Myocardial protection composition and medical application thereof

Abstract

The invention relates to the technical field of medicines, in particular to a myocardial protection composition and medical application thereof.The myocardial protection composition comprises a ginkgo biloba extract, a codonopsis pilosula extract and a croton seed extract, and a compound with a myocardial protection effect is obtained through combination of traditional Chinese medicine ingredients. A new choice is provided for clinical screening and / or preparation of drugs for treating myocardial injury or preventing myocardial infarction.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and in particular to a myocardial protection composition and its medical use. Background Art

[0002] Vascular disease has become the leading cause of death worldwide. Even with active treatment, most patients are still unable to return to normal life, which has placed a serious burden on society. A large number of studies have shown that poor prognosis is closely related to myocardial damage caused by cardiovascular disease and its treatment measures, which is related to oxidative stress damage caused by free radicals, leading to myocardial cell damage and necrosis.

[0003] In the prior art, ACE inhibitors, beta-receptor blockers, diuretics, and aldosterone antagonists are the initial treatment options for myocardial cell injury, but they still fail to achieve the desired goal and have significant side effects. With the in-depth research on the effective ingredients and mechanisms of action of traditional Chinese medicine at home and abroad, more and more traditional Chinese medicine extracts have a certain therapeutic effect on heart failure, providing a new option for myocardial protection.

[0004] Therefore, it is of great practical significance to isolate and extract compounds with cardioprotective effects from traditional Chinese medicines and broaden the clinical drug options. Summary of the invention

[0005] The purpose of the present invention is to provide a myocardial protection composition and its medical use. The myocardial protection composition includes ginkgo extract, codonopsis extract and croton extract, and a compound with myocardial protection effect is obtained by combining traditional Chinese medicine components, thereby broadening the clinical drug selection.

[0006] The present invention solves the technical problem by adopting the following technical solutions:

[0007] In a first aspect, the present invention provides a myocardial protection composition, comprising the following components in parts by weight:

[0008] 10 to 30 parts of croton extract; 10 to 30 parts of Codonopsis pilosula extract; 1 to 10 parts of Ginkgo biloba extract.

[0009] In some embodiments of the present invention, the myocardial protective composition comprises the following components in parts by weight:

[0010] 5 to 25 parts of croton extract; 5 to 25 parts of Codonopsis pilosula extract; 1 to 10 parts of Ginkgo biloba extract.

[0011] In some embodiments of the present invention, the croton extract is crotonin; the Codonopsis pilosula extract is codonoside.

[0012] In some embodiments of the present invention, the ginkgo extract is at least one of bilobalide, ginkgolide A, ginkgolide B, ginkgolide C, and ginkgolide K.

[0013] In some embodiments of the present invention, the ginkgo extract is bilobalide.

[0014] In some embodiments of the present invention, the myocardial protection composition comprises the following components in parts by weight: 10 to 20 parts of crotonin; 10 to 20 parts of codonopsis pilosula; and 1 to 5 parts of bilobalide.

[0015] In some embodiments of the present invention, the myocardial protective composition comprises the following components in parts by weight: 10 to 15 parts of crotonin; 10 to 15 parts of codonopsis pilosula; and 2 to 4 parts of bilobalide.

[0016] In some embodiments of the present invention, the myocardial protection composition comprises the following components in parts by weight: 12 to 15 parts of crotonin; 12 to 15 parts of codonopsis pilosula; and 2 to 3 parts of bilobalide.

[0017] The second aspect of the present invention provides use of a myocardial protective composition in preparing a drug for preventing or treating myocardial injury.

[0018] The third aspect of the present invention provides use of the myocardial protection composition in preparing a medicament for preventing or treating myocardial infarction.

[0019] The present invention has the following beneficial effects: The myocardial protection composition of the present invention has a good myocardial protection effect, and provides a new option for clinical screening and / or preparation of drugs for treating myocardial injury or preventing myocardial infarction. The present invention reduces apoptosis and myocardial infarction reactions caused by myocardial ischemia through the myocardial protection composition, and its therapeutic effect is predictable, and provides a new option for clinical screening and preparation of drugs for treating or preventing myocardial infarction. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the HE image of the myocardial tissue of mice in the blank group B.

[0021] Figure 2 HE images of myocardial tissue of mice in model group B.

[0022] Figure 3 HE images of myocardial tissue of mice in the low-dose group.

[0023] Figure 4 HE images of myocardial tissue of mice in the medium-dose group.

[0024] Figure 5 HE images of myocardial tissue of mice in the low-dose group.

[0025] Figure 6 It is a bar graph of the levels of lactate dehydrogenase (LDH), a marker of myocardial injury, in each group.

[0026] Figure 7 Schematic diagram of cardiomyocyte apoptosis in mice in blank group B.

[0027] Figure 8 Schematic diagram of cardiomyocyte apoptosis in mice in model group B.

[0028] Fig. 9 Schematic diagram of cardiomyocyte apoptosis in mice in the low-dose group.

[0029] Fig.10 Schematic diagram of myocardial cell apoptosis in mice in the medium-dose group.

[0030] Fig.11 Schematic diagram of cardiomyocyte apoptosis in mice in the high-dose group.

[0031] Fig.12 Schematic diagram of TTC detection of myocardial infarction in the hearts of mice in each group.

[0032] Unless stated to the contrary, the terms used in the specification and claims have the following meanings.

[0033] As used herein, the term "prevention" refers to preventing the occurrence of a disease and / or preventing the recurrence of a disease.

[0034] The bilobalide, ginkgolide A, ginkgolide B, ginkgolide C, ginkgolide K, crotonin and codonopsis pilosula described in the present invention can be prepared by separation and purification through existing technologies. DETAILED DESCRIPTION

[0035] The present invention specification describes the specific implementation scheme in detail. Those skilled in the art should recognize that the following implementation scheme is exemplary and cannot be understood as limiting the present invention. For those skilled in the art, without departing from the principle of the present invention, by making several improvements and modifications to the present invention, the technical solutions obtained by these improvements and modifications also fall within the protection scope of the claims of the present invention. The following examples specifically illustrate the beneficial effects of the present invention.

[0036] The solvent composition was DMSO: 0.1% DMEM: 0.5% MC = 50:30:20 (v / v / v). DMSO was purchased from Shanghai Lianshuo Biotechnology Co., Ltd., 0.1% DMEM culture medium was purchased from Shanghai Boyao Biotechnology Co., Ltd., and 0.5% MC (methylcellulose) aqueous solution was prepared by ourselves.

[0037] Lactate dehydrogenase (LDH) kit was purchased from Biosino Biotech Co., Ltd.

[0038] Crotonoside, CAS: 1818-71-9, was purchased from Chengdu Desit Biotechnology Co., Ltd.

[0039] Codonopsis pilosula, CAS: 117278-74-7, was purchased from Chengdu Desit Biotechnology Co., Ltd.

[0040] Bilobalide, CAS: 33570-04-6, was purchased from Chengdu Desit Biotechnology Co., Ltd.

[0041] H9c2(2-1) rat cardiomyocytes were purchased from Biohui Biotechnology (Guangzhou) Co., Ltd.

[0042] Example 1

[0043] Preparation process of the myocardial protective composition solution: weigh 1.4 g, 1.4 g and 0.2 g of crotonin, codonopsis pilosula and biloba lactone respectively, mix them evenly, and obtain the myocardial protective composition.

[0044] Preparation process of myocardial protection composition solution: weigh crotonin, codonopsis pilosula and biloba lactone according to weights of 1.4g, 1.4g and 0.2g respectively, add them into the solvent and mix evenly, and adjust the volume to 20ml to obtain the myocardial protection composition solution.

[0045] Example 2

[0046] Preparation process of the myocardial protective composition: weigh crotonin, codonopsis pilosula and biloba lactone according to weights of 1.4g, 1.3g and 0.3g respectively, add them into the solvent and mix evenly, and adjust the volume to 20ml to obtain the myocardial protective composition solution.

[0047] Example 3

[0048] Preparation process of the myocardial protective composition: weigh crotonin, codonopsis pilosula and biloba lactone according to weights of 1.5g, 1.2g and 0.3g respectively, add them into the solvent and mix evenly, and adjust the volume to 20ml to obtain the myocardial protective composition solution.

[0049] Example 4

[0050] Preparation process of the myocardial protective composition: weigh crotonin, codonopsis pilosula and biloba lactone according to weights of 2g, 1g and 0.3g respectively, add them into the solvent and mix evenly, and adjust the volume to 20ml to obtain the myocardial protective composition solution.

[0051] Example 5

[0052] Preparation process of the myocardial protective composition: weigh crotonin, codonopsis pilosula and biloba lactone according to weights of 1g, 1.7g and 0.3g respectively, add them into the solvent and mix evenly, and adjust the volume to 20ml to obtain the myocardial protective composition solution.

[0053] Example 6

[0054] Preparation process of the myocardial protective composition: weigh crotonin, codonopsis pilosula and biloba lactone according to weights of 1.2g, 1.5g and 0.3g respectively, add them into the solvent and mix evenly, and adjust the volume to 20ml to obtain the myocardial protective composition solution.

[0055] Example 7

[0056] Preparation process of the myocardial protective composition: weigh crotonin, codonopsis pilosula and biloba lactone according to weights of 1.4g, 1.3g and 0.3g respectively, add them into the solvent and mix evenly, and adjust the volume to 20ml to obtain the myocardial protective composition solution.

[0057] Example 8

[0058] Preparation process of the myocardial protective composition: weigh crotonin, codonopsis pilosula and biloba lactone according to weights of 1.3g, 1.5g and 0.2g respectively, add them into the solvent and mix evenly, and adjust the volume to 20ml to obtain the myocardial protective composition solution.

[0059] Comparative Example 1

[0060] Preparation process of myocardial protection composition solution: weigh 1.4 g of crotonin and 1.4 g of codonopsis pilosula respectively, add them into the solvent and mix evenly, and adjust the volume to 20 ml to obtain the myocardial protection composition solution.

[0061] Comparative Example 2

[0062] Preparation process of myocardial protection composition solution: weigh 1.4 g of crotonin and 0.2 g of ginkgo lactone respectively, add them into the solvent and mix evenly, and adjust the volume to 20 ml to obtain the myocardial protection composition solution.

[0063] Comparative Example 3

[0064] Preparation process of myocardial protection composition solution: weigh 1.4 g of codonopsis pilosula and 0.2 g of ginkgo lactone respectively, add them into the solvent and mix evenly, and adjust the volume to 20 ml to obtain the myocardial protection composition solution.

[0065] Comparative Example 4

[0066] Preparation process of myocardial protective drug solution: weigh 1.4 g of crotonoside, add it to the solvent and mix evenly, and adjust the volume to 20 ml to obtain the myocardial protective drug solution.

[0067] Comparative Example 5

[0068] Preparation process of myocardial protective drug solution: weigh 1.4 g of codonopsis pilosula, add it to the solvent and mix evenly, and adjust the volume to 20 ml to obtain the myocardial protective drug solution.

[0069] Comparative Example 6

[0070] Preparation process of myocardial protective drug solution: weigh 0.2 g of ginkgolide, add it to the solvent and mix evenly, and adjust the volume to 20 ml to obtain the myocardial protective drug solution.

[0071] Biological testing experiments

[0072] 1. Myocardial protection cell test - protective effect on adriamycin-induced H9c2 cell injury

[0073] 1. Experimental method of blank group A: H9c2(2-1) rat cardiomyocytes were seeded on a 96-well plate at a density of 7×10 4 Cells / mL were cultured in culture medium for 24 hours, and centrifuged. Cells were pre-incubated with 1 ml of medium for 48 hours, and 20 μL of MTT (5 mg / mL) was added to each well and cultured for 4 hours. The absorbance was measured at 490 nm using an ELISA reader, and the measurement was repeated three times to obtain the average value.

[0074] 2. Experimental methods of model group A:

[0075] H9c2(2-1) rat cardiomyocytes were seeded in 96-well plates at a density of 7×10 per well. 4 Cells / mL were cultured in culture medium for 24 hours, and centrifuged. Cells were pre-incubated with 1 ml of medium for 24 hours, and then incubated with 5.0 μM doxorubicin for 24 hours. 20 μL of MTT (5 mg / mL) was added to each well and cultured for 4 hours. The absorbance was measured at 490 nm using an ELISA reader, and the average value was obtained by repeating the measurement three times.

[0076] 3. Experimental methods of drug administration group:

[0077] H9c2(2-1) rat cardiomyocytes were seeded in 96-well plates at a density of 7×10 per well. 4 Cells / mL were cultured in culture medium for 24 hours, and centrifuged. Each well of cells was pre-incubated with 1 ml of sample solution for 24 hours, and each well of cells was incubated with 5.0 μM doxorubicin for 24 hours, and then 20 μL of MTT (5 mg / mL) was added to each well and cultured for 4 hours. The absorbance was measured at 490 nm using an ELISA reader, and the average value was obtained by repeating the measurement three times.

[0078] The sample solutions are respectively the myocardial protective composition solutions of Examples 1 to 8, the myocardial protective composition solutions of Comparative Examples 1 to 3 or the myocardial protective drug solutions of Comparative Examples 4 to 6.

[0079] 4. Calculation method

[0080] Cell viability (%) = OD (drug group or model group A) / OD (blank group A) × 100%

[0081] Protection rate (%) = (OD (administration group) / OD (model group A) - 1) × 100%

[0082] 5. Experimental results

[0083] The cell viability of each experimental group is shown in the data in Table 1

[0084]

[0085]

[0086] Table 1

[0087] The activity of the cells was determined by the MTT method, and the protective effect of the myocardial protective composition on adriamycin-induced H9c2 cardiomyocyte injury was calculated with the blank group A as the control. From the data in Table 1, it can be seen that the myocardial protective composition of Examples 1 to 8 can significantly improve cell viability on adriamycin-induced H9c2 cardiomyocyte injury, and the protection rate is at least 70%. Comparative Examples 1 to 3 only added two active ingredients, and although their cell viability was improved compared with model group A, the cell viability was not high, and their protective effect on myocardial cell injury was general.

[0088] The test results show that the myocardial protective composition of the present invention has a good myocardial protective effect, and provides a new option for clinical screening and / or preparation of drugs for treating myocardial injury or preventing myocardial infarction.

[0089] 2. Protective effect of myocardial protection composition on myocardial infarction in mice

[0090] 1. Modeling method: Intraperitoneal injection of 1% sodium pentobarbital (60 mg / kg) for anesthesia. Use a 20# cannula needle to perform tracheal puncture between 1-2 tracheal rings under the cricoid cartilage, insert the tracheal tube, and the depth is 1.5 cm. The tracheal tube is connected to the small animal ventilator, with a tidal volume of 0.8-0.9 ml, a respiratory rate of 120 times / min, and an inspiratory-expiratory ratio of 1:1. Cut the intercostal muscles at the third intercostal space on the left side, cut the pleura, enter the chest cavity, and use the intersection between the pulmonary artery cone and the right edge of the left atrial appendage and the line connecting the apex as the course mark of the mouse coronary artery anterior descending branch. Use a 6 / 0 suture to insert the needle 2mm below the root of the left atrial appendage, and exit the needle at the left edge of the pulmonary artery cone. The suture direction is parallel to the lower edge of the left atrial appendage. The sign of successful ligation is the weakening of the movement of the myocardial tissue around the anterior wall of the left ventricle and the apex. The ligature was released after 60 minutes of ischemia, and the mice were reperfused for 120 minutes to establish the myocardial ischemia-reperfusion injury model in mice.

[0091] 2. Grouping and intervention methods

[0092] The SPF-level C57BL / 6 mice after adaptive feeding were randomly weighed and randomly divided into 5 groups, namely, blank group B (normal saline), model group B (myocardial infarction model group, normal saline), high-dose group (myocardial protective composition of Example 1, 90 mg / kg / day), medium-dose group (myocardial protective composition of Example 1, 60 mg / kg / day), and low-dose group (myocardial protective composition of Example 1, 30 mg / kg / day), and the drugs were administered for 5 days.

[0093] 3. Experimental results detection

[0094] 3.1 Experimental process of HE staining: Myocardial tissue slices were baked, soaked in xylene, hydrated with gradient ethanol, stained with hematoxylin for 3 minutes, and counterstained with eosin for 1 minute. The stained slices were dehydrated with pure alcohol, treated with xylene to make the slices transparent, and sealed with neutral gum.

[0095] Figure 1 This is the HE image of the myocardial tissue of mice in the blank group B. Figure 2 HE images of myocardial tissue of mice in model group B. Figure 3 This is the HE image of the myocardial tissue of mice in the low-dose group. Figure 4 HE images of myocardial tissue of mice in the medium-dose group. Figure 5 HE images of myocardial tissue of mice in the low-dose group. Figures 1 to 5 As shown, compared with the blank group, the myocardial fibrosis area in the model group was significantly increased, and the cardioprotective composition can reduce the increase in fibrosis area in mice caused by myocardial infarction.

[0096] Figure 1 The gross structure of the myocardial tissue of mice in the blank group B was normal. Figure 2 This is the myocardial tissue of mice in model group B, in which the swelling of myocardial cells is aggravated. Figure 3 This is the myocardial tissue of mice in the low-dose group. Compared with the model group, myocardial disorder and myocardial cell swelling were alleviated. Figure 4 This is the myocardial tissue of mice in the medium-dose group. Compared with the low-dose group, the degree of myocardial disorder and myocardial cell swelling was alleviated. Figure 5 This is the myocardial tissue of mice in the high-dose group. Compared with the medium-dose group, the degree of myocardial disorder and myocardial cell swelling was alleviated.

[0097] 3.2 Experimental process of LDH level detection: The apical tissue of mice treated for 5 days was minced and digested with appropriate amount of 0.125% trypsin and 0.05% type II collagenase to prepare cell suspension, centrifuged at 1000r / min for 10min, and washed 3 times with PBS. 5 U Penicillin, 1×10 5The cells were cultured in DMEM medium containing U of streptomycin at 37°C and 5% CO2 for 2 h, and the cardiomyocytes were purified by adherent separation method. 5 The cells were inoculated into the culture plate at a density of 100 / ml, and the medium was changed after 48 hours. After culturing for 3 days, the supernatant of the cell culture medium was taken and the corresponding lactate dehydrogenase (LDH) value was determined using a lactate dehydrogenase (LDH) kit.

[0098] Figure 6 The bar graph of the myocardial injury marker lactate dehydrogenase LDH level in each group. Figure 6 It can be seen that the LDH level of the model group was significantly higher than that of each dose group. In the myocardial cells of mice treated with the myocardial protective composition of Example 1, the LDH level was significantly reduced and showed a dose-dependent relationship.

[0099] 3.3 Detection of cardiomyocyte apoptosis: Samples from different groups were cut into 5 μm slices using a freezing slicer and placed on adhesive slides. They were fixed with 4% formaldehyde at room temperature for 20 min, and then washed with PBS three times, 5 min each time. The PBS on the slide was gently shaken off, and when the slide was dry but the tissue was not completely dry, the sample range was carefully marked around each piece of tissue with an immunohistochemistry pen. 0.1% TritonX-100 was used for permeabilization at 4°C for 2 min, and PBS was washed three times, 5 min each time. 50 μL TUNEL staining solution was added, incubated at 37°C for 60 min, and PBS was washed three times, 5 min each time. Under light-proof conditions, PBS was gently shaken off, 50 μL DAPI (1 μg / mL) was added, and after incubation at room temperature for 5 min, the slide was washed three times with PBS, 5 min each time. Finally, the slide was sealed with 50% glycerol, and cell apoptosis was observed by laser confocal microscopy.

[0100] Figure 7 Schematic diagram of cardiomyocyte apoptosis in mice in blank group B. Figure 8 Schematic diagram of cardiomyocyte apoptosis in mice in model group B. Fig. 9 Schematic diagram of cardiomyocyte apoptosis in mice in the low-dose group. Fig.10 Schematic diagram of myocardial cell apoptosis in mice in the medium-dose group. Fig.11 Schematic diagram of cardiomyocyte apoptosis in mice in the high-dose group.

[0101] As for the effects of different interventions on the apoptosis level of myocardial cells in mice, when comparing the apoptosis level of myocardial cells in each group of mice, it was found that the number of apoptotic myocardial cells in the blank group B was small, and almost no apoptosis. The apoptosis rate of the model group B was significantly increased, and the number of apoptotic cells increased significantly. Compared with the model group B, the apoptosis rate of each dose group after treatment with the myocardial protection composition of Example 1 was significantly reduced, showing a dose-dependent relationship, and the number of apoptotic myocardial cells in the medium-dose group and the high-dose group was close to that of the blank group B.

[0102] 3.4 Myocardial infarction TTC detection: After 5 days of drug administration, the hearts of mice in each group were removed, sliced ​​and placed in 2% TTC, incubated at 37°C for 15 minutes, and stained with TTC. TTC staining pictures were taken, and the non-infarcted area was stained red, while the infarcted area was not stained and remained white.

[0103] Fig.12 Schematic diagram of TTC detection of myocardial infarction in the hearts of mice in each group. In the figure, the heart slices of the blank group B are completely red, with no myocardial infarction area. The myocardial infarction area accounts for about 3 / 4 of the heart slices of the model group B, and severe myocardial infarction has occurred. The myocardial infarction area of ​​the low-dose group treated with the myocardial protective composition of Example 1 is significantly smaller than that of the model group B, accounting for about 2 / 5 of the heart slice area. The myocardial infarction area of ​​the medium-dose group treated with the myocardial protective composition of Example 1 is significantly smaller than that of the model group B and the low-dose group, accounting for about 1 / 5 of the heart slice area. The myocardial infarction area of ​​the high-dose group treated with the myocardial protective composition of Example 1 is significantly smaller than that of the model group B, accounting for about 1 / 10 of the heart slice area. Therefore, in each dose group treated with the myocardial protective composition of Example 1, the area of ​​the infarction area of ​​the heart slice is significantly reduced, showing a dose-dependent relationship, and the area of ​​the infarction area of ​​the heart slice of the high-dose group is close to that of the blank group B.

[0104] The present invention reduces cell apoptosis and myocardial infarction reaction caused by myocardial ischemia through the myocardial protection composition, and its therapeutic effect is predictable, which provides a new option for clinical screening and preparation of drugs for treating or preventing myocardial infarction.

[0105] The specification of the present invention describes the specific implementation scheme in detail. Those skilled in the art should recognize that the above implementation scheme is exemplary and cannot be understood as limiting the present invention. For those skilled in the art, without departing from the principle of the present invention, by making several improvements and modifications to the present invention, the technical solutions obtained by these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. A myocardial protective composition, characterized in that: The composition comprises the following components in parts by weight: 10 to 30 parts of croton extract; 10 to 30 parts of Codonopsis pilosula extract; 1 to 10 parts of Ginkgo biloba extract.

2. A myocardial protection composition according to claim 1, characterized in that: The composition comprises the following components in parts by weight: 5 to 25 parts of croton extract; 5 to 25 parts of Codonopsis pilosula extract; 1 to 10 parts of Ginkgo biloba extract.

3. A myocardial protective composition according to claim 1, characterized in that: The croton extract is crotonin; the Codonopsis pilosula extract is codonoside.

4. A myocardial protective composition according to claim 1, characterized in that: The ginkgo extract is at least one of bilobalide, ginkgolide A, ginkgolide B, ginkgolide C and ginkgolide K.

5. A myocardial protective composition according to claim 4, characterized in that: The ginkgo extract is biloba lactone.

6. A myocardial protective composition according to any one of claims 1 to 5, characterized in that: The invention comprises the following components in parts by weight: 10 to 20 parts of crotonin; 10 to 20 parts of codonopsis pilosula; and 1 to 5 parts of bilobalide.

7. A myocardial protective composition according to claim 4, characterized in that: The invention comprises the following components in parts by weight: 10 to 15 parts of crotonin; 10 to 15 parts of codonopsis pilosula; and 2 to 4 parts of bilobalide.

8. A myocardial protective composition according to claim 5, characterized in that: The invention comprises the following components in parts by weight: 12 to 15 parts of crotonin; 12 to 15 parts of codonopsis pilosula; and 2 to 3 parts of bilobalide.

9. Use of the myocardial protective composition according to any one of claims 1 to 8 in the preparation of a medicament for preventing or treating myocardial injury.

10. Use of the myocardial protective composition according to any one of claims 1 to 8 in the preparation of a medicament for preventing or treating myocardial infarction.

Images