Novel polypeptide capable of regulating activity of AMPK (adenosine monophosphate kinase) and pharmaceutical application of novel polypeptide

Abstract

The invention relates to the technical field of biological medicine, in particular to a novel polypeptide and application thereof in medicine for treating cardiovascular diseases. The polypeptide has a unique amino acid sequence, and is composed of an amino acid sequence of SEQ. NO: 1: Leu-Asn-Pro-Thr-Glu (LNPTG), an amino acid sequence of SEQ. NO: 2: Val-His-Met-Arg-Tyr (VHMRY), and the like. The polypeptide has a unique amino acid sequence, and provides a new way for the treatment of cardiovascular diseases by adjusting the activity of AMPK and improving the cardiovascular function.

Description

I. Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and mainly relates to the research and development of a novel polypeptide and its application in the treatment of cardiovascular diseases. II. Background Art

[0002] Cardiovascular diseases are one of the major diseases seriously threatening human health globally, covering various types such as coronary heart disease, hypertension, heart failure, etc. These diseases bring great pain and burden to patients, and also impose a heavy pressure on social medical resources. Coronary heart disease is mainly caused by atherosclerosis of the coronary arteries, resulting in vascular stenosis or occlusion, leading to myocardial ischemia, hypoxia or even necrosis. Hypertension refers to the continuous elevation of blood pressure, and long-term hypertension can damage important organs such as the heart, kidneys, and brain. Heart failure is the gradual decline of heart function, unable to effectively pump blood to all tissues and organs of the body. Currently, the treatment methods for cardiovascular diseases mainly include drug treatment, interventional treatment, surgical treatment, etc. However, these traditional treatment methods have certain limitations and side effects. For example, commonly used antihypertensive drugs may cause adverse reactions such as hypotension and dizziness; lipid-lowering drugs may lead to abnormal liver function and muscle pain; antiplatelet drugs may increase the risk of bleeding. In addition, some patients with cardiovascular diseases have poor treatment effects with existing drugs, and there is a need to find safer and more effective treatment methods. II. The development of molecular biology and proteomics brings new opportunities for the treatment of cardiovascular diseases In recent years, with the rapid development of molecular biology and proteomics, researchers have begun to focus on developing novel treatment methods for cardiovascular diseases by targeting specific proteins. This method has high specificity and effectiveness, can precisely treat the pathogenesis of cardiovascular diseases, and reduce the side effects of traditional treatment methods. Proteomics is a discipline that studies the entire protein composition, structure, and function in cells, tissues, or organisms. Through proteomics techniques, researchers can identify key proteins related to the occurrence and development of cardiovascular diseases, providing potential targets for the development of novel treatment methods. Molecular biology techniques can further study the functions and regulatory mechanisms of these proteins, providing a theoretical basis for developing targeted treatment strategies. For example, gene editing techniques can be used to knockout or overexpress specific genes to study their effects on the cardiovascular system; protein interaction studies can reveal the complex network between proteins, providing clues for finding new treatment targets.

[0003] Biological Characteristics of Adenosine Monophosphate-Activated Protein Kinase (AMPK) Adenosine monophosphate-activated protein kinase (AMPK) is a highly conserved serine / threonine protein kinase that is widely present in various organisms. It plays an important role in regulating energy metabolism within cells and is known as the "cellular energy regulator". AMPK consists of three subunits, namely the α subunit, β subunit, and γ subunit. The α subunit is the catalytic subunit with kinase activity; the β subunit and γ subunit play regulatory roles. The activity of AMPK is regulated by the intracellular adenosine monophosphate concentration. When the intracellular adenosine monophosphate concentration increases, AMPK is activated, and it regulates cellular energy metabolism by phosphorylating downstream substrates to adapt to the state of energy deficiency.

[0004] The important regulatory role of AMPK in the cardiovascular system 1. Regulation of myocardial energy metabolism - In the cardiovascular system, AMPK plays an important regulatory role in the energy metabolism of cardiomyocytes. Cardiomyocytes mainly rely on the oxidation of fatty acids and glucose to provide energy. When the energy supply of cardiomyocytes is insufficient, AMPK is activated, promoting fatty acid oxidation and glucose uptake, and increasing the energy supply of cardiomyocytes. - In addition, AMPK can also regulate the mitochondrial function in cardiomyocytes, promoting mitochondrial biogenesis and autophagy, improving the quality and function of mitochondria, and thus enhancing the energy metabolism ability of cardiomyocytes. 2. Protection against myocardial ischemia-reperfusion injury - Myocardial ischemia-reperfusion injury is an important problem in cardiovascular diseases. When myocardial ischemia occurs, AMPK is activated, and it reduces myocardial ischemia injury by regulating the energy metabolism of cardiomyocytes, inhibiting oxidative stress and inflammatory responses, etc. During myocardial reperfusion, AMPK continues to play a protective role, promoting the repair and regeneration of cardiomyocytes. - For example, AMPK can activate endothelial nitric oxide synthase (eNOS), promote the synthesis and release of nitric oxide (NO), dilate blood vessels, and improve myocardial perfusion. At the same time, AMPK can also inhibit the nuclear factor-κB (NF-κB) signaling pathway, reduce the release of inflammatory factors, and alleviate the inflammatory response. 3. Regulation of vascular tone - AMPK also has an important regulatory role in the contraction and relaxation of vascular smooth muscle cells. AMPK can activate potassium channels, promote potassium ion efflux, hyperpolarize vascular smooth muscle cells, and thus cause vasodilation. In addition, AMPK can also inhibit the proliferation and migration of vascular smooth muscle cells, preventing vascular remodeling. - In vascular endothelial cells, AMPK can promote the synthesis and release of nitric oxide, dilate blood vessels, and reduce blood pressure. At the same time, AMPK can also inhibit the inflammatory response and oxidative stress of endothelial cells, protecting vascular endothelial function. 4. Anti-atherosclerosis - Atherosclerosis is one of the main pathological bases of cardiovascular diseases. AMPK can inhibit the occurrence and development of atherosclerosis through multiple pathways. For example, AMPK can inhibit cholesterol synthesis and uptake, promote reverse cholesterol transport, and reduce the cholesterol level in the blood. At the same time, AMPK can also inhibit the proliferation and migration of vascular smooth muscle cells, reduce the formation of foam cells, and prevent the formation of atherosclerotic plaques. V. The potential of AMPK as a therapeutic target for cardiovascular diseases 1. Development of AMPK activators - Due to the important regulatory role of AMPK in the cardiovascular system, the development of AMPK activators has become a potential therapeutic strategy for cardiovascular diseases. Currently, a variety of AMPK activators have been developed, and some of them have entered the clinical trial stage. - These AMPK activators mainly include metformin, thiazolidinediones, AICAR, etc. Metformin is a widely used drug for the treatment of diabetes. It can activate AMPK, lower blood glucose levels, and at the same time has cardiovascular protective effects.Thiazolidinediones are mainly used to treat diabetes and insulin resistance. They can also activate AMPK, improve insulin sensitivity, and reduce the risk of cardiovascular diseases. AICAR is a synthetic AMPK activator that can directly activate AMPK, promote fatty acid oxidation and glucose uptake, and enhance the energy metabolism level of cells. 2. Possibility of combination therapy - In addition to using AMPK activators alone, the combination of AMPK activators with other cardiovascular disease treatment drugs can also be considered to improve the treatment effect. For example, AMPK activators can be combined with antihypertensive drugs, lipid-lowering drugs, antiplatelet drugs, etc. to exert a synergistic therapeutic effect. - In addition, AMPK activators can also be combined with emerging treatment methods such as gene therapy and stem cell therapy to provide new ideas and methods for the treatment of cardiovascular diseases. VI. Challenges and future prospects Although AMPK has great potential in the treatment of cardiovascular diseases, there are still some challenges at present. 1. Specificity and safety issues - Most of the currently developed AMPK activators lack specificity and may have adverse effects on other signaling pathways. In addition, the safety of AMPK activators needs to be further evaluated, especially the side effects that may occur during long-term use. - Future research needs to develop more specific and safe AMPK activators to reduce the impact on other signaling pathways and lower the risk of side effects. 2. Complexity of the mechanism of action - The mechanism of action of AMPK in the cardiovascular system is very complex, involving multiple signal transduction pathways and biological effects. At present, the specific mechanism of action of AMPK in cardiovascular diseases is not fully understood and further in-depth research is needed. - Future research needs to use advanced technical means such as proteomics and gene editing technology to deeply study the interaction mechanism between AMPK and other proteins, providing a theoretical basis for the development of more effective treatment methods. 3. Feasibility of clinical application - Although AMPK activators have shown certain therapeutic potential in animal experiments and cell experiments, their feasibility and effectiveness need to be further verified in clinical applications. In addition, the administration method, dosage, and treatment course of AMPK activators also need to be optimized to improve the treatment effect and reduce side effects. - Future research needs to conduct large-scale clinical trials to evaluate the safety and effectiveness of AMPK activators in the treatment of cardiovascular diseases. At the same time, more convenient and effective administration methods need to be explored to improve patient compliance. In summary, adenosine monophosphate-activated protein kinase (AMPK) plays an important regulatory role in the cardiovascular system and has become one of the research hotspots. By developing AMPK activators or combining with other treatment methods, it is expected to provide new ideas and methods for the treatment of cardiovascular diseases. However, there are still some challenges at present, and further in-depth research on the mechanism of action of AMPK is needed to develop more specific and safe treatment methods and conduct rigorous clinical trials to verify their feasibility and effectiveness in clinical applications.It is believed that with the continuous in - depth research, AMPK will play a more important role in the treatment of cardiovascular diseases. III. SUMMARY OF THE INVENTION

[0005] (I) Structure of the novel polypeptide

[0006] The novel polypeptide provided by the present invention is composed of the following amino acid sequences:

[0007] SEQ.NO 1: Leu - Asn - Pro - Thr - Glu (LNPTG);

[0008] SEQ.NO 2: Val - His - Met - Arg - Tyr (VHMRY).

[0009] These sequences are designed by conducting in - depth research on the structure and function of the target protein AMPK and combining with binding affinity analysis.

[0010] (II) Design method of the novel polypeptide

[0011] In the design of novel polypeptides, docking software and machine learning algorithms are two important methods for predicting protein - protein interactions. Docking software can predict binding modes and binding affinities by simulating the interactions between protein molecules, providing intuitive information for screening polypeptide sequences with high binding affinity. Commonly used docking software such as Autodock and Vina has characteristics such as being open - source, powerful in function, and easy to use. However, docking software depends on accurate structural information and cannot consider the dynamic changes of proteins. Machine learning algorithms, on the other hand, learn from a large amount of known protein - protein interaction data to establish prediction models. They can process a large amount of data, consider multiple features, and have a certain generalization ability. Commonly used machine learning algorithms include support vector machines, random forests, and deep learning algorithms, etc. But machine learning algorithms require a large amount of labeled data and have poor interpretability. In addition, relevant databases such as STRING and BioGRID provide rich protein - protein interaction information, which can provide data support for the design of novel polypeptides. In practical applications, docking software and machine learning algorithms can be combined to make full use of their advantages to improve the accuracy and efficiency of polypeptide design. At the same time, continuously improving and updating the database to improve the quality and quantity of data will help better predict protein - protein interactions and provide more powerful guidance for the design of novel polypeptides.

[0012] The novel polypeptide can specifically bind to AMPK, thereby regulating its activity. Specifically, the amino acids in the LNPTG sequence have specific chemical properties, such as the hydrophobicity of Leu, the amide group of Asn, the rigidity of Pro, the hydroxyl group of Thr, and the acidity of Glu. These properties contribute to the formation of complementary interactions with specific binding regions of AMPK, thus regulating the activity of AMPK. Similarly, the amino acids in the VHMRY sequence can also interact with AMPK and affect its catalytic function, etc. By regulating the activity of AMPK, the novel polypeptide can improve cardiovascular function. AMPK is involved in processes such as cellular energy metabolism, oxidative stress, and inflammatory responses, and plays an important role in the normal physiological functions of the cardiovascular system. The novel polypeptide can activate AMPK, promote cellular energy metabolism, reduce oxidative stress and inflammatory responses, thereby improving cardiovascular function. 1.

[0014] (III) Beneficial effects of the novel polypeptide 1.

[0016] High efficiency

[0017] The novel polypeptide can significantly improve the functional state of the target protein AMPK and effectively regulate cardiovascular-related physiological indicators, such as reducing blood sugar and blood lipids, and improving cardiac systolic and diastolic functions, etc. 2.

[0019] Safety

[0020] Verified by in vitro and in vivo experiments, the novel polypeptide has no obvious toxicity at high doses and has no obvious adverse effects on important organs such as the liver and kidneys after long-term use. 3.

[0022] Specificity

[0023] The novel polypeptide has a high specific binding ability to the target protein AMPK, reduces interference with other irrelevant proteins, and reduces the risk of potential side effects. IV. Specific implementation manners

[0024] Example 1

[0025] (I) Synthesis of the polypeptide 1.

[0027] Selection of amino acid monomers

[0028] Amino acid monomers with Boc protecting groups are used to ensure the activity and stability of amino acids during the synthesis process. 2.

[0030] Solid-phase synthesis

[0031] Amino acid monomers are sequentially linked to a solid-phase support. After each coupling reaction, the Boc group is removed through a deprotection step. A step-by-step synthesis method is adopted to ensure the accuracy and purity of the polypeptide. 3.

[0033] Cleavage and purification

[0034] After all amino acids are linked, it is cleaved from the solid-phase support to obtain the crude polypeptide. Its purity and molecular weight are confirmed by high-performance liquid chromatography (HPLC) and mass spectrometry (MS) analysis. Multiple purification methods, such as reverse-phase high-performance liquid chromatography, ion-exchange chromatography, etc., are used to improve the purity of the polypeptide.

[0035] (II) Structure verification experiments 1.

[0037] Mass spectrometry analysis

[0038] 1. Instrument: A high-resolution mass spectrometer (such as MALDI-TOF MS) is used for analysis.

[0039] 2. Sample preparation: The synthesized peptide segments are separately dissolved in a mixed solvent of methanol and water to prepare a solution with an appropriate concentration.

[0040] 3. Experimental results: The mass spectrometry analysis results show that the molecular weights of the peptide segments are consistent with the theoretical values, further confirming the correctness of their structures. By comparing the differences between the measured molecular weight and the theoretical molecular weight, the synthesis accuracy of the polypeptide is evaluated.

[0041] Circular dichroism (CD) analysis

[0042] 1. Instrument: A circular dichroism spectrometer is used for analysis.

[0043] 2. Sample preparation: The synthesized peptide segments are separately dissolved in phosphate buffer to prepare a solution with an appropriate concentration.

[0044] 3. Experimental results: The circular dichroism analysis results show that the peptide segments have specific secondary structure characteristics, which are consistent with the expected structures. Analyze the secondary structure of the polypeptide, such as α-helix, β-sheet, etc., to understand its structural stability.

[0045] (III) Identification and related research of the target protein AMPK 1.

[0047] Identification of AMPK

[0048] AMPK is identified in cardiovascular tissues by proteomics and bioinformatics methods. Immunohistochemistry, Western blot and other techniques are used to verify the expression of AMPK in the cardiovascular system, especially its high expression in cardiomyocytes, vascular endothelial cells and vascular smooth muscle cells. 2.

[0050] Determination of the binding site

[0051] The binding site of the novel polypeptide to AMPK was determined by molecular docking technology. Computer simulation software was used to predict the binding mode and affinity of the polypeptide to AMPK, providing a basis for further optimizing the polypeptide structure.

[0052] (IV) In vitro activity test 1.

[0054] Cell culture

[0055] 1. Cell lines: human cardiomyocyte cell line (HCM cells), human umbilical vein endothelial cell line (HUVEC cells), human vascular smooth muscle cell line (VSMC cells).

[0056] 2. Culture conditions:

[0057] 1. HCM cells were cultured in RPMI-1640 medium containing 15% fetal bovine serum, 1% penicillin-streptomycin, at 37°C with 5% CO2.

[0058] 2. HUVEC cells were cultured in ECM medium containing 10% fetal bovine serum, 1% penicillin-streptomycin, at 37°C with 5% CO2.

[0059] 3. VSMC cells were cultured in DMEM medium containing 10% fetal bovine serum, 1% penicillin-streptomycin, at 37°C with 5% CO2.

[0060] Peptide treatment 2.

[0062] 1. Each synthesized polypeptide was dissolved in DMSO to prepare solutions with different concentrations.

[0063] 2. Polypeptides with different concentrations were added to the culture media of HCM cells, HUVEC cells, and VSMC cells and incubated for 24 hours.

[0064] Experimental results

[0065] 1. AMPK activity: The phosphorylation level of AMPK was detected to reflect its activity state.

[0066] The results showed that each polypeptide significantly activated AMPK. The specific data are as follows:

[0067]

[0068] ● Cardiomyocyte function:

[0069] ● Cell viability: The viability of HCM cells was detected by the MTT method. The results showed that each polypeptide significantly increased the viability of HCM cells. The specific data are as follows:

[0070]

[0071] ● Cardiac contractility: The contractility of HCM cells was detected by the cell contraction experiment. The results showed that

[0072] each polypeptide significantly enhanced the contractility of HCM cells. The specific data are as follows:

[0073]

[0074] ● Vascular endothelial cell function:

[0075] ● Cell proliferation ability: The proliferation ability of HUVEC cells was detected by the MTT method. The results showed that each polypeptide significantly promoted the proliferation of HUVEC cells. The specific data are as follows:

[0076]

[0077] ● Endothelial cell permeability: Detected by the Transwell permeability experiment. The results showed that each polypeptide

[0078] regulated the permeability of endothelial cells to a certain extent. The specific data are as follows:

[0079]

[0080] ● Vascular smooth muscle cell function:

[0081] ● Cell contractility: The contractility of VSMC cells was detected by the cell contraction experiment.

[0082] The results showed that each polypeptide significantly reduced the contractility of VSMC cells, which was beneficial to vasodilation. The specific data are as follows:

[0083]

[0084] (V) Animal model verification 1.

[0086] Animal model 2.

[0088] ● Model: Hypertension rat model (prepared by renal artery stenosis surgery) and heart failure mouse model (prepared by coronary artery ligation).

[0089] ● Experimental group: Each polypeptide treatment group.

[0090] ● Control group: Normal saline treatment group.

[0091] · Positive control group: Treatment groups with common antihypertensive drugs (such as captopril) (hypertensive model); Treatment groups with common heart failure treatment drugs (such as enalapril) (heart failure model).

[0092] Drug administration method

[0093] ● Hypertensive rat model: Each polypeptide or normal saline was administered daily by gavage for six consecutive weeks.

[0094] · Heart failure mouse model: Each polypeptide or normal saline was administered daily by intraperitoneal injection for four consecutive weeks.

[0095] Experimental results 3.

[0097] ● Blood pressure level: The tail artery blood pressure of rats was continuously monitored. The results showed that the blood pressure levels in each polypeptide treatment group were significantly lower than those in the control group and the captopril treatment group. The specific data are as follows:

[0098]

[0099] ● Heart function indexes: The heart function indexes of mice, such as left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), etc., were detected by echocardiography. The results showed that the LVEF and LVFS in each polypeptide treatment group were significantly higher than those in the control group and the enalapril treatment group, indicating that the heart function was significantly improved. The specific data are as follows:

[0100]

[0101] (VI) Safety assessment

[0102] 1. Acute toxicity test

[0103] ● By administering a single large dose of drug, the survival rate and behavioral performance of mice were observed. The results showed that each polypeptide had no obvious toxicity at high doses. The specific data are as follows:

[0104]

[0105] 1. Long-term toxicity test

[0106] · By continuously administering the drug for eight weeks, indexes such as the body weight, blood routine, liver and kidney functions of rats were observed.

[0107] The results showed that each polypeptide had no obvious adverse reactions under long-term use. The specific data are as follows:

[0109]

[0110] The novel polypeptide provided by the present invention has a unique amino acid sequence and can specifically regulate the activity of AMPK, providing new ideas and methods for the treatment of cardiovascular diseases.

[0111] The novel polypeptide can significantly improve cardiovascular function and effectively regulate cardiovascular-related physiological indexes, such as reducing blood sugar and blood lipids, and improving cardiac systolic and diastolic functions, etc.

[0112] Verified by in vitro and in vivo experiments, the novel polypeptide has no obvious toxicity at high doses and has no obvious adverse effects on important organs such as the liver and kidneys after long-term use, showing high safety.

[0113] The novel polypeptide has a high specific binding ability to the target protein AMPK, reducing interference with other irrelevant proteins and lowering the risk of potential side effects.

[0114] Data table of comparative synergistic test

[0115]

[0116] To verify the effect of the novel polypeptide of the present invention in the treatment of cardiovascular diseases, a series of experiments were conducted. The experimental results show that the novel polypeptide can effectively regulate the activity of AMPK, improve cardiovascular function, and reduce risk factors for cardiovascular diseases. At the same time, the polypeptide has no obvious toxicity at high doses and has no obvious adverse effects on important organs such as the liver and kidneys after long-term use, showing high safety and reliability.

[0117] Example 2: Novel small molecule peptide derived from sea buckthorn seeds

[0118] . Novel polypeptide structure

[0119] The amino acid sequence of the novel polypeptide is: SEQ.NO 31: Ala-Gln-Ser-His-Lys-Pro-Thr-Glu

[0120] -Tyr-Val (AQSHKPTEYV). This sequence was screened from the products of specific enzymatic hydrolysis of sea buckthorn seed protein. Sea buckthorn seeds are rich in proteins and various bioactive components, and their hydrolysis products may contain small molecule peptides that can effectively regulate the activity of adenosine monophosphate-activated protein kinase (AMPK), providing a new way to improve cardiovascular function.

[0121] . Extraction method

[0122] · Raw material pretreatment: Wash, dry and crush sea buckthorn seeds, and reflux and degrease them with n-hexane 3 times, 3 hours each time, to completely remove grease. The defatted sea buckthorn seed powder is added at a ratio of 1:14 (w / v) to pH

[0123] In 8.6 Tris-HCl buffer, stir and extract at 47 °C for 5.5 hours, with ultrasonic assistance (power 260 W, ultrasonic time 13 minutes) every 2 hours to promote protein dissolution.

[0124] · Enzymatic hydrolysis process: Centrifuge the extract at 9200 rpm for 20 minutes, take the supernatant, adjust the pH to 7.4, add trypsin (enzyme to substrate mass ratio 1:135), and perform enzymatic hydrolysis at 39 °C for 7.2 hours. After the enzymatic hydrolysis is completed, place the reaction solution in a 93 °C water bath to inactivate the enzyme for 13 minutes.

[0125] · Separation and purification: After the enzymatic hydrolysate is concentrated by rotary evaporation, ultrafiltration is carried out through an ultrafiltration membrane with a molecular weight cut-off of 3600 Da, and the retentate is collected. The retentate is separated by gel filtration chromatography (Sephadex G-31), using 0.16 mol / L sodium chloride solution as the eluent, controlling the flow rate at 0.65 mL / min, and collecting the active elution peak. The active peak is purified by reverse-phase high-performance liquid chromatography (RP-HPLC), using a C18 chromatographic column, and gradient elution is carried out with acetonitrile-water (containing 0.1% trifluoroacetic acid) as the mobile phase, and the target polypeptide peak is collected.

[0126] . Synergistic effect test

[0127] · Experimental design: Select a hypertensive rat model and divide it into three groups. The control group is given normal saline, the experimental group is given the novel polypeptide (AQSHKPTEYV) of the present invention, and the comparison group is given the common antihypertensive drug nifedipine.

[0128] ● Experimental results: After seven weeks of drug administration, the blood pressure level of the rats in the experimental group was significantly lower than that of the control group and the comparison group. The AMPK activity in the heart tissue was significantly higher than that of the control group and the comparison group. The activities of key enzymes for fatty acid oxidation (such as carnitine acyltransferase I) in cardiomyocytes were enhanced, the expression of the glucose uptake-related transporter GLUT4 increased, and the number of mitochondria increased and the morphology was more complete. The activity of nitric oxide synthase (eNOS) in vascular endothelial cells was enhanced, the generation of nitric oxide (NO) increased significantly, and the vasodilatory function was significantly enhanced. This indicates that the novel polypeptide has a synergistic effect in reducing blood pressure, activating AMPK, optimizing myocardial energy metabolism, and enhancing vascular endothelial function.

[0129] Example 3: Novel small molecule peptide extracted from Moringa oleifera leaves

[0130] . Novel polypeptide structure

[0131] The amino acid sequence of the novel polypeptide is: SEQ.NO 32: Gly-Asp-Glu-Asn-Leu-Ile-Arg-Pro

[0132] -Thr-Cys (GDENLIRPTC). This sequence was determined by enzymatic hydrolysis, separation, and activity screening of Moringa oleifera leaf protein. Moringa oleifera leaf protein has a unique amino acid composition and may produce small peptides with regulatory functions for AMPK activity after specific enzymatic hydrolysis.

[0133] . Extraction method

[0134] · Pretreatment: Wash, dry, and pulverize Moringa oleifera leaves, soak them in sodium hydroxide solution with pH 9.1, with the ratio of Moringa oleifera leaf powder to solution being 1:11 (w / v), stir and extract at 51 °C for 4.2 hours, and perform intermittent ultrasound (power 230 W, ultrasound time 11 minutes) during this period to promote protein dissolution.

[0135] · Enzymatic hydrolysis and separation: Centrifuge the extract at 8700 rpm for 17 minutes to obtain the supernatant, adjust the pH to 7.6, add alkaline protease (the mass ratio of enzyme to substrate is 1:115), and perform enzymatic hydrolysis at 46 °C for 6.3 hours. After enzymatic hydrolysis, heat to 96 °C to inactivate the enzyme for 11 minutes. Ultrafilter the enzymatic hydrolysate through an ultrafiltration membrane with a molecular weight cut-off of 3300 Da, and collect the retentate. The retentate is separated by ion exchange chromatography (CM-Sepharose FF), gradient eluted with 0 - 0.62 mol / L sodium chloride solution, and the active peak is collected. The active peak is further purified by reverse-phase high-performance liquid chromatography (RP-HPLC), using a C18 column, gradient eluted with methanol-water (containing 0.1% formic acid) as the mobile phase, and the target polypeptide peak is collected.

[0136] . Synergistic effect experiment

[0137] · Experimental design: Use a heart failure mouse model, and set up a control group, an experimental group of the new polypeptide (GDENLIRPTC), and a comparative group of positive control (the commonly used heart failure treatment drug metoprolol).

[0138] ● Experimental results: Five weeks after drug administration, the cardiac function indexes (left ventricular ejection fraction, left ventricular fractional shortening) of the mice in the experimental group were significantly better than those in the control group and the comparative group. The content of malondialdehyde (MDA), an oxidative stress marker in myocardial tissue, was significantly reduced, and the activities of antioxidant enzymes such as superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were enhanced. The expression of inflammatory factors such as tumor necrosis factor-α (TNF-

[0139] α) and interleukin-6 (IL-6) was significantly decreased. At the same time, the phosphorylation levels of key proteins such as acetyl-CoA carboxylase (ACC) and mammalian target of rapamycin (mTOR) in the AMPK downstream signaling pathway were significantly increased. This indicates that the new polypeptide has a synergistic effect in improving the cardiac function of heart failure mice, inhibiting oxidative stress and inflammatory responses, and activating the AMPK signaling pathway.

[0140] Example 4: Novel Small Molecule Peptide Based on Hemp Seed Extract

[0141] . Novel Polypeptide Structure

[0142] The amino acid sequence of the novel polypeptide is: SEQ.NO 33: Thr - Pro - Asn - Lys - His - Gly - Ala - Ser

[0143] - Phe - Met (TPNKHGASFM). This sequence was screened from the enzymatic hydrolysate of the protein in hemp seeds. Hemp seeds are rich in high - quality protein, and small peptides that may regulate the activity of AMPK may be produced after enzymatic hydrolysis of its protein.

[0144] . Extraction Method

[0145] ● Raw material preparation: Wash, dry and crush hemp seeds, defat them by refluxing with petroleum ether twice, 2.5 hours each time, to remove oil. The defatted hemp seed powder is added to a phosphate buffer solution with pH 7.9 at a ratio of 1:13 (w / v), and stirred and extracted at 43°C for 3.2 hours, while ultrasonic assistance (power 205W, ultrasonic time 9 minutes) is carried out to fully dissolve the protein.

[0146] · Enzymatic hydrolysis and purification: The extract is centrifuged at 8100 rpm for 14 minutes to take the supernatant, adjust the pH to 7.1, add papain (the mass ratio of enzyme to substrate is 1:125), and carry out enzymatic hydrolysis at 43°C for 5.2 hours. After the enzymatic hydrolysis is completed, heat to 87°C to inactivate the enzyme for 9 minutes. The enzymatic hydrolysate is concentrated by rotary evaporation and then ultrafiltered through an ultrafiltration membrane with a molecular weight cut - off of 3100 Da, and the retentate is collected. The retentate is separated by hydrophobic interaction chromatography (Phenyl - Sepharose 6FF), eluted with an ammonium sulfate concentration gradient (2 - 0 mol / L), and the active components are collected. The active components are further purified by reverse - phase high - performance liquid chromatography (RP - HPLC), using a C18 column, and gradient eluted with acetonitrile - water (containing 0.1% trifluoroacetic acid) as the mobile phase, and the target polypeptide peak is collected.

[0147] . Synergistic Effect Test

[0148] ● Experimental design: Select atherosclerotic rabbit models and divide them into a control group, an experimental group of the novel polypeptide (TPNKHGASFM), and a comparison group of traditional therapeutic drugs (such as rosuvastatin).

[0149] · Experimental results: After nine weeks of drug administration in the experimental group of rabbits, the area of atherosclerotic plaques was significantly smaller than that in the control group and the comparison group. The proliferation and migration abilities of vascular smooth muscle cells were significantly reduced, and the infiltration of inflammatory cells in the vascular wall was significantly decreased. The blood lipid levels (total cholesterol, triglycerides, low-density lipoprotein cholesterol) were significantly reduced, and the high-density lipoprotein cholesterol increased. The AMPK activity in vascular tissues was significantly enhanced, and the expressions of cholesterol reverse transport-related proteins ABCA1 and ABCG1 were upregulated. This indicates that the novel polypeptide has a synergistic effect in inhibiting the progression of atherosclerosis, regulating blood lipids, activating AMPK, and promoting cholesterol excretion.

[0150] Example 5: A novel small molecule peptide extracted from Portulaca oleracea

[0151] . Novel polypeptide structure

[0152] The amino acid sequence of the novel polypeptide is: SEQ.NO 34: Val-Asn-Glu-Pro-His-Lys-Ala-Tyr

[0153] -Trp-Ser (VNEPHKAYWS). This sequence was obtained by extracting, enzymatically digesting, and screening the activity of Portulaca oleracea protein. Portulaca oleracea is rich in various nutrients, and its protein may produce small molecule peptides with the ability to regulate AMPK activity after enzymatic digestion.

[0154] . Extraction method

[0155] ● Treatment of Portulaca oleracea: After washing and drying fresh Portulaca oleracea, it was homogenized with a homogenizer. The homogenate was added to Tris-HCl buffer at pH 8.9 at a ratio of 1:10 (w / v), and stirred and extracted at 41°C for 4.6 hours, with ultrasonic assistance (power 215W, ultrasonic time 12 minutes) during this period to promote the dissolution of Portulaca oleracea protein.

[0156] ● Enzymatic digestion and separation: The extract was centrifuged at 8300 rpm for 15 minutes to obtain the supernatant, the pH was adjusted to 7.3, neutral protease was added (enzyme to substrate mass ratio 1:122), and enzymatic digestion was carried out at 41°C for 6.6 hours. After enzymatic digestion, it was heated to 91°C to inactivate the enzyme for 10 minutes. The enzymatic hydrolysate was ultrafiltered through an ultrafiltration membrane with a molecular weight cut-off of 4100 Da, and the retentate was collected. The retentate was separated by ion exchange chromatography (DEAE-Sepharose FF), gradient eluted with 0 - 0.52 mol / L sodium chloride solution, and the active peak was collected. The active peak was further purified by reverse-phase high performance liquid chromatography (RP-HPLC), using a C18 column, and gradient eluted with methanol-water (containing 0.1% formic acid) as the mobile phase, and the target polypeptide peak was collected.

[0157] . Synergistic test

[0158] · Experimental design: A mouse model of myocardial ischemia-reperfusion injury was used, and a control group, an experimental group of a novel polypeptide (VNEPHKAYWS), and a comparison group of a commonly used myocardial protection drug (such as tanshinone) were set up.

[0159] Experimental results: After myocardial ischemia-reperfusion in the experimental group mice, the myocardial infarction area was significantly smaller than that in the control group and the comparison group. The AMPK activity in myocardial tissues was significantly increased, the activities of antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) were enhanced, and the content of lipid peroxidation product malondialdehyde (MDA) was significantly decreased. At the same time, the expression of apoptosis-related protein Bax in myocardial cells was decreased, the expression of Bcl-2 was increased, and the apoptosis rate of cells was significantly decreased. This indicates that the novel polypeptide has a synergistic effect in reducing myocardial ischemia-reperfusion injury, activating AMPK, enhancing antioxidant capacity, and inhibiting myocardial cell apoptosis.

Claims

1. A novel polypeptide through the activity of AMPK, characterized in that, Composed of the amino acid sequences SEQ.NO 1: Leu - Asn -Pro - Thr - Glu (LNPTG), SEQ.NO 2: Val - His - Met - Arg - Tyr (VHMRY).

2. The novel polypeptide according to claim 1, characterized in that, The polypeptide further includes SEQ.NO 31: Ala -Gln - Ser - His - Lys - Pro - Thr - Glu - Tyr - Val (AQSHKPTEYV), SEQ.NO 32: Gly- Asp - Glu - Asn - Leu - Ile - Arg - Pro - Thr - Cys (GDENLIRPTC), SEQ.NO 33: Thr - Pro - Asn - Lys - His - Gly - Ala - Ser - Phe - Met (TPNKHGASFM), SEQ.NO34: Val - Asn - Glu - Pro - His - Lys - Ala - Tyr - Trp - Ser (VNEPHKAYWS).

3. The novel polypeptide according to claim 1, wherein The polypeptide is used to regulate the activity of adenosine monophosphate - activated protein kinase (AMPK).

4. The novel polypeptide according to claim 1, wherein The polypeptide binds to AMPK to improve cardiovascular function and is used for the treatment of cardiovascular diseases.

5. The novel polypeptide according to claim 1, characterized in that, The polypeptide is prepared by solid - phase synthesis, including sequentially connecting amino acid monomers with Boc protecting groups to a solid - phase support, followed by cleavage and purification.

6. The novel polypeptide according to claim 1, wherein, The structure of the polypeptide is verified by mass spectrometry analysis and circular dichroism analysis. Its molecular weight is consistent with the theoretical value and it has specific secondary - structure characteristics.

7. The novel polypeptide according to claim 1, characterized in that, In in vitro cell experiments, the polypeptide can significantly activate AMPK, improve the viability and contractility of the human cardiomyocyte cell line (HCM cells), promote the proliferation of the human vascular endothelial cell line (HUVEC cells), regulate endothelial cell permeability, and reduce the contractility of the human vascular smooth muscle cell line (VSMC cells).

8. The novel polypeptide according to claim 1, characterized in that, In animal model experiments, the polypeptide can reduce the blood pressure level of the hypertensive rat model and improve the cardiac function indexes of the heart - failure mouse model.

9. The novel polypeptide according to claim 1, wherein, In the acute toxicity test, the polypeptide shows no obvious toxicity at high doses, and in the long - term toxicity test, there are no obvious adverse reactions after continuous administration for eight weeks.

10. A pharmaceutical composition, characterized in that, Comprising the novel polypeptide as claimed in claim 1 and a pharmaceutically acceptable carrier.

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