Pharmaceutical compositions for treating myocardial infarction, their preparation methods and uses

Abstract

This invention discloses a pharmaceutical composition for treating myocardial infarction, its preparation method, and its uses. The pharmaceutical composition is prepared from components comprising the following active pharmaceutical ingredients: 6.5–7.5 parts by weight of atractylodesin and 2.5–3.5 parts by weight of catechin. The pharmaceutical composition of this invention has a synergistic effect in the treatment of myocardial infarction.

Description

Technical Field

[0001] This invention relates to a pharmaceutical composition for treating myocardial infarction, and also to a method of preparation and use thereof. Background Technology

[0002] Myocardial infarction generally refers to acute myocardial infarction. Acute myocardial infarction is myocardial necrosis caused by acute and persistent ischemia and hypoxia of the coronary arteries. Clinically, it often presents with severe and persistent retrosternal pain that cannot be completely relieved by rest and nitrates, accompanied by elevated serum myocardial enzyme activity and progressive electrocardiographic changes. It can be complicated by arrhythmias, shock, or heart failure, and is often life-threatening.

[0003] Atractylodes lancea, a traditional Chinese medicine, is a dampness-resolving herb. It is the dried rhizome of Atractylodes lancea or Atractylodes dasycarpus, both belonging to the Asteraceae family. Atractylodes lancea extract is an active ingredient in the essential oil contained in the rhizome of Atractylodes lancea.

[0004] Catechin, also known as catechin acid or catechin extract, is a class of phenolic active substances extracted from natural plants such as tea leaves.

[0005] CN107158008A discloses a pharmaceutical composition for treating myocardial infarction, comprising tanshinone compounds and phenolic acid compounds containing one or more substituents. CN105287605B discloses a pharmaceutical composition comprising 0.5-2% protocatechuic acid, 1.5-3.5% cryptotanshinone, 65-85% borneol, and 13-30% eugenol. This pharmaceutical composition has a significant protective effect against hypoxia and reoxygenation of myocardial cells and can reduce the area of ​​myocardial infarction.

[0006] CN114887017A discloses a traditional Chinese medicine composition for treating coronary heart disease by eliminating dampness and resolving phlegm, and its application. The composition includes: 10-30 parts of Magnolia officinalis, 10-30 parts of Atractylodes lancea, 5-15 parts of Citrus reticulata peel, 5-20 parts of Curcuma zedoaria, 10-20 parts of Acorus tatarinowii, and 10-20 parts of Pinellia ternata. This composition can reduce the area of ​​myocardial infarction and myocardial fibrosis in mice with coronary heart disease and phlegm-dampness. CN106727487A discloses the application of atractylodes lancea extract in the preparation of drugs for treating and preventing heart failure.

[0007] CN108853086A discloses the use of catechin and epicatechin compounds for upregulating the expression level of MicroRNA-150. The patent document points out that catechin, epicatechin gallate, gallocatechin, gallocatechin gallate, epicatechin, epicatechin gallate, epigallocatechin and epigallocatechin gallate have the effect of upregulating the expression level of MicroRNA-150 and can be used to treat myocardial hypertrophy, hypertension, myocardial infarction, etc.

[0008] To date, there have been no reports of using atractylodesin and catechin in combination for the preparation of drugs to treat myocardial infarction. Summary of the Invention

[0009] In view of this, the inventors have discovered a pharmaceutical composition through in-depth research and experimentation that has few raw material types and can have a synergistic effect on the treatment of myocardial infarction.

[0010] Based on this, one object of the present invention is to provide a pharmaceutical composition for treating myocardial infarction, said pharmaceutical composition being prepared from a component comprising the following active pharmaceutical ingredients: 6.5 to 7.5 parts by weight of atractylodes lancea and 2.5 to 3.5 parts by weight of catechin.

[0011] According to the pharmaceutical composition of the present invention, preferably, the pharmaceutical composition is prepared from a component comprising the following active pharmaceutical ingredients: 6.8 to 7.3 parts by weight of atractylodes lancea and 2.7 to 3.2 parts by weight of catechin.

[0012] According to the pharmaceutical composition of the present invention, preferably, the pharmaceutical composition is prepared from a component comprising the following active pharmaceutical ingredients: 6.9 to 7.2 parts by weight of atractylodes lancea and 2.9 to 3.1 parts by weight of catechin.

[0013] According to the pharmaceutical composition of the present invention, preferably, the pharmaceutical composition is made only from the following components of active pharmaceutical ingredients: 6.5 to 7.5 parts by weight of atractylodes lancea and 2.5 to 3.5 parts by weight of catechin.

[0014] According to the pharmaceutical composition of the present invention, preferably, the pharmaceutical composition is made only from the following components of active pharmaceutical ingredients: 6.9 parts by weight of atractylodes lancea and 3.0 parts by weight of catechin.

[0015] According to the pharmaceutical composition of the present invention, preferably, the pharmaceutical composition is made only from the following components of active pharmaceutical ingredients: 7.0 parts by weight of atractylodes lancea and 3.0 parts by weight of catechin.

[0016] According to the pharmaceutical composition of the present invention, preferably, the pharmaceutical composition is made only from the following components of active pharmaceutical ingredients: 7.15 parts by weight of atractylodes lancea and 3.0 parts by weight of catechin.

[0017] According to the pharmaceutical composition of the present invention, preferably, the pharmaceutical composition is obtained by mixing atractylodes lancea and catechin.

[0018] Another objective is to provide a pharmaceutical preparation for treating myocardial infarction, said pharmaceutical preparation comprising the pharmaceutical composition as described above and pharmaceutically acceptable excipients.

[0019] Another objective is to provide the use of the pharmaceutical composition as described above in the preparation of a medicament for treating myocardial infarction.

[0020] The traditional Chinese medicine composition of the present invention can synergistically enhance its effects within a specific ratio range. The two components, when combined, show significant efficacy in the treatment of myocardial infarction. According to a preferred embodiment of the present invention, the ratio of the pharmaceutical composition is: 6.9–7.2 parts by weight of atractylodes lancea and 2.9–3.1 parts by weight of catechins. Attached Figure Description

[0021] Figure 1 In this invention, the effect of different concentrations of atractylodes lancea (Atr) on the viability of H9c2 cells was detected using a CCK-8 assay (n=4).

[0022] Figure 2 In this invention, CCK-8 assay was used to detect the effect of different concentrations of catechin on the viability of H9c2 cells (n=4).

[0023] Figure 3 In this invention, the effect of different concentrations of atractylodes lancea (Atr) on the viability of H9c2 cells induced by H2O2 was detected using a CCK-8 assay (n=4).

[0024] Figure 4 In this invention, the effect of different concentrations of catechin on the viability of H9c2 cells induced by H2O2 was detected using a CCK-8 assay (n=4).

[0025] Figure 5 In this invention, the effect of different ratios of atractylodesin (Atr) / catechin on the viability of H9c2 cells induced by H2O2 was detected using a CCK-8 assay (n=3). Detailed Implementation

[0026] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0027] <Pharmaceutical Composition>

[0028] The pharmaceutical composition for treating myocardial infarction of the present invention is prepared from a component comprising the following active pharmaceutical ingredients to obtain atractylodes and catechins. The atractylodes comprises 6.5–7.5 parts by weight, and the catechins comprise 2.5–3.5 parts by weight. Preferably, the pharmaceutical composition is prepared from a component comprising the following active pharmaceutical ingredients: atractylodes comprises 6.8–7.3 parts by weight, and the catechins comprise 2.7–3.2 parts by weight. More preferably, the pharmaceutical composition is prepared from a component comprising the following active pharmaceutical ingredients: atractylodes comprises 6.9–7.2 parts by weight, and the catechins comprise 2.9–3.1 parts by weight. Such a specific formulation of the pharmaceutical composition can synergistically enhance the therapeutic effect on myocardial infarction.

[0029] In some embodiments, the pharmaceutical composition is made solely from the following active pharmaceutical ingredient: 6.5–7.5 parts by weight of atractylodesin and 2.5–3.5 parts by weight of catechin. For example, atractylodesin may be present in quantities of 6.6, 6.7, 6.8, 6.85, 6.9, 6.95, 7.0, 7.1, 7.15, 7.2, 7.3, 7.4, or 7.5 parts by weight, and catechin may be present in quantities of 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, or 3.2 parts by weight.

[0030] According to one specific embodiment of the present invention, the pharmaceutical composition is made only from the following components of active pharmaceutical ingredients: 6.8 parts by weight of atractylodes lancea and 3.0 parts by weight of catechin.

[0031] According to one specific embodiment of the present invention, the pharmaceutical composition is made only from the following components of active pharmaceutical ingredients: 6.9 parts by weight of atractylodes lancea and 3.0 parts by weight of catechin.

[0032] According to one specific embodiment of the present invention, the pharmaceutical composition is made only from the following components of active pharmaceutical ingredients: 7.0 parts by weight of atractylodes lancea and 3.0 parts by weight of catechin.

[0033] According to another specific embodiment of the present invention, the pharmaceutical composition is made only from the following components of active pharmaceutical ingredients: 7.1 parts by weight of atractylodes lancea and 3.0 parts by weight of catechin.

[0034] According to another specific embodiment of the present invention, the pharmaceutical composition is made only from the following components of active pharmaceutical ingredients: 7.2 parts by weight of atractylodes lancea and 3.0 parts by weight of catechin.

[0035] In some embodiments of the present invention, when conducting experiments on the effect of the pharmaceutical composition on the viability of H9c2 cells induced by H2O2, the pharmaceutical composition is prepared as a solution (the solvent is cell culture medium, such as DMEM (1×) cell culture medium), wherein atractylodesin is present in 6.9–7.2 parts by weight, catechin is present in 2.9–3.1 parts by weight, and the total concentration of atractylodesin and catechin is 4–8 μmol / L, preferably 4.5–7 μmol / L, more preferably 5–6 μmol / L. Under such conditions, the synergistic effect of atractylodesin and catechin is better.

[0036] The structural formula of atractylodesin is as follows:

[0037]

[0038] The structural formula of catechins is as follows:

[0039]

[0040] In the prior art, there are reports of individual studies on catechins and atractylodes, but no reports on the synergistic effect of combined use of atractylodes and catechins in the treatment of myocardial infarction. Through extensive research and experimentation, the inventors have discovered that, within a specific ratio range, the combined use of atractylodes and catechins can achieve a synergistic effect in the treatment of myocardial infarction.

[0041] Furthermore, the inventors have discovered that if the ratio of the two ingredients is outside the range specified in this invention, they essentially do not have a synergistic effect and may even exhibit antagonistic effects. For example, when the weight ratio of atractylodesin to catechin is 9:1, the two ingredients show significant antagonistic effects. Currently, the inventors are unclear about the mechanism of synergistic effect between the two ingredients within a specific ratio range.

[0042] <Preparation Method>

[0043] In this invention, atractylodes and catechin are mixed evenly to obtain the pharmaceutical composition.

[0044] <Pharmaceutical Preparations and Uses>

[0045] The present invention also provides a pharmaceutical preparation for treating myocardial infarction, the pharmaceutical preparation comprising the pharmaceutical composition as described above and pharmaceutically acceptable excipients. The dosage form of the preparation is not limited, and may be, for example, tablets, pills, capsules, granules, oral liquids, etc.

[0046] This invention also provides the use of the above-mentioned pharmaceutical composition in the preparation of a medicament for treating myocardial infarction. The present invention has found that atractylodes and catechins alone enhance the viability of H9c2 cells induced by H2O2, but their combined use in a specific ratio has a more significant enhancing effect on the viability of H9c2 cells induced by H2O2, exhibiting a clear synergistic effect.

[0047] The sources of the reagents used in the following examples and comparative examples:

[0048] Atractylodes lancea and catechin were purchased from Nanjing Jingzhu Biotechnology Co., Ltd., and were both powders with a purity >98% (HPLC).

[0049] Example 1

[0050] Seven parts by weight of atractylodes and three parts by weight of catechin were mixed evenly to obtain a pharmaceutical composition.

[0051] Comparative Example 1

[0052] Two parts by weight of atractylodes and eight parts by weight of catechin were mixed evenly to obtain a pharmaceutical composition.

[0053] Comparative Example 2

[0054] Four parts by weight of atractylodes and six parts by weight of catechin were mixed evenly to obtain a pharmaceutical composition.

[0055] Comparative Example 3

[0056] Six parts by weight of atractylodes and four parts by weight of catechin were mixed evenly to obtain a pharmaceutical composition.

[0057] Comparative Example 4

[0058] Eight parts by weight of atractylodes and two parts by weight of catechin were mixed evenly to obtain a pharmaceutical composition.

[0059] Comparative Example 5

[0060] Nine parts by weight of atractylodes and one part by weight of catechin were mixed evenly to obtain a pharmaceutical composition.

[0061] The pharmaceutical compositions obtained in the above examples and comparative examples were subjected to cell experiments.

[0062] 1. Experimental Materials

[0063] Table 1. Experimental Materials and Reagents

[0064] name source DMEM (1×) cell culture medium Hyclone serum Hyclone Penicillin-Streptomycin Solution Hyclone 0.25% pancreatic enzyme cell digestion solution Biosharp CCK-8 reagent kit Biosharp Rat cardiomyocyte line H9c2 Shanghai Fuheng Biotechnology Co., Ltd.

[0065] 2. Experimental Methods

[0066] 2.1 Cell Culture

[0067] Rat cardiomyocyte line H9c2 culture conditions: cultured in DMEM medium containing 10% serum and 1% penicillin-streptomycin solution at 37°C in a 5% CO2 incubator, with the culture medium changed or passaged every 1-2 days.

[0068] 2.1.1 Cell resuscitation

[0069] (1) Thaw the frozen cells in a 37°C water bath until they are covered with ice crystals, then transfer them into an EP tube containing 3 mL of 10% DMEM culture medium and mix well.

[0070] (2) Centrifuge at 1000 rpm for 5 min;

[0071] (3) Discard the supernatant, add 1 mL of culture medium, and mix well;

[0072] (4) Add the cell suspension to a cell culture dish and incubate it in an incubator.

[0073] 2.1.2 Cell passage

[0074] (1) Observe the state under a microscope. When the cell density reaches 80% to 90%, discard the culture medium and wash twice with sterile PBS.

[0075] (2) Add 1 mL of 0.25% trypsin and digest for 1 min, then add 3 mL of 10% DMEM culture medium to stop digestion;

[0076] (3) Centrifuge at 1000 rpm for 5 min;

[0077] (4) Discard the supernatant, add 1 mL of culture medium, gently blow and aspirate to turn the cell cluster into single cells, then add them evenly to 2-3 culture dishes and place them in an incubator for culture.

[0078] 2.1.3 Cell cryopreservation

[0079] (1) Observe the state under a microscope. When the cell density reaches 80-90%, discard the culture medium and wash twice with sterile PBS.

[0080] (2) Add 1 mL of 0.25% trypsin and digest for 1 min, then add 3 mL of 10% DMEM culture medium to stop digestion;

[0081] (3) Centrifuge at 1000 rpm for 5 min;

[0082] (4) Discard the supernatant, add fetal bovine serum:DMSO (9:1) cryopreservation solution, and transfer it into a cryovial;

[0083] (5) Place it in a cryogenic container at room temperature and store it in a -80°C freezer overnight. The next day, store it in liquid nitrogen.

[0084] 2.2 CCK-8 cell proliferation

[0085] (1) Seed the cells in 96-well cell culture plates and culture them in an incubator;

[0086] (2) When the cell density is about 60-70%, discard the culture medium and add different treatment factors to continue culturing;

[0087] (3) Add 10 μL of CCK-8 test reagent to each well and incubate for 2 hours;

[0088] (4) The absorbance at 450 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader.

[0089] 3. Experimental Results

[0090] 3.1 Effects of different concentrations of hydrogen peroxide (H2O2) on the viability of H9c2 cells were detected using a CCK-8 assay.

[0091] To determine the effective concentration of H2O2 on rat cardiomyocytes H9c2, H9c2 cells were first treated with different concentrations of H2O2 (0 μmol / L, 25 μmol / L, 50 μmol / L, 100 μmol / L, 200 μmol / L, 400 μmol / L, 800 μmol / L, and 1600 μmol / L) for 4 h. The effect of different concentrations of H2O2 on H9c2 cell viability was detected by the CCK-8 assay. The results showed that, compared with the control group, H2O2 concentrations of 800 μmol / L and 1600 μmol / L significantly inhibited H9c2 cell viability (P<0.01).

[0092] Since the concentration difference between 400 μmol / L and 800 μmol / L was significant, the concentration difference between groups was reduced, and H9c2 cells were treated with different concentrations of H2O2 (0 μmol / L, 400 μmol / L, 500 μmol / L, 600 μmol / L, 700 μmol / L, and 800 μmol / L) for 4 h. The effect of different concentrations of H2O2 on H9c2 cell viability was detected using the CCK-8 assay. The results showed that, compared with the control group, H2O2 concentrations of 700 μmol / L and 800 μmol / L significantly inhibited H9c2 cell viability (P<0.01). Subsequent experiments used 700 μmol / L H2O2 as the treatment concentration.

[0093] 3.2 The effects of different concentrations of atractylodesin (Atr) and catechin on the viability of H9c2 cells were detected using a CCK-8 assay.

[0094] To determine the effective concentrations of atractylodesin and catechin on H9c2 cells, H9c2 cells were treated with different concentrations of atractylodesin and catechin (0 μmol / L, 5 μmol / L, 10 μmol / L, 20 μmol / L, 40 μmol / L, and 80 μmol / L) for 24 h. The effects of different concentrations of atractylodesin and catechin on H9c2 cell viability were detected by the CCK-8 assay. The results showed that, compared with the control group, none of the concentrations of atractylodesin and catechin inhibited H9c2 cell viability (see [link to study].) Figure 1 and Figure 2 ). Figure 1 and Figure 2 In the graph, the horizontal axis “CON” represents the control group, and the unit of the horizontal axis values ​​is μmol / L. The vertical axis represents “cell viability %”.

[0095] 3.3 The effects of different concentrations of atractylodesin (Atr) and catechin on H2O2-induced H9c2 cell viability were detected using a CCK-8 assay.

[0096] To determine the effects of atractylodesin and catechin on H2O2-induced H9c2 cell viability, H9c2 cells were first pretreated with 700 μmol / L H2O2 for 4 h, and then treated with different concentrations of atractylodesin and catechin (0 μmol / L, 5 μmol / L, 10 μmol / L, 20 μmol / L, 40 μmol / L, and 80 μmol / L) for 24 h. The effects of different concentrations of atractylodesin and catechin on H2O2-induced H9c2 cell viability were detected using the CCK-8 assay. The results showed that, compared with the control group, the viability of H9c2 cells in the H2O2 group was significantly reduced (P<0.01); 5, 10, and 20 μmol / L atractylodes lancea extracts all inhibited H2O2-induced H9c2 cell damage (P<0.05); 5, 10, 20, and 40 μmol / L catechins all inhibited H2O2-induced H9c2 cell damage (P<0.05, P<0.01). Figure 3 and Figure 4 Therefore, 5 μmol / L atractylodesin and catechin were selected as the treatment concentrations in subsequent experiments. Figure 3 and Figure 4 In the above, compared with the control group (CON), "**" indicates P < 0.01; compared with the model group (H2O2), "##" indicates P < 0.01; and compared with the model group (H2O2), "#" indicates P < 0.05. The horizontal axis "CON" represents the control group, and "H2O2" represents the H9c2 cell model group induced by H2O2.

[0097] Figure 3 In the figure, the horizontal axis “H2O2+5Atr” indicates that H9c2 cells were pretreated with H2O2 using 5 μmol / L atractylodesin, and so on. Figure 4 In the figure, the horizontal axis “H2O2+5Cat” indicates that H9c2 cells were pretreated with H2O2 at a concentration of 5 μmol / L catechins, and so on.

[0098] 3.4 Effects of different ratios of atractylodesin (Atr) / catechin on H2O2-induced H9c2 cell viability were detected using a CCK-8 assay.

[0099] Atractylodes lancea (Atr) and catechin (total concentration 5 μmol / L) were mixed in different proportions, and the effects of different combinations on H2O2-induced H9c2 cell viability were examined. Figure 5It can be seen that, compared with the use of the two drugs alone, most of the different ratios significantly improved the H2O2-induced H9c2 cell activity. Among them, the weight ratio of atractylodes lancea (Atr) to catechin (Catechin) was 7:3, which is the best effect of Example 1. However, in Comparative Example 5, under the condition of a weight ratio of atractylodes lancea to catechin of 9:1, compared with atractylodes lancea and catechin alone, the activity of H2O2-induced H9c2 cells was not only not improved, but was significantly reduced, that is, atractylodes lancea and catechin have obvious antagonistic effects at this ratio.

[0100] Figure 5 In the comparison with the control group (CON), "**" indicates P < 0.01; compared with the model group (H2O2), "##" indicates P < 0.01; compared with the model group (H2O2), "#" indicates P < 0.05. Compared with Example 1 group, "&&" indicates P < 0.01, and "&" indicates P < 0.05.

[0101] Figure 5 In the text, “CON” represents the control group, “H2O2” represents the H2O2-induced H9c2 cell model group, “Atr” represents the experiment using atractylodes alone on H2O2-induced H9c2 cells, and “Cat” represents the experiment using catechin alone on H2O2-induced H9c2 cells.

[0102] Table 2

[0103] serial number The weight ratio of atractylodesin to catechin Example 1 7:3 Comparative Example 1 2:8 Comparative Example 2 4:6 Comparative Example 3 6:4 Comparative Example 4 8:2 Comparative Example 5 9:1

[0104] This invention is not limited to the above-described embodiments. Any modifications, improvements, or substitutions that can be conceived by those skilled in the art without departing from the essential content of this invention fall within the scope of this invention.

Claims

1. A pharmaceutical composition for treating myocardial infarction, characterized in that, The pharmaceutical composition is made solely from the following active pharmaceutical ingredients: 6.9 to 7.2 parts by weight of atractylodes lancea and 3.0 parts by weight of catechin.

2. The pharmaceutical composition according to claim 1, characterized in that, The pharmaceutical composition is made solely from the following active pharmaceutical ingredients: 6.9 parts by weight of atractylodes lancea and 3.0 parts by weight of catechin.

3. The pharmaceutical composition according to claim 1, characterized in that, The pharmaceutical composition is made solely from the following active pharmaceutical ingredients: 7.0 parts by weight of atractylodes lancea and 3.0 parts by weight of catechin.

4. The pharmaceutical composition according to claim 1, characterized in that, The pharmaceutical composition is made solely from the following active pharmaceutical ingredients: 7.15 parts by weight of atractylodes lancea and 3.0 parts by weight of catechin.

5. The method for preparing the pharmaceutical composition according to any one of claims 1 to 4, characterized in that, Atractylodes lancea and catechin are mixed to obtain the pharmaceutical composition.

6. A pharmaceutical preparation for treating myocardial infarction, characterized in that, The pharmaceutical preparation comprises the pharmaceutical composition according to any one of claims 1 to 4 and pharmaceutically acceptable excipients.

7. Use of the pharmaceutical composition according to any one of claims 1 to 4 in the preparation of a medicament for treating myocardial infarction.

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