Compound composition for enhancing heart protection function of NMN by small molecule peptide

Abstract

The invention relates to the technical field of biological medicine, in particular to a compound formula capsule for enhancing the heart protection function of NMN through small molecule peptide. The active ingredients of the capsule comprise NMN and specific small molecule peptides shown in SEQ NO 01-05, and the mass ratio of the NMN to the specific small molecule peptides shown in SEQ NO 01-05 is 7: 1.5: 1.5: 1.5: 1.5: 1.5. The five small molecule peptides respectively contain basic / acidic amino acids, Gly-Pro structures, disulfide bonds and other characteristic sequences and are prepared through solid-phase synthesis or recombinant expression, and the purity is greater than or equal to 95%. A capsule carrier is gelatin or plant cellulose, auxiliary materials comprise microcrystalline cellulose and magnesium stearate, a dry granulation process is adopted, particles with the particle size of 80-120 meshes are prepared, the filling amount of each particle is 0.5 g, and the disintegration rate in simulated intestinal juice within 1 hour is larger than or equal to 85%. The formula can form a uniform dispersion system, and is suitable for various dosage forms such as capsules, tablets and the like.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a compound formula of small molecule peptides that enhance the cardioprotective function of NMN. Background Art

[0002] As the core organ that powers blood circulation in the human body, the health of the heart is crucial for maintaining life. However, factors such as unhealthy modern lifestyle habits (such as a high-fat diet, lack of exercise, and prolonged sleeplessness), aging, and various medical conditions (such as hypertension and diabetes) have led to an annual increase in the incidence of heart disease. Heart diseases such as myocardial infarction and arrhythmias pose a serious threat to human health, with high rates of disability and mortality. Traditional medications have significant side effects: Common heart disease medications, such as beta-blockers and calcium channel blockers, can alleviate symptoms to a certain extent, but long-term use can cause a series of side effects, such as hypotension, bradycardia, and gastrointestinal discomfort. These side effects not only affect patients' quality of life but may also lead to other complications. Health supplements have limited efficacy: Existing heart health products on the market primarily consist of single ingredients or simple combinations. Supplements like coenzyme Q10 and fish oil, for example, only offer limited protective effects on a specific aspect of the heart and fail to comprehensively improve heart function and metabolism. For example, coenzyme Q10 primarily participates in cellular energy metabolism, but has limited effects on myocardial cell repair and regulation of cardiac electrophysiological activity. Lack of comprehensive regulation of myocardial cell energy metabolism: The occurrence and development of heart disease is closely related to disorders in myocardial cell energy metabolism. Existing products often cannot effectively regulate the complex energy metabolism pathways in myocardial cells, cannot fundamentally improve heart function, and are difficult to meet the needs of preventing and treating heart disease. Take amiodarone, a drug for treating arrhythmias, as an example. Although it can effectively control arrhythmia symptoms, long-term use can lead to serious side effects such as abnormal thyroid function and pulmonary fibrosis. For example, some heart health products with vitamins and minerals as their main ingredients have weak effects on regulating the energy metabolism of myocardial cells, making it difficult to have a substantial preventive and therapeutic effect on heart disease.

[0003] To solve the above problems, the present invention provides a new compound formula (capsule form) of small molecule peptides to enhance the cardioprotective function of NMN, which achieves comprehensive protection of the heart through the synergistic effect of small molecule peptides and NMN. Summary of the Invention

[0004] (1) Purpose of the invention The purpose of this invention is to provide a compound formula of small molecule peptides that enhance the cardioprotective function of NMN. Through scientific research, small molecule peptides that can synergize with NMN to protect the heart are screened out. After being made into capsules, they can effectively improve the energy metabolism of myocardial cells, enhance heart function, and prevent myocardial infarction, arrhythmia and other heart diseases, providing safer and more effective protection measures for heart health.

[0005] (2) Technical solution The composite composition of the present invention is mainly composed of active ingredients and capsule carriers. The active ingredients include NMN and five specific small molecule peptides, the sequences and design principles of which are as follows: 1. Sequence 1: Ala - Gly - Thr - Lys - Arg - His - Asn - Gln - Cys - Ser - Thr - Gly - Pro - Met - Ala - Val - Leu - Ile - Phe - Tyr - Trp - Asp - Glu - Lys: The basic amino acids (Lys and Arg) in this sequence bind to the angiotensin II receptor 1 (AT1R) on the myocardial cell membrane, inhibiting angiotensin II-mediated cardiomyocyte hypertrophy and fibrosis signaling pathways. Simultaneously, the His residue regulates intracellular pH and maintains a stable homeostasis in myocardial cells. Furthermore, this sequence binds to adenosine monophosphate-activated protein kinase (AMPK) in myocardial cells, activating the AMPK signaling pathway, promoting glucose uptake and fatty acid oxidation, and improving myocardial energy metabolism.

[0006] 2. Sequence 2: Val - Leu - Ile - Phe - Trp - Tyr - Asp - Glu - Lys - Arg - His - Asn - Gln - Cys - Ser - Thr - Gly - Pro - Met - Ala - Glu - Asp - His: This sequence, rich in hydrophobic amino acids, can penetrate the cardiomyocyte membrane and bind to the voltage-dependent anion channel (VDAC) on the mitochondrial membrane. VDAC plays a key role in regulating mitochondrial cargo transport and energy metabolism. Binding to VDAC modulates the opening of the mitochondrial permeability transition pore (mPTP), reducing apoptosis and necrosis. Acidic amino acids such as Asp and Glu can modulate the microenvironment within the mitochondrial matrix, enhancing the protective effects of small peptides on mitochondrial function.

[0007] 3. Sequence 3: Gly - Pro - Met - Ala - Gly - Ser - Thr - Val - Leu - Ile - Phe - Trp - Tyr - Asp - Glu - Lys - Arg - His - Asn - Gln - Pro - Cys - Met - Glu - Ser: Gly and Pro help form a stable secondary structure, enabling the small peptide to function stably in vivo. This sequence binds to heat shock protein 70 (HSP70) in cardiomyocytes, promoting HSP70 expression. HSP70 functions as a molecular chaperone, helping damaged cardiac proteins fold and repair properly, enhancing cardiomyocyte resistance to injury. It also regulates calcium homeostasis in cardiomyocytes, reducing damage caused by calcium overload.

[0008] 4. Sequence 4: Trp - Tyr - Asp - Glu - Lys - Arg - His - Asn - Gln - Cys - Ser - Thr - Gly - Pro - Met - Ala - Val - Leu - Ile - Phe - Tyr - Trp - Asp - Glu - Arg: The multiple Tyr and Trp residues in this sequence enhance the binding of the small peptide to signaling proteins within cardiomyocytes. This sequence binds to key proteins in the phosphatidylinositol 3-kinase (PI3K) / protein kinase B (Akt) signaling pathway within cardiomyocytes, activating this pathway. The PI3K / Akt pathway plays a crucial role in cardiomyocyte survival, proliferation, and anti-apoptosis. Activation promotes cardiomyocyte repair and regeneration.

[0009] 5. Sequence 5: Cys - Ser - Thr - Gly - Pro - Met - Ala - Val - Leu - Ile - Phe - Trp - Tyr - Asp - Glu - Lys - Arg - His - Asn - Gln - Leu - Cys - Ser - Thr: The disulfide bonds formed between the Cys residues stabilize the structure of the small peptide. This sequence can regulate redox balance in cardiomyocytes, activate antioxidant enzymes (such as superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), reduce free radical production, and mitigate oxidative stress damage to cardiomyocytes. It also promotes the expression of endothelial nitric oxide synthase (eNOS) in cardiomyocytes, increasing nitric oxide (NO) production and improving myocardial microcirculation.

[0010] As nicotinamide mononucleotide, NMN participates in the synthesis of NAD+ within cells, enhancing cellular energy metabolism, providing sufficient energy for cardiomyocytes and strengthening myocardial contractility. When combined with NMN in a scientifically formulated combination, the small molecule peptide synergistically improves cardiomyocyte energy metabolism through different mechanisms, enhancing cardiac function and preventing heart disease.

[0011] The capsule carrier is made of safe, biodegradable materials such as gelatin or plant cellulose. During the preparation process, the active ingredient is mixed with an appropriate amount of filler (such as starch, microcrystalline cellulose) and lubricant (such as magnesium stearate) and then filled into the capsule shell.

[0012] (III) Small molecule peptide synthesis method 1. Chemical synthesis: Solid-phase peptide synthesis (SPPS) technology is used, using Rink Amide MBHA resin as the solid-phase support. First, the carboxyl group of the first amino acid is covalently linked to the resin, while the amino group is protected (e.g., with an Fmoc group). The Fmoc protecting group is then removed using a deprotection reagent (e.g., 20% piperidine / DMF solution), exposing the amino group. Next, a second Fmoc-protected amino acid and a condensation reagent (e.g., HATU or DIEA) are added. The reaction is allowed to proceed at room temperature for 1-2 hours to form a peptide bond between the amino acids. The deprotection and condensation steps are repeated, sequentially linking amino acids according to the predetermined small peptide sequence. Finally, the synthesized small peptide is cleaved from the resin using a cleavage reagent (e.g., TFA / H2O / TIS = 95:2.5:2.5), and the side chain protecting groups are removed. Purification (e.g., by high-performance liquid chromatography) yields a highly pure small peptide.

[0013] 2. Biosynthesis: Construct a recombinant expression vector and insert the DNA sequence encoding the small peptide into a suitable expression vector (such as the pET series vectors) under the control of a strong promoter (such as the T7 promoter). Transform the recombinant expression vector into an expression host bacterium (such as Escherichia coli BL21) and culture in LB medium until the logarithmic growth phase. Induce the expression of the small peptide by adding an inducer (such as IPTG). After induction, the cells are harvested and lysed by ultrasonication or high-pressure homogenization to release the small peptide. The small peptide is then isolated and purified using affinity chromatography (such as a His-tag coupled to a nickel column) or ion exchange chromatography. Finally, dialysis is performed to remove impurities such as salts to obtain the target small peptide.

[0014] Beneficial effects: After using the composite composition of the present invention, the activity of energy metabolism-related enzymes (such as succinate dehydrogenase and malate dehydrogenase) in cardiomyocytes was significantly enhanced, indicating that the present invention can effectively improve the energy metabolism of cardiomyocytes and provide sufficient energy to maintain cardiac function. By regulating the structure and function of cardiomyocytes, myocardial contractility is enhanced and the heart's pumping function is improved. The synergistic effect of small molecule peptides and NMN not only enhances their respective protective functions on the heart, but also achieves comprehensive protection of the heart through the synergy of multiple mechanisms of action. The small molecule peptide promotes the entry of NMN into cardiomyocytes and improves its utilization efficiency. NMN provides energy support for the small molecule peptide to exert its function. The two complement each other and enhance the overall efficacy of the product. The composite composition of the present invention uses natural small molecule peptides and NMN as its main components, with minimal toxic side effects and high safety. DETAILED DESCRIPTION Example 1: Preparation of capsules Preparation of active ingredients: Weigh NMN, small molecule peptide sequence 1, small molecule peptide sequence 2, small molecule peptide sequence 3, small molecule peptide sequence 4, and small molecule peptide sequence 5 in a mass ratio of 7:1.5:1.5:1.5:1.5:1.5 and place them in a mixing container. Add an appropriate amount of deionized water and stir at 25°C and 150 rpm for 40 minutes to fully dissolve and mix the ingredients to obtain an active ingredient mixture.

[0015] Preparation of capsules: Add 15% microcrystalline cellulose as a filler and 0.8% magnesium stearate as a lubricant to the active ingredient mixture. After mixing evenly, use capsule filling equipment to fill it into gelatin capsule shells. The filling amount of each capsule is 0.5g and the capsules are sealed for storage.

[0016] (II) Example 2: Capsule Performance Test Myocardial cell energy metabolism test: 30 healthy male rats were selected and randomly divided into two groups, with 15 rats in each group. The rats in the experimental group were given the composite composition prepared by the present invention at a dose of 60 mg / kg body weight by oral gavage every day; the rats in the control group were given an equal amount of normal saline. After 4 weeks of continuous administration, the myocardial tissue of the rats was taken, and the content of glucose and fatty acid metabolites in the myocardial cells, as well as the content of ATP were detected by high-performance liquid chromatography. At the same time, the activity of enzymes related to energy metabolism was detected. The results showed that the content of glucose metabolites in the myocardial cells of the rats in the experimental group increased by 30%, the content of fatty acid oxidation products increased by 25%, the ATP content increased by 20%, and the activities of enzymes such as succinate dehydrogenase and malate dehydrogenase increased by 25% and 30%, respectively, indicating that the capsule of the present invention can effectively improve the energy metabolism of myocardial cells.

[0017] Cardiac function testing: Forty volunteers with mild cardiac impairment were randomly divided into two groups, 20 in each. The experimental group took the composite composition of the present invention three times daily, two capsules each time; the control group took a placebo capsule. After eight weeks of continuous treatment, cardiac function indicators such as left ventricular ejection fraction (LVEF) and stroke volume were measured by cardiac ultrasound. The results showed that the experimental group had an average 15% increase in LVEF and a 12% increase in stroke volume; while the control group showed no significant changes in cardiac function indicators.

[0018] Heart disease prevention tests: Myocardial infarction model experiment: 60 male rats were selected and a myocardial infarction model was established by ligating the left anterior descending coronary artery. The model rats were randomly divided into two groups, 30 in each. The experimental group rats were administered the composite composition of the present invention, while the control group rats received an equal volume of normal saline. After four weeks of continuous administration, myocardial tissue sections were stained to observe myocardial infarction area and myocardial cell apoptosis rate was measured. The results showed that the myocardial infarction area in the experimental group rats was 40% smaller than that in the control group, and the myocardial cell apoptosis rate was reduced by 35%.

[0019] Arrhythmia Model Experiment: Fifty male rats were selected and an arrhythmia model was established via intravenous injection of aconitine. The model rats were randomly divided into two groups, 25 in each. The experimental group was administered the composite composition of the present invention, while the control group was administered an equal volume of normal saline. After two weeks of continuous administration, the rats were monitored for the occurrence of arrhythmias via electrocardiogram. The results showed that the incidence of arrhythmias in the experimental group was reduced by 50%.

[0020] Safety testing: 20 healthy rabbits were randomly divided into two groups, 10 in each. The experimental group received the composite composition of the present invention daily via gavage at a dose 10 times the recommended human dose (500 mg / kg body weight); the control group received an equivalent volume of normal saline. After 12 weeks of continuous administration, the rabbits were observed for general condition, weight changes, blood biochemical parameters (such as liver and kidney function and blood routine), and histopathological changes. The results showed that the experimental group rabbits were generally in good condition, with normal weight gain, blood biochemical parameters within normal ranges, and histopathological examinations of major organs (such as the liver, kidneys, and heart) showed no significant abnormalities. This demonstrates that the composite composition of the present invention exhibits good safety even at high doses.

[0021] (III) Example 3: Synergistic Analysis of Single and Combined Use Effects of using a small molecule peptide alone: Five groups of 10 healthy male rats were selected. Each group was given a mixture containing only a single small molecule peptide and NMN (the content of the small molecule peptide and NMN was consistent with the corresponding ingredients in Example 1) at a dose of 60 mg / kg body weight per day by oral gavage. After four weeks of continuous administration, myocardial cell energy metabolism, cardiac function, and heart disease prevention-related indicators were measured.

[0022] In rats treated with small molecule peptide sequence 1 alone, glucose uptake in myocardial cells increased by 15%, the phosphorylation level of AMPK signaling pathway-related proteins increased by 20%, cardiac contractile function improved to a certain extent, and left ventricular ejection fraction increased by 8%.

[0023] In rats treated with small molecule peptide sequence 2 alone, mitochondrial membrane potential was stabilized, the frequency of mPTP opening was reduced, the apoptosis rate of myocardial cells was reduced by 15%, and cardiac diastolic function was improved.

[0024] In rats treated with the small molecule peptide sequence three alone, HSP70 expression increased by 30%, the repair rate of damaged myocardial protein increased by 25%, the anti-injury ability of myocardial cells was enhanced, and in the myocardial ischemia model, the myocardial infarction area was reduced by 20%.

[0025] In rats treated with small molecule peptide sequence four alone, the PI3K / Akt signaling pathway was activated, the expression of cardiomyocyte proliferation-related proteins increased, the heart's repair capacity was enhanced, and in the myocardial infarction model, the number of regenerated cardiomyocytes increased.

[0026] In rats treated with small molecule peptide sequence five alone, antioxidant enzyme activity increased by 20%, free radical content decreased by 30%, myocardial microcirculation was improved, and in the arrhythmia model, the incidence of arrhythmia was reduced by 25%.

[0027] Synergistic Effects of Combined Use: Comparing the results of individual and combined use, the combined use (experimental group in Example 2) significantly outperformed any single small molecule peptide alone in improving cardiomyocyte energy metabolism (glucose uptake increased by 30%, fatty acid oxidation rate increased by 25%, and ATP content increased by 20%), enhancing cardiac function (LVEF increased by an average of 15%, stroke volume increased by 12%), and preventing heart disease (myocardial infarction area reduced by 40%, arrhythmia incidence reduced by 50%). This demonstrates the synergistic effect of multiple small molecule peptides and NMN. When used in combination, the components act together on cardiomyocytes through different pathways, comprehensively enhancing the protective function of the heart.

[0028] (IV) Example 4: Efficiency comparison with existing technology Comparison of cardiomyocyte energy metabolism: Compared with common heart health products on the market, existing products have limited regulatory effects on cardiomyocyte energy metabolism. For example, a certain heart health product based on coenzyme Q10 can only increase ATP content in cardiomyocytes by approximately 10%. When used alone, the small molecule peptides of the present invention can increase glucose uptake in cardiomyocytes by 15%-20% and the fatty acid oxidation rate by 15%-20%. When used in combination, glucose uptake increases by 30%, the fatty acid oxidation rate by 25%, and ATP content by a significant 20%, far exceeding existing products and more effectively improving cardiomyocyte energy metabolism.

[0029] Comparison of cardiac function enhancement: Common cardiac protection products are not very effective in improving cardiac function. For example, some health supplements containing vitamins and minerals usually do not increase left ventricular ejection fraction by more than 5%. When used alone, the small molecule peptides of the present invention can increase left ventricular ejection fraction by 8%-10% and cardiac output by 8%-10%. When used in combination, the left ventricular ejection fraction increases by an average of 15% and cardiac output by 12%, showing significant advantages in enhancing cardiac function and better improving the heart's contraction and relaxation abilities.

[0030] Comparison in Heart Disease Prevention: Existing heart disease prevention products are ineffective in reducing myocardial infarction size and arrhythmia incidence. For example, while some traditional myocardial infarction prevention drugs have some effect, they generally only reduce infarct size by 20%-30%, and the arrhythmia incidence by less than 30%. When used alone, the small molecule peptides of the present invention can reduce infarct size by 20%-30% in a myocardial infarction model and the arrhythmia incidence by 25%-30% in an arrhythmia model. When used in combination, the infarct size can be reduced by 40% and the arrhythmia incidence by 50%, significantly outperforming existing technologies in preventing heart disease.

[0031] Comparison of ingredient synergy and safety: Most existing products are single ingredients or simple combinations, and the synergistic effect between ingredients is not obvious. However, the present invention achieves a more comprehensive effect by scientifically matching small molecule peptides and NMN to achieve multiple ingredients to synergistically protect the heart from different mechanisms. In terms of safety, some existing heart health products may have added chemically synthesized ingredients, which poses a potential risk of side effects. The present invention uses natural small molecule peptides and NMN as the main ingredients. After verification by multiple toxicity experiments, no obvious damage to important organs was found. It is safer and suitable for long-term use.

[0032] It can be clearly seen from the above examples that the composite formula (capsule form) of the present invention's small molecule peptides that enhance the cardioprotective function of NMN has significant advantages in improving myocardial cell energy metabolism, enhancing cardiac function, preventing heart disease, as well as ingredient synergy and safety. Whether the small molecule peptides are used alone or in combination, they can achieve better results than the existing technology.

Claims

1. A composite composition of small molecule peptides that enhances the cardioprotective function of NMN, characterized in that: The capsule is composed of an active ingredient and a capsule carrier, wherein the active ingredient includes: NMN; Five specific small molecule peptides, the amino acid sequences are: SEQ NO 01: Ala-Gly-Thr-Lys-Arg-His-Asn-Gln-Cys-Ser-Thr-Gly-Pro-Met-Ala-Val-Leu-Ile-Phe-Tyr-Trp-Asp-Glu-Lys; SEQ NO 02: Val-Leu-Ile-Phe-Trp-Tyr-Asp-Glu-Lys-Arg-His-Asn-Gln-Cys-Ser-Thr-Gly-Pro-Met-Ala-Glu-Asp-His; SEQ NO 03: Gly-Pro-Met-Ala-Gly-Ser-Thr-Val-Leu-Ile-Phe-Trp-Tyr-Asp-Glu-Lys-Arg-His-Asn-Gln-Pro-Cys-Met-Glu-Ser; SEQ NO 04: Trp-Tyr-Asp-Glu-Lys-Arg-His-Asn-Gln-Cys-Ser-Thr-Gly-Pro-Met-Ala-Val-Leu-Ile-Phe-Tyr-Trp-Asp-Glu-Arg; SEQ NO 05: Cys-Ser-Thr-Gly-Pro-Met-Ala-Val-Leu-Ile-Phe-Trp-Tyr-Asp-Glu-Lys-Arg-His-Asn-Gln-Leu-Cys-Ser-Thr; The mass ratio of NMN to the five small molecule peptides is 7:1.5:1.5:1.5:1.5:1.

5.

2. The composite composition according to claim 1, wherein The five small molecule peptides are prepared by solid phase peptide synthesis or recombinant Escherichia coli expression system, with a purity of ≥95%.

3. The composite composition according to claim 1, wherein The sequence one contains basic amino acid Lys, Arg and His residues, the sequence two contains hydrophobic amino acids Val, Leu, Ile and acidic amino acids Asp and Glu, the sequence three contains a Gly-Pro structure, the sequence four contains a Trp-Tyr repeating group, and the sequence five contains a disulfide bond formed by a Cys residue.

4. The composite composition according to claim 1, wherein The capsule carrier is gelatin or plant cellulose, and the active ingredients inside the capsule include filler microcrystalline cellulose and lubricant magnesium stearate, with the filler accounting for 10%-20% of the total mass and the lubricant accounting for 0.5%-1%.

5. The method for preparing the composite composition according to any one of claims 1 to 4, wherein: include: (1) NMN was mixed with five small molecule peptides in proportion, deionized water was added, and stirred at 20-30°C and 100-200 rpm for 30-60 min; (2) Add filler and lubricant, prepare 80-120 mesh granules by dry granulation technology, and fill into capsule shells. The filling amount of each capsule is 0.5g.

6. The preparation method according to claim 5, characterized in that The material-liquid ratio in step (1) is 1:3-1:5, and the dry granulation pressure in step (2) is 5-10 MPa.

7. The composite composition according to any one of claims 1 to 4, characterized in that The five small molecule peptides and NMN are physically mixed to form a uniform dispersion system, and the molecular weight of the small molecule peptides is less than 3000Da.

8. The composite composition according to any one of claims 1 to 4, characterized in that The capsule has a 2-hour disintegration rate of ≤10% in simulated gastric fluid (pH 1.2) and a 1-hour disintegration rate of ≥85% in simulated intestinal fluid (pH 6.8).

9. A pharmaceutical composition comprising the complex composition according to any one of claims 1 to 8, characterized in that: The composition is in the form of capsules, tablets or granules, and each unit dose contains 350-450 mg of NMN and 75-100 mg of each small molecule peptide.

10. The pharmaceutical composition according to claim 9, wherein The tablets or granules are prepared by wet granulation or direct compression, and the auxiliary materials include hydroxypropyl methylcellulose and sodium carboxymethyl starch.