Use of small molecule active oligopeptides in the preparation of products for improving skeletal muscle dysfunction and / or enhancing skeletal muscle contractile function

Abstract

The application discloses application of a small-molecule active oligopeptide in preparation of a product for improving skeletal muscle dysfunction and / or enhancing skeletal muscle contraction function, and an amino acid sequence of the small-molecule active oligopeptide is Leu-Pro-Phe. The small-molecule active oligopeptide can improve skeletal muscle dysfunction and complications caused by acute and chronic alcoholism, relieve muscle weakness, muscle spasm, muscle pain, muscle atrophy and muscle dissolution, and is suitable for enhancing normal skeletal muscle function and has a potential therapeutic effect on muscle dysfunction caused by other reasons. The small-molecule active oligopeptide is safe, has no toxic side effect and has a remarkable curative effect; has a small molecular weight, can be directly absorbed without digestion after oral administration; and has a simple preparation process and is suitable for industrialized production.

Description

Technical Field

[0001] This invention relates to the application of a small molecule active oligopeptide in the preparation of products that improve skeletal muscle dysfunction and / or enhance skeletal muscle contractile function. Background Technology

[0002] In Chinese culinary culture, alcohol and its associated culture hold a significant place. Drinking is often a daily habit, with some people experiencing significant addiction and withdrawal symptoms. Furthermore, with increasing life burdens, people tend to relieve stress through alcohol. Modern medical research has found that moderate alcohol consumption can promote metabolism and improve blood circulation, but excessive alcohol intake not only leads to alcoholic liver disease but also impairs skeletal muscle contraction function, potentially inducing chronic alcoholic myopathy, manifested as decreased muscle tone, muscle atrophy, pain, and even muscle breakdown. In addition, acute alcohol poisoning can cause short-term muscle weakness, increasing the risk of falls and injuries, thus endangering health and life.

[0003] The effects of alcohol consumption on skeletal muscle function are related to factors such as muscle calcium flux, ATP hydrolysis, nerve conduction, blood supply, and endocrine hormones. Studies have shown that ethanol can reduce calcium flux in muscle cells, hindering muscle fiber activation and thus reducing muscle contractile ability. Furthermore, alcohol poisoning can impair energy metabolism processes within muscle cells, and the lack of ATP affects muscle contractile force and speed. Alcohol consumption can also lead to hormonal imbalances; long-term alcohol consumption has a sustained impact on sex hormone levels, and testosterone has been found to enhance muscle contractile performance. Therefore, ethanol can also affect muscle function through the secretion of endocrine hormones.

[0004] In recent years, people have gradually realized the importance of improving alcohol-induced skeletal muscle dysfunction. Although many hangover remedies have appeared on the market, most of them are used to promote alcohol metabolism and protect the liver; currently, there are no products available to improve skeletal muscle dysfunction. Summary of the Invention

[0005] The primary objective of this invention is to overcome the shortcomings and deficiencies of existing technologies and provide an application of a small molecule active oligopeptide in the preparation of products that improve skeletal muscle dysfunction and / or enhance skeletal muscle contractile function. This small molecule active oligopeptide has a significant effect in improving skeletal muscle dysfunction and enhancing skeletal muscle contractile function.

[0006] The objective of this invention is achieved through the following technical solution: the application of small molecule active oligopeptides in the preparation of products that improve skeletal muscle dysfunction and / or enhance skeletal muscle contractile function, wherein the amino acid sequence of the small molecule active oligopeptides is Leu-Pro-Phe.

[0007] The skeletal muscle dysfunction mentioned can be caused by acute or chronic alcohol poisoning.

[0008] The enhancement of skeletal muscle contraction function can be applied to normal skeletal muscle.

[0009] The small molecule active oligopeptides include their modified derivatives or salt-forming compounds.

[0010] The active small molecule oligopeptides can be derived from the enzymatic hydrolysis products of corn protein, or they can be prepared by methods such as biosynthesis, fermentation synthesis, heterologous expression, and chemical synthesis.

[0011] This invention also protects the use of the small molecule active oligopeptide in the preparation of a product, the function of which is at least one of the following 1)-8): 1) Improves muscle weakness; 2) Improves muscle spasms; 3) Improves muscle pain; 4) Improves muscle atrophy; 5) Improves rhabdomyolysis; 6) Enhances energy metabolism in muscle cells; 7) Enhances the activity of respiratory chain complexes; 8) Promotes ATP production in mitochondria.

[0012] The product in question is a pharmaceutical product.

[0013] The drug may also contain necessary excipients.

[0014] The excipients include, but are not limited to, at least one of the following: sustained-release agents, fillers, binders, humectants, disintegrants, absorption promoters, surfactants, lubricants, preservatives, antioxidants, colorants, and sweeteners.

[0015] The formulation types of the medicine include, but are not limited to, oral formulations such as tablets, capsules, powders, pills, granules, and oral liquids, as well as injections.

[0016] The product may also contain other hangover-relieving and liver-protecting ingredients, including but not limited to taurine, silymarin, biphenyl diester, vitamins, and amino acids.

[0017] This invention also protects a product for improving skeletal muscle dysfunction and / or enhancing skeletal muscle contractile function, wherein the active ingredient is the aforementioned small molecule active oligopeptide.

[0018] The skeletal muscle dysfunction mentioned can be caused by acute or chronic alcohol poisoning.

[0019] The enhancement of skeletal muscle contraction function can be applied to normal skeletal muscle.

[0020] The small molecule active oligopeptides include their modified derivatives or salt-forming compounds.

[0021] This invention also protects a product whose active ingredient is the aforementioned small molecule active oligopeptide, and the product has the function of at least one of the following 1)-8): 1) Improves muscle weakness; 2) Improves muscle spasms; 3) Improves muscle pain; 4) Improves muscle atrophy; 5) Improves rhabdomyolysis; 6) Enhances energy metabolism in muscle cells; 7) Enhances the activity of respiratory chain complexes; 8) Promotes ATP production in mitochondria.

[0022] The product in question is a pharmaceutical product.

[0023] The drug may also contain necessary excipients.

[0024] The excipients include, but are not limited to, at least one of the following: sustained-release agents, fillers, binders, humectants, disintegrants, absorption promoters, surfactants, lubricants, preservatives, antioxidants, colorants, and sweeteners.

[0025] The formulation types of the medicine include, but are not limited to, oral formulations such as tablets, capsules, powders, pills, granules, and oral liquids, as well as injections.

[0026] The product may also contain other hangover-relieving and liver-protecting ingredients, including but not limited to taurine, silymarin, biphenyl diester, vitamins, and amino acids.

[0027] Compared with the prior art, the present invention has the following beneficial effects: This invention provides the application of a small molecule active oligopeptide with the amino acid sequence Leu-Pro-Phe in products for improving skeletal muscle dysfunction and / or enhancing skeletal muscle contractile function. This small molecule active oligopeptide can improve skeletal muscle dysfunction and its complications caused by acute and chronic alcohol poisoning, relieving muscle weakness, muscle spasms, muscle pain, muscle atrophy, and rhabdomyolysis. It is also suitable for enhancing normal skeletal muscle function and has potential therapeutic effects for muscle dysfunction caused by other reasons. This small molecule active oligopeptide is safe and has no toxic side effects, with significant therapeutic effects; its small molecular weight allows for rapid and direct absorption after oral administration without digestion; and its simple preparation process makes it suitable for industrial production. Attached Figure Description

[0028] Figure 1 This is the peak diagram of the parent ion of the small molecule active oligopeptide Leu-Pro-Phe.

[0029] Figure 2 This is a peak diagram of secondary fragment ions of the small molecule active oligopeptide Leu-Pro-Phe.

[0030] Figure 3 This is a quantitative standard curve of the small molecule active oligopeptide Leu-Pro-Phe.

[0031] Figure 4 This is a graph showing the safety results of the small molecule active oligopeptide Leu-Pro-Phe.

[0032] Figure 5 This is a diagram showing the experimental results of how the small molecule active oligopeptide Leu-Pro-Phe improves the motility of zebrafish embryos. Detailed Implementation

[0033] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] Example 1: Preparation and amino acid sequence identification of the small molecule active oligopeptide Leu-Pro-Phe (LPF) The preparation of active small molecule oligopeptides involved the following steps: Corn gluten powder was added to an alkaline alcohol solution (prepared by mixing 0.1 mol / L sodium hydroxide solution with 95% (v / v) ethanol at a volume ratio of 45:55), with a material-to-liquid ratio of 1:67 (g / L). Extraction was performed by ultrasonication (500 W, 50 kHz) for 3 hours at 55℃. After extraction, the supernatant was collected by centrifugation, and an equal volume of deionized water was added to adjust the pH to 6.4. After stirring, 1 / 5 volume of 2% (v / v) sodium chloride solution was added to precipitate the corn gluten. The precipitate was collected, dried in a 60℃ oven, and then ground and sieved to obtain concentrated corn gluten powder. 100 g of the concentrated corn gluten powder was weighed, dissolved in water to a substrate concentration of 10%, and 1% alkaline protease (2709 protease, derived from Bacillus licheniformis, Nanning Dongheng Huadao Biotechnology) was added. The pH was adjusted to 8.5, and hydrolysis was performed at 55℃ for 10 hours, followed by protease inactivation at 95℃ for 10 minutes. After cooling to room temperature, centrifuge and collect the supernatant. Dissolve the precipitate in water until the substrate concentration reaches 8%, then add 2% of a complex protease (Protamex, derived from Bacillus spp., Merck, Germany), adjust the pH to 7.0, hydrolyze at 45°C for 5 hours, inactivate the protease at 95°C for 10 minutes, cool to room temperature, centrifuge and collect the supernatant. Combine the two supernatants. Spray-dry the filtrate to obtain corn protein peptides.

[0035] The above-mentioned corn protein peptides were dissolved in water and subjected to Q... Sepharose ion exchange chromatography, Sephadex G 25 Gel Chromatography and RP HPLC separation and purification yielded a single characteristic peptide. The chemical structure of the characteristic peptide was confirmed using a liquid chromatography-mass spectrometry (LC-MS) system under the following conditions: triple quadrupole LC-MS (AB SCIEX QTRAP 5500); mobile phase consisting of 0.1% (v / v) formic acid aqueous solution (A) and acetonitrile (B); elution method: 0–1.00 min 5.0% B, 1.00–1.50 min 5.0–10.0% B, 1.50–7.50 min 10.0–40.0% B, 7.50–8.50 min 40.0–90.0% B, 8.50–10.00 min 90% B, 10.00–10.50 min 90.0–5.0% B, 10.50–13.00 min 5.0% B; flow rate 0.10 mL / min; injection volume 1 μL; column temperature 40℃; column type HSS. T3 (1.0 × 100 mm, Waters, USA).

[0036] Analysis confirmed that the characteristic peptide of the obtained single component was indeed Leu-Pro-Phe (LPF), and its parent ion peak diagram was as follows. Figure 1 The peak diagram of secondary fragment ions is as follows Figure 2 Quantitative determination using a standard curve showed that the LPF content was not less than 0.0186%. Figure 3 (and Table 1).

[0037] Table 1 Content of small molecule active oligopeptide LPF

[0038] Example 2: Small molecule active oligopeptide LPF enhances energy metabolism in muscle cells. Skeletal muscle contraction requires ATP hydrolysis for energy, therefore the inventors investigated the effect of the oligopeptide LPF on ATP levels in skeletal muscle cells. C2C12 mouse myoblasts, induced by horse serum, can differentiate into retractable myotubes and express characteristic myoproteins, which can be used for in vitro experimental studies of skeletal muscle.

[0039] C2C12 mouse myoblasts were purchased from ATCC (catalog number CRL-1772) and cultured in an ESCO cell culture incubator at 37°C, 5% CO2, and 70-80% humidity. The cell culture medium was DMEM supplemented with 10% fetal bovine serum.

[0040] C2C12 mouse myoblasts were seeded in 6-well plates. When the confluence reached approximately 80%, differentiation was induced using horse serum. The procedure was as follows: the original culture medium was discarded, and the cells were washed twice with sterile 1×PBS. 2 mL of differentiation medium (DMEM medium supplemented with 2% horse serum) was added, and the plates were then incubated. The differentiation medium was changed every 24 hours after the start of differentiation induction. Cell morphology was observed under a microscope regularly during the differentiation induction period until distinct tubular structures of myotubes appeared, indicating successful differentiation.

[0041] After successful differentiation, the muscle cells were treated with ethanol and oligopeptides for 6 hours, and then the muscle cells were harvested. The intracellular ATP content was detected using an ATP assay kit (catalog number S0027) from Shanghai Beyotime Biotechnology Co., Ltd. The principle is as follows: When firefly luciferase catalyzes the production of fluorescence from luciferin, ATP is required as energy. Within a certain concentration range, the production of fluorescence is directly proportional to the concentration of ATP. Therefore, the ATP content in the reaction system can be calculated by detecting the fluorescence signal.

[0042] The ATP assay procedure is as follows: Remove the drug-treated culture medium, wash cells twice with sterile 1×PBS, add 200 μL of lysis buffer to each well, lyse on ice for 15 minutes, centrifuge at 12000g for 5 minutes at 4ºC, and collect the supernatant as the test sample. Add 100 μL of ATP detection working solution to an EP tube and incubate at room temperature for 3 minutes to remove background ATP interference. Add 20 μL of the test sample, mix quickly with a pipette, and detect using a Promega Glomax chemiluminescence analyzer, recording the RUL. Prepare a standard curve according to the protocol provided in the manufacturer's instructions and calculate the ATP content in the test sample. Simultaneously, determine the protein content in the remaining sample using the BCA method and standardize the ATP content calculation.

[0043] The inventors first examined the effects of different concentrations of LPF (5-40 μM) on intracellular ATP in normal muscle cells. The results are shown in Table 2. LPF (20 and 40 μM) significantly increased intracellular ATP levels. This demonstrates that oligopeptides promote energy metabolism in muscle cells under normal conditions. The inventors then observed the effect of the oligopeptide on cellular ATP under ethanol loading conditions. The results are shown in Table 3. Compared with the normal group, ethanol (300 mM) loading reduced the intracellular ATP level from 59.9 nmol / mg protein to 31.8 nmol / mg protein, while LPF (20 and 40 μM) significantly restored the intracellular ATP content, with 40 μM LPF almost restoring ATP to normal levels. Therefore, the oligopeptide provided by this invention can restore the energy metabolism disorder caused by ethanol, providing the energy required for muscle cell contraction.

[0044] Table 2. Effects of LPF on intracellular ATP levels in normal C2C12 cells.

[0045] Note: Compared to Control, * p <0.05, *** p <0.001.

[0046] Table 3. Effects of LPF on ATP levels in C2C12 cells treated with ethanol.

[0047] Note: Compared to Control, *** p <0.001; compared to Ethanol, ### p <0.001.

[0048] Example 3: Small molecule active oligopeptide LPF increases the activity of the mitochondrial respiratory chain complex in muscle cells. Mitochondria are crucial sites for aerobic respiration and the tricarboxylic acid cycle, serving as the cell's energy factories. The tricarboxylic acid cycle breaks down pyruvate into carbon dioxide and water, releasing a large amount of ATP; this process is accomplished by a series of respiratory chain complexes on the inner and outer mitochondrial membranes. Given that oligopeptides can effectively restore muscle cell energy levels induced by ethanol loading, the inventors investigated whether this is related to their influence on the activity of mitochondrial respiratory chain complexes.

[0049] The activity of mitochondrial respiratory chain complexes was determined using kits from Nanjing Jiancheng Biotechnology Institute. The product information is as follows: Complex I kit (NADH-CoQ reductase, catalog number: A089-1-1); Complex II kit (succinate-CoQ reductase, catalog number: A089-2-1); Complex III kit (CoQ-cytochrome C reductase, catalog number: A089-3-1); Complex IV kit (ferrocytochrome C oxidoreductase, catalog number: A089-4-1); Complex V kit (FOF1-ATPase / ATP synthase, catalog number: A089-5-1).

[0050] The detection process is as follows: After successful differentiation of C2C12 cells, they were treated with ethanol (300 mM) and oligopeptides for 6 hours, and the cells were harvested. Cells were lysed using a freeze-thaw cycle of -80℃ to 25℃ three times, and the supernatant was collected after centrifugation as the test sample. Following the instructions, the test sample was mixed with the reaction system and immediately placed in an automated microplate reader to measure the absorbance at 340 nm. The initial absorbance value and the absorbance value after 5 minutes of reaction were recorded, and the difference ΔA340 was calculated. Simultaneously, a background control well was set as required. The activity of the complex in the sample was calculated using the following formula: .

[0051] Meanwhile, the test sample was used as a negative control in accordance with the above method to remove non-specific signals from the sample.

[0052] Finally, the specific activity of the complex = the total activity of the complex - the non-specific activity.

[0053] The results are shown in Table 4. Ethanol (300 mM) significantly reduced the activity of mitochondrial respiratory chain complexes in C2C12 cells, while the oligopeptide LPF had a good protective effect on all five complexes and significantly enhanced their activity. Therefore, the oligopeptide provided by this invention can enhance respiratory chain activity, improve mitochondrial energy metabolism, and promote ATP production. Table 4. Activity of mitochondrial respiratory chain complex in C2C12 cells

[0054] Note: Compared to Control, *** p <0.001; compared to Ethanol, # p <0.05, ## p <0.01, ### p <0.001.

[0055] Example 4: Small molecule active oligopeptide LPF improves zebrafish embryonic motility. The inventors evaluated the kinesiogenic effect of the oligopeptide LPF using zebrafish as a model animal. Five-day-old post-fertilization zebrafish embryos were placed in 12-well plates, 25 embryos per well. A normal control group and three LPF dosage groups (10 μM, 100 μM, and 1 mM) were established. The normal control group was given zebrafish embryo culture medium, while the other groups were given zebrafish embryo culture medium containing the corresponding drug concentrations (H-Buffer, 59.88 mM NaCl, 2.281 mM NaHCO3, 0.6707 mM KCl, 1.059 mM CaCl2). The culture medium was changed every 24 hours, and the treatment lasted for 48 hours. The survival rate of the zebrafish was recorded. The results are as follows: Figure 4 As shown, LPF concentrations below 1 mM have almost no toxicity to zebrafish embryos. This demonstrates that LPF has a high safety profile and a wide therapeutic window.

[0056] Subsequently, based on the aforementioned safe dosage of LPF, the inventors established an ethanol loading model using zebrafish embryos to study the effect of LPF on ethanol-stimulated zebrafish embryos. Five-day-old zebrafish embryos were randomly selected and placed in 12-well plates, with 25 embryos per well and 3 replicates per group. Groups were set up as a normal group, an ethanol group, and two LPF dosage groups (10 μM and 100 μM). The normal group received embryo culture medium, while the other groups received 2% (v / v) ethanol. The treatment lasted 32 hours to establish the ethanol loading model. Afterward, the normal and ethanol groups were replaced with normal embryo culture medium, while the two LPF groups were given zebrafish culture medium at concentrations of 10 μM and 100 μM, respectively. The embryos were cultured for another 48 hours, and their motility was collected for analysis. The experimental results are as follows: Figure 5 As shown, ethanol significantly shortened the movement distance of zebrafish and inhibited the movement of zebrafish embryos, while administration of LPF significantly alleviated the movement disorders in zebrafish embryos. Furthermore, LPF also significantly increased the ATP content in zebrafish tissues (see Table 5), promoting energy biosynthesis. Therefore, the oligopeptides provided by this invention can enhance the contractility of muscle fibers and promote the movement of zebrafish embryos by improving energy metabolism within muscle cells.

[0057] Table 5. Effects of LPF on ATP levels in zebrafish treated with ethanol.

[0058] Note: Compared to Control, *** p <0.001; compared to Ethanol, # p <0.05.

[0059] Example 5 Mouse suspension experiment Ethanol has a significant muscle relaxant effect, leading to weakness in the limbs, which is particularly pronounced during acute alcohol poisoning. Therefore, the inventors also evaluated the effect of the oligopeptide LPF on ethanol-induced muscle relaxation and weakness in a mouse model.

[0060] Male 7-week-old Kunming mice (purchased from Guangdong Provincial Experimental Animal Center) were used in the experiment. The housing conditions were: 23±2℃, humidity 55±5%, lighting time 12 hours / day (7:00~19:00), and free access to water. The experiment was conducted after one week of housing.

[0061] Mice were randomly divided into a control group (Control), an ethanol model group (Ethanol), and an oligopeptide LPF treatment group (LPF), with 15 mice in each group. LPF (50 mg / kg) was administered by gavage 3 hours before the ethanol loading, while the other groups received an equal volume of physiological saline. The ethanol loading test was then performed as follows: a 25% ethanol solution was prepared and administered by gavage (12 mL / kg) to all groups except the control group. Suspension tests were conducted 1, 3, and 5 hours after the ethanol loading, and the suspension time of the mice was recorded. The suspension test was performed as follows: the mouse's forearm was placed on a horizontally open stainless steel wire with a diameter of 2 mm, and the suspension duration was measured; a measurement of less than 10 seconds was considered muscle relaxation.

[0062] The results are shown in Table 6. Compared with the normal control group, the suspension time of mice in the ethanol model group was shorter. After 1 hour of ethanol loading, 40% of the mice had a suspension time of less than 10 seconds, indicating a decrease in skeletal muscle contraction function. Even after 5 hours, 6.7% of the mice still had a suspension time of less than 10 seconds. In contrast, compared with the ethanol model group, the suspension time of mice in the oligopeptide LPF treatment group was significantly increased. After 1 hour of ethanol loading, only 20% of the mice had a suspension time of less than 10 seconds, and their recovery speed was higher than that of the model group. After 5 hours, all mice had a suspension time of more than 10 seconds. Therefore, oligopeptide LPF shortened the muscle tone recovery time in mice subjected to ethanol loading. Table 6 Suspension time of mice in each group

[0063] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. The application of small molecule active oligopeptides in the preparation of products for improving skeletal muscle dysfunction, characterized in that, The amino acid sequence of the small molecule active oligopeptide is Leu-Pro-Phe, and the skeletal muscle dysfunction is myasthenia gravis caused by acute or chronic alcohol poisoning.

2. The application according to claim 1, characterized in that, The product is a pharmaceutical product; The drug also contains necessary excipients.

Images