoresene B solution. Then mix the 10 μM daphnetin solution and the 10 μM dracoresene B solution in equal volumes, and that's it. At this time, the concentration of daphnetin in the composition is 5 μM, and the concentration of dracoresene B is 5 μM.

[0039] Comparative Example 8 A method for preparing a composition with anti-doxorubicin cardiotoxicity, the steps are as follows: Dilute the 20 mM daphnetin stock mother solution 2000x with the basal medium to obtain a 10 μM daphnetin solution; dilute the 20 mM dracoresene B stock solution 500x with the basal medium to obtain a 40 μM dracoresene B solution. Then mix the 10 μM daphnetin solution and the 40 μM dracoresene B solution in equal volumes, and that's it. At this time, the concentration of daphnetin in the composition is 5 μM, and the concentration of dracoresene B is 20 μM.

[0040] Comparative Example 9 A method for preparing a composition with anti-doxorubicin cardiotoxicity, the steps are as follows: Dilute the 20 mM daphnetin stock mother solution 1000x with the basal medium to obtain a 20 μM daphnetin solution; dilute the 20 mM dracoresene B stock solution 2000x with the basal medium to obtain a 10 μM dracoresene B solution. Then mix the 20 μM daphnetin solution and the 10 μM dracoresene B solution in equal volumes, and that's it. At this time, the concentration of daphnetin in the composition is 10 μM, and the concentration of dracoresene B is 5 μM.

[0041] Comparative Example 10 A method for preparing a composition with anti-doxorubicin cardiotoxicity, the steps are as follows: Dilute the 20 mM daphnetin stock mother solution 1000x with the basal medium to obtain a 20 μM daphnetin solution; dilute the 20 mM dracoresene B stock solution 1000x with the basal medium to obtain a 20 μM dracoresene B solution. Then mix the 20 μM daphnetin solution and the 20 μM dracoresene B solution in equal volumes, and that's it. At this time, the concentration of daphnetin in the composition is 10 μM, and the concentration of dracoresene B is 10 μM.

[0042] Comparative Example 11 A preparation method of a composition with anti-doxorubicin myocardial toxicity, the steps are as follows: Dilute the 20 mM luteolin stock mother liquor 1000x with the basal medium to obtain a 20 μM luteolin solution; dilute the 20 mM loureirin B stock solution 500x with the basal medium to obtain a 40 μM loureirin B solution. Then mix the 20 μM luteolin solution and the 40 μM loureirin B solution in equal volumes, and that's it. At this time, the concentration of luteolin in the composition is 10 μM, and the concentration of loureirin B is 20 μM.

[0043] Control Example 12 A preparation method of a composition with anti-doxorubicin myocardial toxicity, the steps are as follows: Dilute the 20 mM luteolin stock mother liquor 500x with the basal medium to obtain a 40 μM luteolin solution; dilute the 20 mM loureirin B stock solution 2000x with the basal medium to obtain a 10 μM loureirin B solution. Then mix the 40 μM luteolin solution and the 10 μM loureirin B solution in equal volumes, and that's it. At this time, the concentration of luteolin in the composition is 20 μM, and the concentration of loureirin B is 5 μM.

[0044] Control Example 13 A preparation method of a composition with anti-doxorubicin myocardial toxicity, the steps are as follows: Dilute the 20 mM luteolin stock mother liquor 500x with the basal medium to obtain a 40 μM luteolin solution; dilute the 20 mM loureirin B stock solution 1000x with the basal medium to obtain a 20 μM loureirin B solution. Then mix the 40 μM luteolin solution and the 20 μM loureirin B solution in equal volumes, and that's it. At this time, the concentration of luteolin in the composition is 20 μM, and the concentration of loureirin B is 10 μM.

[0045] Control Example 14 A preparation method of a composition with anti-doxorubicin myocardial toxicity, the steps are as follows: Dilute the 20 mM luteolin stock mother liquor 500x with the basal medium to obtain a 40 μM luteolin solution; dilute the 20 mM loureirin B stock solution 500x with the basal medium to obtain a 40 μM loureirin B solution. Then mix the 40 μM luteolin solution and the 40 μM loureirin B solution in equal volumes, and that's it. At this time, the concentration of luteolin in the composition is 20 μM, and the concentration of loureirin B is 20 μM.

[0046] Test Example 1 Verification of the synergistic effect of luteolin and loureirin B based on cell viability.

[0047] (1) CCK8 cell viability experiment Experimental method: H9c2 cells were seeded in a transparent 96-well plate at a density of 5000 cells / well. After adherent culture for 24 h, a normal group, a model group and a drug administration group were set up.

[0048] Normal group: After washing with PBS buffer, the liquid in the wells was replaced with high-glucose DMEM complete medium. Model group: After washing with PBS buffer, the liquid in the wells was replaced with high-glucose DMEM complete medium containing 0.5 μM doxorubicin. Drug administration group: After washing with PBS buffer, the liquid in the wells was replaced with the solution prepared in Example 1 or Comparative Examples 1-14 containing 0.5 μM doxorubicin.

[0049] Subsequently, the cells were placed in an incubator and incubated for 24 h. CCK8 solution was added to each well at a concentration of 10% (100 μL / well), and the cells were continuously incubated in the incubator. After 0.5 - 1 h, the absorbance at 580 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader.

[0050] Experimental results: As Figure 1 shown, it can be seen from Figure 1 that the combined administration of 5 μM diosmetin and 10 μM loureirin B (i.e., Example 1) can significantly improve the survival rate of cardiomyocytes damaged by doxorubicin, and the cell survival rate increased from 53.07 ± 6.17% to 95.20 ± 9.32%.

[0051] (2) Synergy analysis Based on the Synergyfinder program, the combination of the two compounds showed good synergy in the cell viability index. Commonly used reference models include the Highest Single Agent (HSA) model and the Loewe isobole model. A parameter value between -10 and 10 indicates that the interaction between the two may be additive; a parameter value greater than 10 indicates that the interaction between the two drugs may be synergistic.

[0052] The analysis results are as Figure 2 shown, Figure 2 showing that the synergy score of the Loewe model is 13.69 and the synergy score of the HSA model is 12.86, both higher than the synergy threshold of 10, confirming the existence of a synergistic protective effect between 5 μM diosmetin and 10 μM loureirin B.

[0053] Test Example 2 Verification of the synergistic effect of diosmetin and loureirin B based on cell phenotype analysis. The specific experimental method is as follows: 1. Preparation of drug and staining working solution: Drug preparation: Doxorubicin was dissolved in DMSO to prepare a 1 mM stock solution, and diluted with high-glucose DMEM complete medium to a 0.5 μM doxorubicin solution before use; Weigh a certain amount of cyanidin chloride, dissolve it in DMSO to prepare a 200 mM mother solution, and dilute it to a 200 μM cyanidin chloride solution before use.

[0054] Staining working solution preparation: Mitochondrial superoxide indicator (MitoSOX Red) was dissolved in 13 μL DMSO to prepare a 5 mM stock solution, and diluted to a 0.5 μM working solution before use. Hoechst 33342 was diluted with sterile PBS to a 3 μg / mL stock solution, and diluted to a 1 μg / mL working solution before use. The above working solutions were aliquoted and stored at -20°C.

[0055] 2. Cell staining and high-content imaging verification of mitochondrial reactive oxygen species level: According to the effective drug concentrations obtained from the CCK8 cell viability experiment, a normal group, a model group, a positive drug group and a sample group were set respectively. Staining and high-content imaging were performed 24 h after drug administration.

[0056] Normal group: After washing with PBS buffer, the liquid in the well was replaced with high-glucose DMEM complete medium; Model group: After washing with PBS buffer, the liquid in the well was replaced with high-glucose DMEM complete medium containing 0.5 μM doxorubicin; Positive drug group: After washing with PBS buffer, the liquid in the well was replaced with a cyanidin chloride solution containing 0.5 μM doxorubicin; Sample group: After washing with PBS buffer, the liquid in the well was replaced with the solution prepared in Example 1, Comparative Example 1 or Comparative Example 5 containing 0.5 μM doxorubicin.

[0057] After the arrival time, the cell wells were washed with PBS, and the operation was carried out in the dark. MitoSOX Red working solution was added to cover the cells, and incubated at 37°C for 30 min. After washing with PBS, a sufficient amount of Hoechst 33342 was added to counterstain the cell nuclei, and incubated at 37°C for 13 min. After the incubation, the cells were washed with PBS, and a sufficient amount of high-glucose DMEM complete medium was added to each well to cover the cells. After adjusting the exposure time and Z-axis time to appropriate imaging parameters with ImageXpress Mico Confocal, high-content time-series imaging was performed under a 40× objective using the DAPI / TRITC fluorescence double channel, and the average fluorescence value of mitochondrial reactive oxygen species and the positive cell rate were statistically calculated.

[0058] 3. Experimental results: The results are as Figures 3 - 4 shown, from Figures 3 - 4It can be seen that in the detection of mitochondrial reactive oxygen species (ROS) levels, compared with the normal group, the model group showed significant ROS accumulation (1.0×10 8 ), and an increase in the positive cell rate (94.3%) ( p <0.01), indicating successful model establishment; compared with the model group, the positive drug group could significantly reduce the average fluorescence value of mitochondrial ROS (1.7×10 7 ) and the positive cell rate (23.5%) ( p <0.01); the average fluorescence value of mitochondrial ROS in Example 1 of the sample group (2.2×10 7 ) and the positive cell rate (28.2%) were the lowest ( p <0.01), indicating that the combined drug group of 5 μM diosmetin and 10 μM loureirin B could significantly reduce the ROS level and the positive cell rate.

[0059] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than limiting the protection scope of the present invention. Any simple modification or equivalent replacement of the technical solution of the present invention by those of ordinary skill in the art shall not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A composition with anti-doxorubicin myocardial toxicity, characterized in that, The composition comprises diosmetin and dracorhodin B.

2. The composition according to claim 1, wherein The composition comprises 5 μM diosmetin and 8 - 12 μM dracorhodin B.

3. The composition according to claim 2, wherein The composition comprises 5 μM diosmetin and 10 μM dracorhodin B.

4. A method for preparing a composition according to any one of claims 1-3, characterized in that, It includes the following steps: (1) First, dissolve diosmetin and dracorhodin B in solvents respectively, and perform ultrasonic treatment to obtain a diosmetin solution and a dracorhodin B solution; (2) Then, dilute the diosmetin solution and the dracorhodin B solution respectively, and mix the two to obtain the product.

5. The preparation method according to claim 4, characterized in that, In step (1), the temperature of the ultrasonic treatment is 20 - 30 °C, the ultrasonic power is 80 - 120 W, and the time of the ultrasonic treatment is 4 - 6 min; stirring is carried out before the ultrasonic treatment, the rotation speed of the stirring is 1400 - 1600 rpm, and the time of the stirring is 20 - 40 s.

6. The preparation method according to claim 4, characterized in that, The solvent in step (1) is dimethyl sulfoxide; in step (2), the dilution is carried out using a basal medium.

7. A preparation with anti-doxorubicin myocardial toxicity, characterized in that, It includes the composition according to any one of claims 1 - 3 or the composition prepared by the preparation method according to any one of claims 4 - 6 and pharmaceutically acceptable excipients.

8. The preparation according to claim 7, characterized in that, The excipients include fillers, surfactants, binders, antioxidants, preservatives, flavoring agents, lubricants, coating agents or coloring agents.

9. The preparation according to claim 7, characterized in that, The type of the preparation is capsule, tablet, granule, pellet, freeze-dried powder or injection.

10. Use of a composition according to any one of claims 1 - 3 or a preparation according to any one of claims 7 - 9 in the preparation of a drug for anti-doxorubicin cardiotoxicity.

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