A pharmaceutical composition for alleviating oxidative stress associated with myocardial ischemia-reperfusion injury and application thereof

Abstract

The application provides a pharmaceutical composition for alleviating myocardial ischemia-reperfusion injury related oxidative stress and application thereof, and active ingredients of the composition comprise ilexin A, isochlorogenic acid C and isohyperoside, the active ingredients jointly act to achieve inhibition of myocardial ischemia-reperfusion injury related oxidative stress, alleviation of myocardial cell damage and improvement of mitochondrial function.

Description

Technical Field

[0001] This invention relates to the field of biomedicine and natural medicine application technology, and in particular to a pharmaceutical composition for alleviating oxidative stress associated with myocardial ischemia-reperfusion injury and its application. Background Technology

[0002] Myocardial ischemia-reperfusion injury (MIRI) is a common secondary pathological injury that occurs frequently during reperfusion therapy, interventional cardiology, and surgical procedures following acute myocardial infarction. Restoring blood flow is essential for saving ischemic myocardium, but the reperfusion process can induce oxidative stress, excessive accumulation of reactive oxygen species, decreased mitochondrial membrane potential, membrane lipid peroxidation, and cell death, causing secondary myocardial injury and becoming a key risk factor affecting the prognosis of cardiovascular disease patients.

[0003] Existing research confirms that the occurrence and development of myocardial infarction (MIRI) are closely related to oxidative stress, lipid peroxidation, and damage to cell membrane and mitochondrial function. Screening for antioxidant strategies that can intervene in multiple aspects is of great value for the clinical prevention and treatment of MIRI. Mountain green tea contains a variety of natural antioxidant active ingredients, among which holmarin A, isochlorogenic acid C, and isoquercitrin have complementary chemical types and mechanisms of action, all of which have a basis for myocardial protection.

[0004] Existing technologies have many shortcomings: there are limited effective intervention options for MIRI, and there is a lack of combination drug regimens with natural active ingredients that are clearly defined in composition, source, and easy to control in terms of quality; a single active ingredient can only act on some pathological aspects of MIRI and cannot achieve multi-target synergistic intervention; there is a lack of systematic efficacy evaluation of the combination of the above three ingredients to prevent and treat MIRI, and there is no efficacy comparison data with the whole formulation of Shanlvcha capsules, which cannot provide experimental support for the development of related precise drug combinations. Summary of the Invention

[0005] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0006] A pharmaceutical composition is provided for alleviating oxidative stress associated with myocardial ischemia-reperfusion injury. The active ingredients of the composition include holmine A, isochlorogenic acid C, and isoquercitrin. The active ingredients work together to inhibit oxidative stress associated with myocardial ischemia-reperfusion injury, reduce myocardial cell damage, and improve mitochondrial function.

[0007] In some embodiments, the wintergreen glycoside A, isochlorogenic acid C, and isoquercitrin are all derived from the extraction, separation, and purification products of green tea, or are chemically synthesized, or are equivalent compounds with the same structure.

[0008] In some embodiments, the molar ratio of holmarin A, isochlorogenic acid C, and isoquercitrin is 1:(0.5-5):(0.5-5).

[0009] In some embodiments, the molar ratio of holmarin A, isochlorogenic acid C, and isoquercitrin is 1:2:2.

[0010] In some embodiments, in the in vitro cell experimental system, the working concentration of holly A is 20 μM, the working concentration of isochlorogenic acid C is 40 μM, and the working concentration of isoquercitrin is 40 μM.

[0011] In some embodiments, the composition further comprises at least one of a pharmaceutically acceptable carrier, excipient, diluent, stabilizer, or solvent.

[0012] In some embodiments, the dosage form of the pharmaceutical composition is selected from one or more of the following: injection, lyophilized powder for injection, tablets, capsules, granules, and oral liquids.

[0013] The use of the pharmaceutical composition in the preparation of drugs or formulations for the prevention, mitigation or adjuvant treatment of myocardial ischemia-reperfusion injury-related oxidative stress, research compositions or cell-protective drugs.

[0014] Application of the pharmaceutical composition in the preparation of a drug for reducing oxygen-glucose deprivation / reoxygenation-induced cardiomyocyte damage.

[0015] Its effects include: improving cardiomyocyte vitality, reducing lactate dehydrogenase, malondialdehyde and reactive oxygen species levels, increasing superoxide dismutase and glutathione levels, and improving mitochondrial membrane potential.

[0016] Compared with existing technologies, the beneficial effects of this invention are as follows: The pharmaceutical composition of this invention for alleviating oxidative stress related to myocardial ischemia-reperfusion injury addresses the shortcomings of existing combined drug formulations of natural active ingredients, such as ambiguous composition and difficulty in quality control. It constructs a triple combination using three clearly defined active ingredients from *Mountain Green Tea*, ensuring controllable sources and easy quality standardization, reflecting technological optimization from complex extracts to clearly defined ingredient combinations. It overcomes the limitations of single-drug intervention in MIRI pathological processes; the three different types of ingredients have complementary effects, enabling simultaneous intervention at multiple targets and stages related to MIRI pathological processes, resulting in a more comprehensive intervention effect. It fills the technological gap in the combined use of these three ingredients for the protection against OGD / R-induced cardiomyocyte damage. Experimental screening has determined a safe and effective concentration, and the safety and efficacy of the dosing regimen are supported by solid experimental evidence. The combined protective effect is superior to that of single ingredients and the complete *Mountain Green Tea* capsule formulation, showing more significant improvements in key indicators such as cell viability, LDH, and MDA, and exhibiting stronger protective effects. The administration is convenient, providing a clear compatibility basis for subsequent related experiments, a scientific basis for the precise development of *Mountain Green Tea* active ingredients, and facilitating industrial transformation. Attached Figure Description

[0017] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof.

[0018] Figure 1 The graph shows the results of the detection of CCK-8 cytotoxicity of holmarin A, isochlorogenic acid C, isoquercitrin and mountain green tea capsules on H9C2 cells.

[0019] Figure 2 The figure shows the results of screening for the optimal therapeutic concentrations of holly A, isochlorogenic acid C, isoquercitrin, and mountain green tea capsules under the OGD / R model.

[0020] Figure 3 The figure shows the effects of single administration and triple combination administration of holly A, isochlorogenic acid C, isoquercitrin, and mountain green tea capsules on the viability of H9C2 cells after OGD / R injury.

[0021] Figure 4 The graph shows the detection results of LDH, SOD, MDA, and GSH for each group.

[0022] Figure 5 Images and results of fluorescence detection of ROS and JC-1 in each group. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. Of course, the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention. Experimental methods and reagent preparation methods not specifically specified in the embodiments of this invention are all conventional cell biology experimental operating procedures in the art; the cell lines, reagents, consumables, and instruments used can all be obtained through legitimate commercial channels.

[0024] Example 1: Evaluation of the cytotoxicity of holmarin A, isochlorogenic acid C and isoquercitrin on H9C2 cells.

[0025] H9C2 cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin and streptomycin at 37°C and 5% CO2.

[0026] Wintergreen A, isochlorogenic acid C, isoquercitrin and mountain green tea capsules were prepared with DMSO stock solutions. Before use, they were diluted with complete culture medium to the corresponding working concentration to ensure that the final concentration of DMSO in the drug delivery system did not exceed 0.1%.

[0027] H9C2 cells were seeded in 96-well plates and cultured for 24 h. Then, they were treated with different concentrations of holly A, isochlorogenic acid C, isoquercitrin, and mountain green tea capsules. After treatment, CCK-8 working solution was added according to the CCK-8 kit instructions and incubated for 1-2 h. The absorbance was measured at 450 nm and cell viability was calculated.

[0028] The results are as follows Figure 1 As shown, Figure 1 The image shows the results of CCK-8 cytotoxicity assays on H9C2 cells by holly A, isochlorogenic acid C, isoquercitrin, and mountain green tea capsules. Holly A showed no significant cytotoxicity to H9C2 cells in the concentration range of 0-40 μM, isochlorogenic acid C showed no significant cytotoxicity in the concentration range of 0-80 μM, isoquercitrin showed no significant cytotoxicity in the concentration range of 0-80 μM, and the mountain green tea capsule group (IHM) also showed no significant cytotoxicity in the concentration range of 0-160 μg / mL. Therefore, the above concentration ranges with no significant cytotoxicity were selected for subsequent OGD / R model drug administration experiments.

[0029] Example 2: Establishment of an OGD / R-induced H9C2 cell damage model and screening of optimal therapeutic concentration

[0030] H9C2 cells were seeded in 96-well plates and OGD / R treatment was performed when the cells reached 70%-80% confluence. The OGD / R model was established as follows: the original culture medium was discarded, the cells were gently washed with PBS, and then glucose-free and serum-free culture medium was added. The cells were then placed in a 95% N2, 5% CO2 hypoxic incubator for 6 hours. Subsequently, the culture medium was replaced with normal complete culture medium and reoxygenated under normoxic conditions for 8 hours.

[0031] Under OGD / R model conditions, cells were treated with different non-toxic concentrations of holly A, isochlorogenic acid C, isoquercitrin, and mountain green tea capsules, and cell viability was evaluated using the CCK-8 assay. Results are as follows: Figure 2 As shown, Figure 2 The figure shows the results of screening for the optimal therapeutic concentrations of holly A, isochlorogenic acid C, isoquercitrin, and mountain green tea capsules under the OGD / R model. Holly A showed the best protective effect against OGD / R-damaged H9C2 cells at 20 μM, while isochlorogenic acid C and isoquercitrin both showed the best protective effect at 40 μM. The mountain green tea capsule group (IHM) showed the best protective effect at 80 μg / mL. Therefore, the above optimal concentrations were selected for subsequent combination therapy and comparative experiments.

[0032] Example 3: Effect of triple therapy on the viability of H9C2 cells after OGD / R injury

[0033] The experimental groups included: normal control group, OGD / R model group, OGD / R + holly A group (20 μM), OGD / R + isochlorogenic acid C group (40 μM), OGD / R + isoquercitrin group (40 μM), OGD / R + holly A + isochlorogenic acid C + isoquercitrin combined group (20 μM + 40 μM + 40 μM), and OGD / R + mountain green tea capsule group (IHM, 80 μg / mL).

[0034] The three components were prepared as stock solutions with DMSO and then added to the culture system at the working concentrations mentioned above. In the combined group, the three components were mixed and administered together. The mountain green tea capsule group (IHM) was added to the culture system at a working concentration of 80 μg / mL as a comparative control for the whole mountain green tea preparation. After treatment, cell viability in each group was detected by the CCK-8 assay.

[0035] The results are as follows Figure 3 As shown, Figure 3 The figure shows the effects of single and triple therapy administration of holmine A, isochlorogenic acid C, isoquercitrin, and *Salvia miltiorrhiza* capsules on the viability of H9C2 cells after OGD / R injury. Compared with the normal control group, cell viability in the OGD / R model group was significantly decreased. Compared with the OGD / R model group, each single drug group and the *Salvia miltiorrhiza* capsule group all improved cell viability to varying degrees. The triple therapy group showed the highest degree of cell viability recovery, which was not only better than the corresponding single drug treatments but also better than the *Salvia miltiorrhiza* capsule group, suggesting that the combined use of the three drugs has a better protective effect against OGD / R-induced H9C2 cell injury.

[0036] Example 4: Effects of triple therapy on cell damage and oxidative stress indicators.

[0037] The cells were grouped and treated according to Example 3. After treatment, cells or cell supernatant were collected, and the levels of LDH, SOD, MDA, and GSH were measured according to the kit instructions.

[0038] The results are as follows Figure 4 , Figure 4The figures show the results of LDH, SOD, MDA, and GSH detection in each group. After OGD / R treatment, the levels of LDH and MDA in H9C2 cells increased, while the levels of SOD and GSH decreased, indicating that cell damage in the model group was aggravated and accompanied by significant oxidative stress. Compared with the OGD / R model group, holly A, isochlorogenic acid C, isoquercitrin, and mountain green tea capsules, administered alone, could reduce LDH and MDA levels to varying degrees and increase SOD and GSH levels. The triple combination therapy group showed the most significant improvement in the above indicators, with LDH decreasing to 190.33±17.18 U / L, SOD increasing to 15.3931±0.4701 U / mgprot, MDA decreasing to 1.2213±0.0997 nmol / mgprot, and GSH increasing to 85.0480±6.3165 μmol / gprot, all of which were significantly improved compared with the OGD / R model group (all P<0.0001). The above results indicate that triple therapy can effectively reduce OGD / R-induced cell damage and alleviate oxidative stress, as shown in Tables 1, 2, 3 and 4 below.

[0039] Table 1: Effects of triple therapy on LDH levels in H9C2 cells

[0040]

[0041] Table 2: Effects of triple therapy on SOD levels in H9C2 cells

[0042]

[0043] Table 3: Effects of triple therapy on MDA levels in H9C2 cells

[0044]

[0045] Table 4: Effects of triple therapy on GSH levels in H9C2 cells

[0046]

[0047] Example 5: Effect of triple therapy on intracellular ROS levels.

[0048] The cells were grouped and treated according to Example 3. After treatment, the cells were stained with DCFH-DA fluorescent probes, observed and images were acquired under a confocal microscope, and the intracellular ROS fluorescence intensity of each group was analyzed.

[0049] The results are as follows Figure 5 As shown, Figure 5The images show the ROS and JC-1 fluorescence detection results for each group. Compared with the normal control group, the intracellular ROS fluorescence intensity in the OGD / R model group was significantly increased, from 1.0000±0.3715 to 8.5898±1.7986 (P<0.0001), indicating that OGD / R treatment can induce a large accumulation of reactive oxygen species in cells. Compared with the OGD / R model group, the ROS fluorescence intensity in the holly A group, isochlorogenic acid C group, isoquercitrin group, and mountain green tea capsule group decreased to 3.6800±1.5896, 4.6438±1.4462, 4.8890±1.4318, and 3.2983±1.8922, respectively. Among them, the mountain green tea capsule group showed a significant difference compared with the model group (P<0.0001), indicating that it can also reduce the accumulation of reactive oxygen species. The ROS fluorescence intensity in the triple-drug combination group further decreased to 2.5979±1.5265, which was significantly lower than that in the OGD / R model group (P<0.0001), and showed the lowest ROS value among all treatment groups. Overall, the triple-drug combination group showed a better effect in inhibiting reactive oxygen species accumulation than the mountain green tea capsule group. (See Table 5 below.)

[0050] Table 5: Effects of triple therapy on intracellular ROS fluorescence intensity in H9C2 cells

[0051]

[0052] The above results indicate that triple therapy can more effectively inhibit the accumulation of reactive oxygen species induced by OGD / R, thereby reducing oxidative stress damage.

[0053] Example 6: Effect of triple therapy on mitochondrial membrane potential.

[0054] The cells were grouped and treated according to Example 3. After treatment, JC-1 fluorescent probe staining was performed, and images were observed and acquired under a confocal microscope. The red / green fluorescence ratio was analyzed to evaluate changes in mitochondrial membrane potential. As shown in Table 6 below, compared with the normal control group, the JC-1 red / green fluorescence ratio in the OGD / R model group cells decreased significantly, from 4.3875±1.3351 to 1.2157±0.1766 (P<0.0001), indicating a decrease in mitochondrial membrane potential and significant mitochondrial dysfunction. Compared with the OGD / R model group, the JC-1 red / green fluorescence ratios of the holly A group, isochlorogenic acid C group, isoquercitrin group, mountain green tea capsule group, and triple combination therapy group increased to 3.1678±0.7422, 2.2741±1.2936, 2.2665±0.8118, 2.6743±0.8630, and 3.4594±0.9385, respectively. Among these, the holly A group (P=0.0057) and the triple combination therapy group (P=0.0013) showed statistically significant differences compared with the model group. The numerical trends showed that the triple combination therapy group exhibited the best recovery effect in red / green fluorescence ratio, suggesting that the combined use of the three components has a better protective effect in maintaining mitochondrial membrane potential stability and improving OGD / R-induced mitochondrial dysfunction.

[0055] Table 6: Effects of triple therapy on mitochondrial membrane potential in H9C2 cells

[0056] The above results collectively demonstrate that holly A, isochlorogenic acid C, and isoquercitrin have a clear combined protective effect in the H9C2 cell OGD / R injury model. This drug composition can improve cardiomyocyte viability, reduce lactate dehydrogenase, malondialdehyde, and reactive oxygen species levels, increase superoxide dismutase and glutathione levels, and improve mitochondrial membrane potential. Furthermore, when holly A, isochlorogenic acid C, and isoquercitrin are used in combination at their respective optimal working concentrations, their protective effect against OGD / R-induced cardiomyocyte injury is superior to that of the respective single administrations, and the overall numerical trend is superior to that of the mountain green tea capsule group.

[0057] Based on the overall numerical trends of multiple cell protection indicators, the triple combination is superior to the mountain green tea capsule group. Therefore, it can be used to prepare drugs, formulations, or research compositions that alleviate myocardial ischemia-reperfusion injury-related oxidative stress and cell damage.

[0058] The combined administration method is simple and can provide a clear compatibility basis for subsequent pharmacodynamic, mechanistic and animal experimental studies, and provide a basis for the precise compatibility development of active ingredients of green tea.

[0059] The triple combination therapy group further reduced intracellular ROS accumulation and more significantly improved the decrease in the JC-1 red / green fluorescence ratio, indicating that it has a better protective effect on mitochondrial function.

[0060] The three components, administered alone or in combination with the green tea capsules, all improved cell damage caused by OGD / R to some extent. The triple combination therapy group showed the greatest improvement in cell viability recovery, suggesting that it has a better combined protective effect.

[0061] Furthermore, through H9C2 cell experiments, the concentration ranges of holmine A, isochlorogenic acid C, and isoquercitrin that have no significant toxicity to cells, as well as the optimal therapeutic concentration under the OGD / R model, were screened, thus providing a clear experimental basis for the combined drug administration regimen.

[0062] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A pharmaceutical composition for alleviating oxidative stress associated with myocardial ischemia-reperfusion injury, characterized by, The active ingredients of this composition include holmine A, isochlorogenic acid C, and isoquercitrin. These active ingredients work together to inhibit oxidative stress associated with myocardial ischemia-reperfusion injury, reduce myocardial cell damage, and improve mitochondrial function.

2. The pharmaceutical composition of claim 1, wherein, The holmine A, isochlorogenic acid C, and isoquercitrin are all derived from the extraction, separation, and purification products of mountain green tea, or are chemically synthesized holmine A, isochlorogenic acid C, and isoquercitrin, or are equivalent compounds with the same structure as holmine A, isochlorogenic acid C, and isoquercitrin, respectively.

3. The pharmaceutical composition according to claim 2, characterized in that, The molar ratio of holmarin A, isochlorogenic acid C, and isoquercitrin is 1:(0.5-5):(0.5-5).

4. The pharmaceutical composition according to claim 3, characterized in that, The molar ratio of holmarin A, isochlorogenic acid C, and isoquercitrin is 1:2:

2.

5. The pharmaceutical composition according to claim 1, characterized in that, In the in vitro cell experiment system, the working concentration of holly A was 20 μM, the working concentration of isochlorogenic acid C was 40 μM, and the working concentration of isoquercitrin was 40 μM.

6. The pharmaceutical composition according to claim 1, characterized in that, The composition further comprises at least one of a pharmaceutically acceptable carrier, excipient, diluent, stabilizer, or solvent.

7. The pharmaceutical composition according to claim 6, characterized in that, The dosage form of the pharmaceutical composition is selected from one or more of the following: injection, lyophilized powder for injection, tablets, capsules, granules, and oral liquids.

8. Use of the pharmaceutical composition according to any one of claims 1 to 7 in the preparation of a medicament or formulation for the prevention, mitigation or adjunctive treatment of myocardial ischemia-reperfusion injury-related oxidative stress.

9. Use of the pharmaceutical composition according to any one of claims 1 to 7 in the preparation of a drug for reducing oxygen-glucose deprivation / reoxygenation-induced cardiomyocyte injury.

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