Application of ganoderan peptide and composition thereof in preparation of myocardial infarction protection medicine

Abstract

The invention relates to the technical field of biological medicines, in particular to application of ganoderan peptide and a composition thereof in preparation of myocardial infarction protection medicines. The ganoderan peptide provided by the invention has a protective effect on cardiac fibrosis after myocardial infarction, and other components in the composition can further cooperate with the ganoderan peptide component, so that the protective effect of the composition on acute myocardial infarction is improved.

Description

Technical Field

[0001] The present invention relates to the field of biological medicine technology, and particularly to the application of Ganoderma lucidum polysaccharide peptide and its composition in the preparation of drugs for protecting against myocardial infarction. Background Art

[0002] Ganoderma lucidum polysaccharide peptide (GLPP) is a polysaccharide-polypeptide complex obtained by extracting and purifying Ganoderma lucidum fruit bodies or mycelia, with dual biological activities of both polysaccharides and polypeptides. The polysaccharide part is mainly composed of monosaccharides such as glucose and mannose, while the polypeptide contains various amino acids, and its structure is closely related to its function. Ganoderma lucidum polysaccharide peptide has a large molecular weight and a complex structure, and is one of the core components for Ganoderma lucidum to exert its pharmacological effects.

[0003] Ganoderma lucidum is an aerobic fungus belonging to the family Polyporaceae, also known as Linzhongling, Ganoderma lucidum herb or Qiongzhen, and is a precious traditional Chinese medicinal material in China, with effects such as replenishing qi in the middle-jiao, nourishing yin and strengthening the body, strengthening the healthy qi and consolidating the constitution, and prolonging life. Ganoderma lucidum contains various bioactive components and has multiple functions such as enhancing immune function, regulating blood sugar level, anti-tumor and reducing blood pressure. Modern scientific tests have proved that Ganoderma lucidum polysaccharide peptide (GLPP) is one of the main active components of Ganoderma lucidum. Ganoderma lucidum polysaccharide peptide is extracted and purified from Ganoderma lucidum cultivated with mushroom grass, and is a mixture composed of peptide polysaccharide, glucan, heteropolysaccharide, etc., with a wide range of pharmacological activities, such as anti-tumor, antioxidant, anti-fibrotic, anti-aging, anti-atherosclerotic, lipid-lowering, blood sugar-lowering, immune regulation and liver protection activities. However, how to further expand its application scope and explore new pharmacological activities has become one of the key problems that need to be urgently solved in the current development process of Ganoderma lucidum.

[0004] It is worth noting that the Chinese invention patent with the document number CN118902078A discloses a Ganoderma lucidum polysaccharide peptide complex based on an in vitro biological model and its preparation method. The complex is composed of the following raw materials: 10-30 parts of Ganoderma lucidum polysaccharide peptide, 1-10 parts of taurine, 1-15 parts of creatine, 1-15 parts of fermented soy protein, and 0.05-0.5 parts of astaxanthin. The technical solution of this patent shows that, compared with pure Ganoderma lucidum polysaccharide peptide, this complex has stronger antioxidant and anti-inflammatory abilities, and is more significant in promoting immunity. Therefore, how to further enhance its pharmacological activity by means of the synergistic effect of Ganoderma lucidum polysaccharide peptide and other active ingredients is also an important problem that needs to be solved urgently at present. Summary of the Invention

[0005] To solve the above technical problems, the object of the present invention is to provide the application of ganoderma polysaccharide peptide and its composition in the preparation of drugs for protecting against myocardial infarction. The ganoderma polysaccharide peptide has a protective effect on cardiac fibrosis after myocardial infarction, and other components in the composition can further cooperate with the ganoderma polysaccharide peptide component to improve its protective effect on acute myocardial infarction.

[0006] To achieve the above technical effects, the present invention adopts the following technical solutions:

[0007] In the first aspect, the present invention provides the application of a ganoderma polysaccharide peptide in the preparation of drugs for protecting against myocardial infarction.

[0008] Preferably, the myocardial infarction is acute myocardial infarction.

[0009] In the second aspect, the application of a composition of ganoderma polysaccharide peptide in the preparation of drugs for protecting against myocardial infarction, and the composition is a combination of any two or more of ganoderma polysaccharide peptide, esculentoside A, and curcumol.

[0010] Preferably, when the composition contains ganoderma polysaccharide peptide and esculentoside A, the mass ratio of ganoderma polysaccharide peptide to esculentoside A is 1:0.1 - 10.

[0011] Preferably, when the composition contains ganoderma polysaccharide peptide and curcumol, the mass ratio of ganoderma polysaccharide peptide to curcumol is 1:0.05 - 5.

[0012] Preferably, when the composition contains ganoderma polysaccharide peptide, esculentoside A, and curcumol, the mass ratio of ganoderma polysaccharide peptide, esculentoside A, and curcumol is 1:0.1 - 5:0.05 - 3.

[0013] Furthermore, the dosage form of the drug for protecting against myocardial infarction is any one of tablets, capsules, injections, oral liquid preparations, or granules.

[0014] Furthermore, the administration method of the drug is oral administration, intravenous injection, intramuscular injection, or subcutaneous injection.

[0015] Furthermore, the myocardial infarction is acute myocardial infarction.

[0016] Furthermore, the drug is used to reduce the degree of myocardial fibrosis, and / or reduce the infarct area, and / or relieve myocardial ischemia after myocardial infarction.

[0017] Furthermore, the drug is used for: inhibiting drugs for myocardial cell fibrosis, and / or inhibiting the secretion of inflammatory factors after myocardial infarction.

[0018] Compared with the prior art, the beneficial effects of the present invention are:

[0019] Myocardial infarction is a common and serious heart disease. The occurrence of cardiac fibrosis and impaired cardiac function after myocardial infarction are important causes of patient death. In the past few decades, Ganoderma lucidum polysaccharide peptide, as a natural product, has attracted extensive attention due to its excellent pharmacological activities and biological functions. Research has shown that Ganoderma lucidum polysaccharide peptide has antioxidant, anti-inflammatory, and anti-fibrotic properties. It can play a protective role in the kidneys, liver, especially the cardiovascular system through multiple mechanisms such as inhibiting the production of inflammatory mediators, reducing the generation of reactive oxygen species, improving the oxidative stress imbalance of the body or cells, and inhibiting apoptosis. The application of Ganoderma lucidum polysaccharide peptide in the kidneys, cardiovascular system, etc. has made certain progress, but the current research on Ganoderma lucidum polysaccharide peptide in cardiac fibrosis after myocardial infarction is still relatively limited.

[0020] The present invention provides the application of Ganoderma lucidum polysaccharide peptide and its composition in the preparation of drugs for protecting against myocardial infarction. Especially for acute myocardial infarction, it provides a new drug option for the treatment of myocardial infarction. The present invention further proves through animal experiments that Ganoderma lucidum polysaccharide peptide has a protective effect on cardiac fibrosis after myocardial infarction, can reduce the degree of myocardial fibrosis, and can effectively improve the structure and function of the heart after myocardial infarction.

[0021] At the same time, the present invention further provides a composition containing Ganoderma lucidum polysaccharide peptide. The other components (esculentoside A, curcumol) in this composition can act synergistically with the Ganoderma lucidum polysaccharide peptide component to further improve the protective effect against acute myocardial infarction, including reducing the infarct area, alleviating myocardial ischemia after myocardial infarction, etc., and has a more significant therapeutic effect than using Ganoderma lucidum polysaccharide peptide alone.

[0022] The Ganoderma lucidum polysaccharide peptide and its composition of the present invention can not only inhibit myocardial cell fibrosis, but also inhibit the secretion of inflammatory factors after myocardial infarction, intervene and treat myocardial infarction from multiple aspects, and have a good comprehensive therapeutic effect. At the same time, the Ganoderma lucidum polysaccharide peptide and its composition can be prepared into drugs for protecting against myocardial infarction, and their dosage forms and administration methods are diverse, which can meet the needs and usage scenarios of different patients. Description of the Drawings

[0023] Figure 1 Results of the detection of the cardiac function of each group of mice provided in Example 2 of the present invention;

[0024] Figure 2 Results of the Masson staining experiment of the myocardial tissue of each group of mice provided in Example 2 of the present invention. Detailed Embodiments

[0025] The following examples are only used to more clearly illustrate the technical solutions of the present invention. Therefore, they are only examples and cannot be used to limit the protection scope of the present invention. For those skilled in the art, any equivalent modifications and substitutions to the following examples are also within the scope of the present invention. Therefore, equivalent transformations and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention. For those conditions not specified in the examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. All reagents or instruments not specified by the manufacturer can be obtained as conventional products through commercial purchase.

[0026] To better illustrate the present invention, numerous specific details are given in the following detailed implementation manners. Those skilled in the art should understand that the present invention can also be implemented without certain specific details. In other embodiments, methods, means, equipment, and steps well-known to those skilled in the art are not described in detail to highlight the gist of the present invention.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art. Without special instructions, the units used in this specification are all international standard units, and the numerical values and numerical ranges appearing in the present invention should be understood to include the numerical values and numerical ranges that are inevitable errors in industrial production.

[0028] Example 1

[0029] This example provides a preparation method of ganoderma polysaccharide peptide, specifically as follows:

[0030] S1: Select high-quality Ganoderma lucidum fruiting bodies, pulverize them and pass through a 100-mesh sieve.

[0031] S2: Reflux extract with 75% ethanol at 70°C for 2 times, 2 hours each time, to remove fat-soluble impurities, then discard the extract and retain the residue for polysaccharide peptide extraction;

[0032] S3: Decoct and extract the defatted residue according to a solid-liquid ratio of 1:30, 2 hours each time, repeat the extraction 3 times, combine the water extracts, and concentrate under reduced pressure to 1 / 5 of the original volume to obtain a crude polysaccharide peptide solution;

[0033] S4: Add a chloroform-n-butanol (4:1) mixture to the aforementioned crude polysaccharide peptide solution at 1 / 4 of the volume of the crude extract, shake vigorously and then centrifuge, repeat 3 times to remove proteins, then use a 10 kDa ultrafiltration membrane to concentrate the dialysis solution, retain the high molecular weight components, and concentrate and freeze-dry the obtained high molecular weight components to obtain high-purity ganoderma polysaccharide peptide.

[0034] Example 2

[0035] Based on the ganoderma lucidum polysaccharide peptide provided in the foregoing Example 1, this example provides an application of the ganoderma lucidum polysaccharide peptide prepared by the foregoing method in administering to myocardial infarction mice, specifically as follows:

[0036] 2.1 Experimental method:

[0037] SPF-grade C57BL / 6 male mice (8 weeks old, 22 - 25 g) were selected as experimental animals, with 10 mice in each group. The animals were housed separately in a constant temperature (22 ± 2 °C) and humidity (55 ± 5%) environment, with a 12 h light / dark cycle, and free access to food and water. The experimental groups were divided as follows:

[0038] Sham group: Sham operation, only threading the suture without ligation after thoracotomy, and suturing the thoracic cavity layer by layer.

[0039] Sham + GLPP group: Sham operation, without ligating the coronary artery after thoracotomy, and administering GLPP by gavage after the operation. The administration dose was 50 mg / kg / d, and the administration was continuous for 28 d;

[0040] MI model group: The mice were anesthetized and fixed supine, an incision was made in the middle of the neck, the trachea was separated and an endotracheal tube was inserted, and a small animal ventilator was connected for respiratory support. Then the thoracic cavity was opened along the 3rd - 5th intercostal space on the left margin of the sternum to expose the heart. A suture was passed through the full thickness of the myocardium about 2 - 3 mm below the left atrial appendage, and the left anterior descending branch of the coronary artery was ligated to cause myocardial ischemia, and then the chest was sutured layer by layer.

[0041] MI + GLPP group: The modeling method was the same as that of the MI group, and the administration scheme was the same as that of the Sham + GLPP group;

[0042] At 48 h after the operation, mice with EF < 40% were considered to have successfully modeled by echocardiography screening.

[0043] 2.2 Experimental results and analysis:

[0044] 2.2.1 Echocardiographic detection of cardiac function

[0045] At 28 days after administration, the cardiac function of the mice was detected using an echocardiograph. The experimental results are as Figure 1 shown:

[0046] The experimental results include the cardiac echocardiogram images (upper row) of each group of mice, which are used to observe the overall cardiac structure (such as myocardial wall thickness, tissue uniformity), evaluate the structural damage of myocardial infarction and the intervention effect of GLPP; and the cardiac activity waveform diagram (lower row) of the mice, which is used to evaluate the cardiac function state (such as rhythm, contractility), and reflect the improvement effect of GLPP on electrophysiological or mechanical functions. The experimental results show that:

[0047] Sham + GLPP group: There was no significant abnormality compared with the Sham group, indicating that GLPP has no adverse effect on normal cardiac function.

[0048] MI group: Significant infarcted areas appeared in the myocardial tissues of this group, showing structural disorder, cavities, and disordered fiber arrangement. At the same time, typical infarct characteristics such as thinning of the myocardial wall and uneven echogenicity were shown.

[0049] MI+GLPP group: Compared with the MI group, the infarcted area was reduced and the myocardial fibers were arranged more orderly, suggesting that GLPP can improve myocardial structural damage by reducing tissue necrosis and promoting repair. At the same time, the thickness and structural uniformity of the myocardial wall in this group were restored, indicating that GLPP has a protective effect on the overall structure of the heart.

[0050] 2.2.2 Results of HE staining experiment

[0051] The specific steps of HE staining include:

[0052] Fixation and sectioning: Take myocardial tissues, fix them with formalin, dehydrate them, and then embed them in paraffin and cut into thin slices (such as transverse / longitudinal sections);

[0053] Staining: Hematoxylin stains the cell nuclei (blue), and eosin stains the cytoplasm and interstitium (pink);

[0054] Mounting and observation: After dehydration and clearing, mount the sections with neutral balsam and evaluate the myocardial structure (such as infarcted area, fiber arrangement) under a microscope.

[0055] The experimental results are as Figure 2 shown, and these experimental results show that:

[0056] Sham+GLPP group: The staining results were similar to those of the Sham group, indicating that GLPP has no additional adverse effects on normal myocardium. The comparison between the MI group and the MI+GLPP group showed that GLPP can relieve the structural damage caused by myocardial infarction by reducing myocardial necrosis and protecting fiber arrangement.

[0057] These experimental results visually verified the inhibitory effect of GLPP on myocardial fibrosis after myocardial infarction, highlighting the potential value of GLPP in myocardial protection.

[0058] 2.2.3 Evaluate the pathological changes and fibrosis degree of myocardial tissues from a histological perspective.

[0059] In a mouse myocardial infarction (MI) model, detecting the expression levels of fibrosis-related proteins (such as FN, CoIII, α-SMA) by immunohistochemistry (IHC) or Western blot is an important method to evaluate the degree of myocardial fibrosis. Therefore, the following experiment evaluated the degree of myocardial fibrosis by detecting the protein expression levels in the infarcted areas and adjacent normal myocardial tissues of mice in each group. The experimental results are shown in Table 1:

[0060] Detection results of proteins related to myocardial fibrosis degree in each group of mice

[0061]

[0062] Note: * indicates p < 0.05 compared with the Sham group; # indicates p < 0.05 compared with the MI group.

[0063] The above experimental results show that:

[0064] Expressions of α-SMA, COLIII, and FN: There were no significant differences in protein levels between the Sham group and the Sham+GLPP group (without * or # markings), indicating that GLPP treatment has no pro-fibrotic effect on normal myocardial tissue, GLPP has good safety under non-pathological conditions, and does not activate fibrosis-related pathways.

[0065] The three indicators in the MI group were significantly higher than those in the Sham group. For α-SMA: The expression level in the MI group (1.935) was significantly higher than that in the Sham group (0.915, *p < 0.05), suggesting enhanced activation of myofibroblasts, which is the core driving factor of fibrosis. For COLIII and FN: The expression levels in the MI group (2.512, 2.786) were significantly higher than those in the Sham group (1.023, 1.213, *p < 0.05), indicating excessive deposition of extracellular matrix (ECM) and aggravated myocardial fibrosis. The experimental results show that myocardial infarction successfully induced pathological fibrosis, manifested as activation of fibroblasts and increased ECM synthesis.

[0066] The experimental results of the MI+GLPP group show that:

[0067] For α-SMA: The MI+GLPP group (0.913) was significantly lower than the MI group (1.935, #p < 0.05), and returned to the Sham level, indicating that GLPP effectively inhibits the activation of myofibroblasts.

[0068] For COLIII and FN: The MI+GLPP group (1.456, 1.172) was significantly lower than the MI group (2.512, 2.786, #p < 0.05), and FN was even lower than the Sham group, indicating that GLPP significantly reduces ECM deposition and may promote its degradation.

[0069] The experimental results of this group show that GLPP exhibits a potent anti-fibrotic effect in the myocardial infarction model, possibly by inhibiting the activation of fibroblasts and ECM synthesis.

[0070] In summary, all the indicators in the MI+GLPP group returned to the levels of the Sham group (p>0.05), indicating that GLPP can significantly inhibit the fibrotic process, and its mechanism of action may be related to inhibiting the transformation of myofibroblasts (α-SMA↓) and reducing collagen synthesis (COLIII / FN↓).

[0071] Example 3

[0072] Based on the Ganoderma lucidum polysaccharide peptide provided in Example 1 and the application of the Ganoderma lucidum polysaccharide peptide provided in Example 2, this example further provides a Ganoderma lucidum polysaccharide peptide composition, specifically:

[0073] Composition I: Ganoderma lucidum polysaccharide peptide and esculentoside A are combined in a mass ratio of 1:1;

[0074] Control group IA: Only esculentoside A;

[0075] Composition II: Ganoderma lucidum polysaccharide peptide and curcumol are combined in a mass ratio of 1:1;

[0076] Control group IIA: Only curcumol;

[0077] Composition III: Ganoderma lucidum polysaccharide peptide, curcumol and esculentoside A are combined in a mass ratio of 1:1:1;

[0078] Control group IIIA: Curcumol and esculentoside A are combined in a mass ratio of 1:1:1;

[0079] Dissolve the above compositions and controls with normal saline, conduct experiments according to the experimental method in Example 2 above, with the dosage of 50 mg / kg / d, shorten the administration cycle, and continuously administer for 14 days after modeling, n = 5. After the administration is completed, detect the proteins related to the degree of myocardial fibrosis in mice. The experimental results are shown in Table 2:

[0080] Table 2 Detection results of proteins related to the degree of myocardial fibrosis in mice in each group

[0081]

[0082]

[0083] Note: * indicates p<0.05 compared with the Sham group; # indicates p<0.05 compared with the MI group.

[0084] The above experimental results show that

[0085] Under the condition of shortening the administration cycle, GLPP alone exerts an anti-fibrotic effect by inhibiting the activation of fibroblasts and ECM synthesis, but has a weak regulation on COLIII, while the composition treatment group shows an obvious synergistic effect, including:

[0086] Composition I (GLPP + esculentoside A), in this group, α-SMA (1.203) was slightly better than GLPP alone (1.261), but the difference was not significant, suggesting that esculentoside A might enhance the inhibition of myofibroblasts; FN (1.682) was significantly lower than that in the MI + GLPP group (1.734), indicating that esculentoside A had a synergistic effect by promoting FN degradation or inhibiting its synthesis.

[0087] Composition II (GLPP + curcumol), in this group, COLIII (1.552) had no significant difference from GLPP alone (1.615), but FN (1.635) was further reduced, suggesting that curcumol might target the FN-specific pathway (such as integrin signaling).

[0088] Control group IIIA (curcumol + esculentoside A), in this group, FN (2.277) was significantly higher than all GLPP-containing groups, indicating that when GLPP was absent, the combination of the two drugs could not effectively reverse fibrosis.

[0089] Composition III (GLPP + curcumol + esculentoside A), all indicators in this group were the lowest (α-SMA 1.020, COLIII 1.186, FN 1.231), significantly better than other composition groups: its α-SMA inhibition rate reached 37.3% (vs the MI group), approaching the Sham level; FN (1.231) was even lower than that in the Sham group (1.225), suggesting that the combination of the three drugs might over-activate the ECM degradation pathway.

[0090] Based on the above experimental results, the inventor believes that GLPP played a core therapeutic role in the mouse myocardial infarction model. GLPP alone could significantly inhibit α-SMA and FN and was the basic component for anti-fibrosis. At the same time, esculentoside A could enhance the inhibition of FN; curcumol: might have an auxiliary effect by anti-inflammatory or promoting ECM degradation; the combination of the three drugs (Composition III), by integrating multiple pathways (inhibiting activation, anti-inflammatory, promoting degradation), had the best effect. The above combination method was expected to become an efficient treatment plan for anti-fibrosis after myocardial infarction.

[0091] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the purpose and scope of the technical solutions of the present invention, and they should all be covered by the scope of the claims of the present invention. The technologies, shapes, and structures not described in detail in the present invention are all well-known technologies.

Claims

1. Use of Ganoderma lucidum polysaccharide peptide in the preparation of a drug for protecting against myocardial infarction.

2. Use of a composition of Ganoderma lucidum polysaccharide peptide in the preparation of a drug for protecting against myocardial infarction, wherein the composition is a combination of any two or more of Ganoderma lucidum polysaccharide peptide, esculentoside A, and curcumol.

3. The application according to claim 2, wherein: When the composition contains Ganoderma lucidum polysaccharide peptide and esculentoside A, the mass ratio of Ganoderma lucidum polysaccharide peptide to esculentoside A is 1:0.1 - 10.

4. The application according to claim 2, wherein: When the composition contains Ganoderma lucidum polysaccharide peptide and curcumol, the mass ratio of Ganoderma lucidum polysaccharide peptide to curcumol is 1:0.05 - 5.

5. The application according to claim 2, characterized in that: When the composition contains Ganoderma lucidum polysaccharide peptide, esculentoside A, and curcumol, the mass ratio of Ganoderma lucidum polysaccharide peptide, esculentoside A, and curcumol is 1:0.1 - 5:0.05 - 3.

6. The application according to any one of claims 1 to 2, characterized in that: The dosage form of the drug for protecting against myocardial infarction is tablet, capsule, injection, oral liquid preparation, or granule.

7. The application according to any one of claims 1 to 2, characterized in that: The administration method of the drug is oral administration, intravenous injection, intramuscular injection, or subcutaneous injection.

8. The application according to any one of claims 1 to 2, characterized in that: The myocardial infarction is acute myocardial infarction.

9. The application according to any one of claims 1 to 2, characterized in that: The drug is used to reduce the degree of myocardial fibrosis, and / or reduce the infarct area, and / or relieve myocardial ischemia after myocardial infarction.

10. The application according to any one of claims 1 to 2, characterized in that, The drug is used for: inhibiting myocardial cell fibrosis, and / or inhibiting the secretion of inflammatory factors after myocardial infarction.

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