Heptapeptide with muscle building function as well as preparation and application of heptapeptide

Abstract

The invention discloses a heptapeptide with a muscle building function as well as preparation and application thereof, and belongs to the technical field of biological medicines. The amino acid sequence of the heptapeptide SR7 is Ser-Ser-Gly-Lys-Phe-Leu-Arg, and the amino acid sequence of the heptapeptide SR7 is as shown in the specification. The heptapeptide can be obtained through artificial synthesis or directional enzymolysis of wheat protein. Functional verification shows that the heptapeptide SR7 and the wheat peptide obtained through directional enzymolysis have the muscle building function which is mainly reflected in enhancing the exercise ability, including increasing the total exercise distance, the moving speed and the activeness frequency; and increase muscle strength and muscle mass. And the food-borne bioactive peptide is high in biological safety. Therefore, the compound can be applied to preparation of medicines for improving sarcopenia or health-care foods for increasing muscle. The invention provides a novel active substance with a muscle building function, and the novel active substance has a good market prospect and application potential.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a heptapeptide with muscle-building function, and a preparation method and application thereof. Background Art

[0002] Sarcopenia (also known as muscle loss, muscle attenuation syndrome, Sarcopenia) refers to the age-related loss of muscle mass, and there is also a decrease in muscle strength and / or a decline in physical function. According to the 2019 Asian Sarcopenia Working Guidelines, the assessment of sarcopenia mainly includes three aspects: ① muscle mass ② muscle strength and ③ muscle function performance. When ①+② or ①+③ or ①+②+③ are met, it can be concluded that sarcopenia has been suffered. Sarcopenia is an important cause of falls and disability and an inflection point for terminal functional decline. The incidence of sarcopenia increases with age. Therefore, strengthening the prevention and treatment of sarcopenia may be a very important task in the field of geriatric medicine.

[0003] Dietary protein has the most direct impact on muscle tissue protein synthesis, can provide the amino acids required for protein anabolism, and can stimulate protein anabolism. In recent years, bioactive peptides prepared from food proteins by enzymatic hydrolysis have become a hot topic in scientific research due to their good absorbability, unique physiological activity, and safety and non-toxic side effects.

[0004] For example, patent document CN118852406A discloses five ovalbumin peptides with muscle-building effects obtained by separating from ovalbumin enzymatic hydrolysis products, namely ADHPFLF (Leu-Ala-Asp-His-Pro-Phe-Leu-Phe), CPLSILT (Cys-Pro-Leu-Ser-Ile-Leu-Thr), SFSLASRL (Ser-Phe-Ser-Leu-Ala-Ser-Arg-Leu), SRPILPIYLK (Ser-Arg-Pro-Ile-Leu-Pro-Ile-Tyr-Leu-Lys) and YQIGLFR (Tyr-Gln-Ile-Gly-Leu-Phe-Arg).

[0005] Wheat peptides are made from wheat protein (gluten powder) through bio-enzymatic hydrolysis, refining, spray drying and other processes. Due to the rich biological functions of wheat peptides, they have important research value in improving sarcopenia in the elderly. At present, most of the research on the function of wheat peptides is on mixed peptides. In-depth exploration of efficient peptides with muscle-building function will provide new solutions for the intervention of sarcopenia. Summary of the invention

[0006] The purpose of the present invention is to provide a natural small molecule bioactive peptide with muscle-building effect, and to apply the peptide to the development of drugs or health foods for improving sarcopenia.

[0007] To achieve the above object, the present invention adopts the following technical solution: The present invention uses LC-MS / MS peptide mapping technology to analyze the peptide sequence in wheat peptides, and then uses molecular docking technology to explore the interaction between the peptide segment and fibroblast growth factor receptor 1 (FGFR1), and screens out a candidate peptide segment. After mass spectrometry identification, its amino acid sequence is Ser-Ser-Gly-Lys-Phe-Leu-Arg (SSGKFLR), and its molecular weight is 793.445 Da, which is named SR7. Further, through artificial synthesis of the heptapeptide SR7, functional verification found that the peptide segment has muscle-building function.

[0008] Therefore, the present invention provides a biologically active heptapeptide SR7, wherein the amino acid sequence of the heptapeptide SR7 is Ser-Ser-Gly-Lys-Phe-Leu-Arg.

[0009] The present invention also provides a method for preparing the heptapeptide SR7. The heptapeptide SR7 is prepared by solid phase synthesis. The specific method includes: adopting Fmoc solid phase synthesis strategy, using Fmoc protected amino acids as raw materials, selecting Wang resin as a solid phase carrier, sequentially introducing arginine, leucine, phenylalanine, lysine, glycine, serine, and serine residues to extend the peptide chain from the C-terminus to the N-terminus, and solid phase synthesizing the heptapeptide SR7.

[0010] The heptapeptide SR7 can also be obtained by enzymolysis of wheat gluten. Specifically, water and wheat protein (gluten powder) are added into a reactor at a mass ratio of 15:1. After the feeding is completed, the pH of the feed solution is adjusted to 8.0 ± 0.2, and alkaline protease (accounting for 1.0% of the total weight of gluten powder) is added for enzymolysis for 60 minutes. After the end, neutral protease (accounting for 1.5% of the total weight of gluten powder) is added for enzymolysis for 30 minutes. After the enzymolysis is completed, flavor protease (accounting for 0.5% of the total weight of gluten powder) is used for 30 minutes, and then the enzyme is inactivated at 100°C for 30 minutes. Subsequently, the wheat peptide powder is concentrated and dried to obtain the wheat peptide powder, which contains the heptapeptide SR7.

[0011] The present invention also provides the use of the heptapeptide SR7 in the preparation of a drug or muscle-building food for treating sarcopenia. The present invention's research shows that the heptapeptide SR7 has a muscle-building effect. In a sarcopenia model, intervention with the heptapeptide SR7 can significantly improve athletic ability, which is mainly reflected in the significant increase in total movement distance, movement speed, and activity frequency, as well as a significant increase in activity and a significant increase in activity range. Therefore, it can be applied to the development of related products for improving sarcopenia or increasing muscle mass and muscle strength.

[0012] Furthermore, the manifestations of sarcopenia include: decreased athletic ability, decreased muscle mass and muscle strength.

[0013] Another object of the present invention is to provide a wheat peptide with muscle-building function. The preparation method of the wheat peptide comprises: mixing water and wheat gluten at a mass ratio of 15-20:1, adjusting the pH of the slurry to 8.0±0.2, adding 1.0% of alkaline protease accounting for the total weight of the gluten for enzymolysis for 60 minutes, then adding 1.5% of neutral protease accounting for the total weight of the gluten for enzymolysis for 30 minutes, then adding 0.5% of flavor protease accounting for the total weight of the gluten for 30 minutes, and finally inactivating the enzyme, concentrating, and drying to obtain the wheat peptide.

[0014] The wheat peptide contains the heptapeptide SR7. The research of the present invention shows that the wheat peptide has a significant muscle-building effect. In a sarcopenia model, after intervention with the wheat peptide, muscle strength and muscle mass can be significantly improved, and blood lipid levels can be significantly improved to regulate lipid metabolism.

[0015] Therefore, the present invention provides the use of the wheat peptide in the preparation of a medicine for treating sarcopenia or a muscle-building food.

[0016] The present invention provides a pharmaceutical composition for treating sarcopenia, comprising an effective dose of heptapeptide SR7 or the wheat peptide, wherein the amino acid sequence of the heptapeptide SR7 is Ser-Ser-Gly-Lys-Phe-Leu-Arg.

[0017] The heptapeptide SR7 or the wheat peptide in the pharmaceutical composition provided by the present invention can be used as the only active ingredient that exerts a muscle-building effect, or can be compounded with other active ingredients with muscle-building function. The active ingredients with muscle-building function can be, but are not limited to, plant extracts, probiotics and other protein peptides.

[0018] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is any preparation or carrier medium that can deliver an effective dose of the active substance of the present invention, does not interfere with the biological activity of the active substance, and has no toxic side effects on the host or subject.

[0019] Furthermore, the pharmaceutically acceptable carrier includes: one or more of a filler, a wetting agent, a disintegrant, a binder or a lubricant.

[0020] The present invention uses the heptapeptide SR7 or the wheat peptide as a main active ingredient, adds a pharmaceutically acceptable carrier, and prepares a preparation according to a preparation preparation method recorded in pharmacy.

[0021] Furthermore, the pharmaceutical composition may be in the form of, but not limited to, an oral preparation. Specifically, the pharmaceutical composition may be in the form of, but not limited to, an oral liquid, a capsule, a microcapsule powder, a tablet, a granule, or an emulsion.

[0022] The present invention also provides a health food for muscle growth, comprising heptapeptide SR7 or the wheat peptide as an active ingredient, and a food-based acceptable auxiliary material, wherein the amino acid sequence of the heptapeptide SR7 is Ser-Ser-Gly-Lys-Phe-Leu-Arg. The food-based acceptable auxiliary material is a food-grade auxiliary material that can deliver an effective dose of the active substance of the present invention and does not interfere with the biological activity of the active substance.

[0023] Furthermore, the dosage form of the health food is beverage, oral liquid, capsule, microcapsule powder, tablet, granule or emulsion.

[0024] The present invention has the following beneficial effects: The present invention provides a heptapeptide SR7 with muscle-building function and a wheat peptide containing the peptide segment, and the peptide segment can be obtained by artificial synthesis or directed enzymatic hydrolysis of wheat protein. Functional verification of animal models shows that the heptapeptide SR7 and the wheat peptide containing the peptide segment have muscle-building function, which is mainly reflected in enhancing athletic ability, including increasing total movement distance, movement speed and activity frequency; as well as increasing muscle strength and muscle mass. Moreover, food-borne bioactive peptides have high biological safety. Therefore, the heptapeptide SR7 and the wheat peptide containing the peptide segment can be used to prepare drugs for improving sarcopenia or health foods for muscle building. The present invention provides a new active substance with muscle-building function, which has good market prospects and application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The effect of wheat peptide containing SR7 on the grip strength of sarcopenic mice.

[0026] Figure 2 The effect of wheat peptide containing SR7 on liver index of sarcopenic mice.

[0027] Figure 3 The effect of wheat peptide containing SR7 on the renal index of sarcopenic mice.

[0028] Figure 4This is the effect of wheat peptide containing SR7 on the gastrocnemius muscle index of sarcopenic mice.

[0029] Figure 5 The effect of wheat peptide containing SR7 on the soleus index of sarcopenic mice.

[0030] Figure 6 The effect of wheat peptide containing SR7 on total cholesterol in the serum of sarcopenic mice.

[0031] Figure 7 The effect of wheat peptide containing SR7 on serum triglycerides in sarcopenic mice.

[0032] Figure 8 The effect of wheat peptide containing SR7 on low-density lipoprotein cholesterol in the serum of sarcopenic mice.

[0033] Fig. 9 The effect of wheat peptide containing SR7 on high-density lipoprotein cholesterol in the serum of sarcopenic mice.

[0034] Fig.10 To compare the effects of wheat peptides and wheat oligopeptides on arm muscle strength.

[0035] Fig.11 This is the secondary mass spectrum of the heptapeptide SR7.

[0036] Fig.12 Schematic diagram of the binding of the heptapeptide SR7 to FGFR1.

[0037] Fig.13 To compare the effects of heptapeptide SR7 and hexapeptide QR6 on the movement distance of zebrafish.

[0038] Fig.14 To compare the effects of heptapeptide SR7 and hexapeptide QR6 on the movement speed of zebrafish.

[0039] Fig.15 To compare the effects of heptapeptide SR7 and hexapeptide QR6 on the activity frequency in zebrafish locomotor ability.

[0040] Fig.16 To compare the effects of heptapeptide SR7 and hexapeptide QR6 on the movement trajectory of zebrafish.

[0041] Fig.17 To compare the effects of heptapeptide SR7 and wheat peptide on the movement distance of zebrafish.

[0042] Fig.18 To compare the effects of heptapeptide SR7 and wheat peptide on the movement speed of zebrafish.

[0043] Fig.19To compare the effects of heptapeptide SR7 and wheat peptide on the activity frequency in zebrafish locomotor ability.

[0044] Fig. 20 To compare the effects of heptapeptide SR7 and wheat peptide on the movement trajectory of zebrafish. DETAILED DESCRIPTION

[0045] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the inventive method, steps or conditions all belong to the scope of the present invention.

[0046] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0047] Wheat protein (gluten powder) was purchased from Binzhou Zhongyu Food Co., Ltd.; alkaline protease (derived from Bacillus licheniformis) was purchased from Angel Enzyme (Yichang) Co., Ltd.; neutral protease (derived from Bacillus subtilis) was purchased from Nanning Pangbo Bioengineering Co., Ltd.; flavor protease was purchased from Angel Enzyme (Yichang) Co., Ltd. Wheat oligopeptides were purchased from Zhongshi Duqing (Shandong) Biotechnology Co., Ltd.

[0048] Example 1: Preparation of wheat peptides Water and wheat protein (gluten) were added into the reactor at a mass ratio of 15:1. After the feeding was completed, the pH of the feed solution was adjusted to 8.0 ± 0.2, and alkaline protease (accounting for 1.0% of the total weight of gluten) was added for enzymatic hydrolysis for 60 min. After the end, neutral protease (accounting for 1.5% of the total weight of gluten) was added for enzymatic hydrolysis for 30 min. After the enzymatic hydrolysis was completed, flavor protease (accounting for 0.5% of the total weight of gluten) was used for 30 min, and then the enzyme was inactivated at 100 ° C for 30 min. The feed solution was then concentrated and spray-dried to obtain wheat peptide powder.

[0049] Example 2: Effects of wheat peptides on sarcopenia in mice (1) Mouse experiment design SPF male C57BL / 6 mice aged 2 months (weighing about 20 g) were used in the experiment. All procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals of Zhejiang Chinese Medical University and approved by the Animal Ethics Committee of Zhejiang Chinese Medical University (No. 20231204-05). After one week of adaptive feeding, 36 mice were randomly divided into 3 groups (12 mice in each group): ① blank control (NC) group, ② model (MC) group, and ③ wheat peptide (0.70 mg / g, WP) group. The experiment lasted for 6 weeks. In the first 3 weeks, mice in the MC and WP groups were subcutaneously injected with dexamethasone solution (5 mg / kg) at a fixed time every day, and the NC group was given sterile saline for 3 consecutive weeks (volume 0.1 mL / 10g). After 3 weeks, the WP group was gavaged with wheat peptide (0.70 mg / g), and the NC and MC groups were gavaged with the same volume of sterile saline (volume 0.1 mL / 10g) for 3 consecutive weeks. The body weight and food intake of mice were recorded at a fixed time every week during the experiment.

[0050] (2) Mouse grip strength measurement After the test, the mouse forelimb grasping strength was assessed using a mouse grasping strength tester. During the test, the mouse's tail was lifted and induced to grasp a rigid grid attached to a digital force gauge. The tail of each mouse was gently pulled back, and the tension reading of the digital force gauge was defined as the grasping force before the mouse released the grid. Three consecutive tests were performed for each mouse, and the average grasping force value was calculated.

[0051] (3) Collection of serum and tissue samples After the experiment, the mice were fasted but not watered for 12 h, blood was collected from the eyeballs, and after standing for 1-2 h until stratification, the blood was centrifuged in a low-temperature high-speed centrifuge for 15 min (4°C, 3000 rpm) to separate the serum, which was stored at -80°C for later use. After blood collection, the mice were killed by cervical dislocation, and the liver, kidney, gastrocnemius and soleus muscle tissues were accurately removed and weighed to calculate the organ ratio. The samples were then stored in an ultra-low temperature refrigerator at -80°C.

[0052] (4) Determination of biochemical indicators Serum total cholesterol (T-CHO), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were measured according to the kit instructions (Nanjing Jiancheng Bioengineering Institute Kit).

[0053] (5) Results analysis Muscle atrophy often leads to reduced muscle mass and impaired muscle function. Skeletal muscle mass is mainly regulated by the balance between muscle protein synthesis and muscle protein degradation. When this balance is broken, muscle mass loss occurs and may lead to muscle atrophy. Muscle mass and muscle strength occupy a very important core position in the evaluation of sarcopenia. Therefore, the effect of wheat peptides on the grip strength of sarcopenic mice was determined. The results are as follows: Figure 1 Compared with the NC group, the grasping force of mice in the MC group was significantly reduced by 18.00% ( P <0.001). Compared with the MC group, the grasping strength of mice in the WP group was significantly increased by 20.49% ( P <0.001), indicating that WP intervention may have a better effect in improving muscle strength.

[0054] The results of wheat peptides on the organ indexes of sarcopenic mice are as follows Figure 2-Figure 5 As shown in Figure 2, there were no significant differences in liver index and kidney index among the NC, MC, and WP groups ( P >0.05), indicating that dexamethasone had no significant effect on the liver and kidneys, and WP intervention had no significant effect on the liver and kidneys. Compared with the MC group, the gastrocnemius index and soleus index of the WP group increased by 45.79% ( P =0.3312) and 112.03% ( P <0.01), indicating that WP intervention also has a good effect in improving muscle mass.

[0055] Metabolic disorders and metabolites of lipids in muscles may exacerbate the reduction of skeletal muscle mass. Therefore, the effects of wheat peptides on blood lipid levels in sarcopenic mice were determined. Figure 6-Figure 9 There was no significant difference in TCHO, TG and LDL-C levels among the NC, MC and WP groups ( P >0.05). Compared with the NC group, the HDL-C level in the MC group was significantly reduced by 15.47% ( P <0.05); compared with the MC group, the HDL-C level in the WP group increased significantly by 21.14% ( P <0.01). The results showed that wheat peptide intervention could significantly improve the blood lipid level of sarcopenic mice and regulate lipid metabolism.

[0056] Example 3: Comparison of wheat peptides and wheat oligopeptides on mouse arm muscle strength (1) Mouse experiment design The experiment selected 48 ICR male mice, weighing 20 ± 2g, free access to standard pellet feed and drinking water, maintaining an ambient temperature of 25 ± 2℃, and using them after adaptive feeding for one week under a light cycle of 12h:12h. The experiment was approved by the Animal Ethics Committee of Zhejiang Chinese Medical University (No. 20230206-07). The mice were randomly divided into 3 groups, namely, a blank group (CON), a wheat peptide group (WP), and a wheat oligopeptide (WOP) group, with 16 mice in each group, 4 mice in a cage, and 4 cages in each group. The wheat peptide group with a concentration of 0.1 mg / g of wheat peptide prepared in Example 1 was gavaged daily, and the wheat oligopeptide group was given 0.1 mg / g of wheat oligopeptide. The blank group was gavaged with the same volume of sterile saline (gavage volume of 0.01 mL / g. bw) daily for 30 consecutive days.

[0057] (2) Grip force measurement On the 29th day of the experiment, the grip strength of the mouse forelimbs was tested using a grip strength test system (Model-RX-5, Aikoh Engineering, Nagoya, Japan). When the mouse's forepaws grasped the available grid in the instrument, the mouse's tail was lifted and gently pulled upward, and the instrument automatically displayed the maximum force value. The grip strength of the forelimbs of each mouse was measured three times and the average value was taken.

[0058] Grip strength mainly depends on the strength of the forearm flexor and extensor muscles. The force-generating parts of grip strength mainly include the forearm flexor and extensor muscles, in addition to the wrist flexor, superficial flexor and deep flexor muscles. To a certain extent, the arm muscle strength can be evaluated by the size of the grip strength. The results of comparing wheat peptides and wheat oligopeptides on the arm muscle strength of mice are as follows: Fig.10 As shown in the figure, compared with the blank group (CON) and wheat oligopeptide (WOP), wheat peptide (WP) significantly improved the grip strength ( P <0.001). This indicates that wheat peptides are significantly better than wheat oligopeptides in improving arm muscle strength.

[0059] Example 4: Screening of active peptides In this example, wheat peptides were subjected to LC-MS / MS peptide spectrum analysis, and the peptide segments of wheat peptides were sorted according to the screening conditions, and then molecular docking was further used to determine the final theoretical effective peptide segments according to the scores. The specific analysis process is as follows: (1) Wheat peptide sequence identification The wheat peptide sample was dissolved in NH4HCO3 solution, dithiothreitol solution was added, and the sample was reduced in a 56 ℃ water bath for 1 h. Then iodoacetamide solution was added and reacted for 40 min in the dark. After desalting, the solvent was evaporated, and then 10 μL of mobile phase A (0.1% formic acid) was used to dissolve the sample into a liquid injection bottle. LC-MS / MS analysis was then performed.

[0060] Chromatographic conditions: analytical column (Acclaim PepMap RPLC C18, 150×150 mm, 3 μm); mobile phase A (0.1% formic acid); mobile phase B (0.1% formic acid and 80% acetonitrile); flow rate (600 nL / min). Gradient elution program: 0-2 min, 4% B -8% B; 2-45 min, 8% B-40% B; 45-55 min, 40% B-60% B; 55-56 min, 60% B-95% B; 56-66 min, 95% B.

[0061] Mass spectrometry conditions: Full scan MS using Orbitrap for primary scanning, scan range (100-1500 m / z), resolution (70,000), maximum ion introduction time (100 ms), automatic gain control (3×10 6 ); The top 20 parent ions that met the MS / MS fragmentation conditions were fragmented using high-energy collision dissociation and scanned with Orbitrap, with a resolution of 17,500, a maximum ion introduction time of 50 ms, and automatic gain control (1×10 5 The raw data obtained by mass spectrometry were analyzed for peptide sequence using PEAKSStudio's De novo (de novo sequencing) software.

[0062] (2) Screening of active peptides with potential muscle-building function Fibroblast growth factor receptor 1 (FGFR1) is a transmembrane protein composed of an extracellular domain, a transmembrane domain and an intracellular domain, and belongs to the receptor tyrosine kinase family. FGFR1 plays an important role in muscle development (such as promoting muscle cell hypertrophy and differentiation) and muscle regeneration. Therefore, the wheat peptide segment was molecularly docked with FGFR1 to screen small molecule peptide segments that may have muscle hypertrophy function.

[0063] According to the average local confidence (ALC) greater than 95%, the peak area greater than 2×10 6 , PeptideRanker score greater than 0.8 was used to screen and obtain qualified peptides. The peptides were then docked with fibroblast growth factor receptor 1 (5AM6).

[0064] First, the crystal structure of FGFR1 (5AM6) was downloaded from the PDB protein database. The active center was defined by removing water molecules and adding hydrogen atoms from the receptor target using Discovery Studio software. The structure of the peptide segment of the selected wheat peptide was constructed by Discovery Studio, and its energy was minimized by the CHARMm force field, and these peptides were defined as ligands. The constructed peptide was docked with FGFR1 by CDOCKER to simulate the binding mode, site and amino acid residues with the lowest binding energy and the highest binding degree, and screened according to the binding energy and the number of hydrogen bonds. Finally, one heptapeptide (SSGKFLR) and one hexapeptide (QWLQPR) were determined, as shown in Table 1. The target peptide content of the wheat peptide prepared in Example 1 was determined, and the SSGKFLR content (on a dry basis) was 8 mg / 100 g.

[0065] Table 1 Peptides with potential muscle-building function in wheat peptides Peptide sequence ALC score (%) length Mass-to-charge ratio (m / z) Charge number (z) Relative abundance Molecular weight (Da) PeptideRanker Score Docking energy (kcal / mol) SSGKFLR 98.9 7 397.728 2 4.24E+06 793.445 0.76 -99.04 QUR 96.9 6 414.228 2 2.34E+06 826.445 0.77 -68.82 The secondary mass spectrum of the heptapeptide SR7 is as follows Fig.11 As shown in the figure, the fragmentation ions of peptides include: N-terminal fragment ions (a, b, c types) and C-terminal fragment ions (x, y, z types). The side chains of a, y, and z-type ions break to form d, v, and w-type ions, respectively. In addition, there are internal ions formed by the breakage of both ends. The b and y series ions are the most common. The primary structure of the peptide can be analyzed and inferred based on the b or y series fragment ions of the peptide. 397.728 m / z is the [M+H] + The ion signal has a charge number (z) of 2 and a molecular weight of 793.445 Da. The heptapeptide was further analyzed by secondary mass spectrometry using in-source collision-induced dissociation technology (e.g. Fig.10 As shown in FIG. 1 ), the primary structure of the heptapeptide was determined to be Ser-Ser-Gly-Lys-Phe-Leu-Arg.

[0066] The 2D and 3D images of the molecular docking of SR7 and FGFR1 are shown in the figure. Fig.12As shown. Analysis of chemical bonds revealed that SR7 was mainly bound to FGFR1 through van der Waals forces, hydrogen bonds (including conventional hydrogen bonds and carbon-hydrogen bonds), and electrostatic interactions (salt bridges and attractive charges), with a docking energy of -99.04 kcal / mol. SR7 formed four van der Waals forces with amino acid residues ASN568, GLY567, LEU494, and ALA564, 12 hydrogen bonds with GLU571, LYS566, LYS482, SER565, GLU633, LYS510, TYR563, ASP634, and THR632, and five hydrophobic interactions with GLU571, LYS482, GLU633, and ASP634.

[0067] The molecular docking results show that SR7 can bind to FGFR1 at the molecular docking level and has potential muscle-building function.

[0068] Shenzhen Borunsida Biotechnology Co., Ltd. was commissioned to synthesize peptides SSGKFLR and QWLQPR with a purity of ≥98% for subsequent functional verification.

[0069] Example 5: Comparison of the muscle-building effects of heptapeptide SR7 and hexapeptide QR6 on zebrafish sarcopenia Dexamethasone is a synthetic glucocorticoid with anti-inflammatory, anti-allergic and anti-shock effects. However, long-term injection of dexamethasone can cause side effects such as weight gain, muscle atrophy and cardiac fat accumulation. Currently, dexamethasone is widely used to establish animal models of muscle atrophy. Changes in motor behavior are the main indicator of muscle capacity.

[0070] (1) Zebrafish husbandry and embryo collection Wild-type AB strain zebrafish were used in this study. AB zebrafish (4 months old) were kept in a circulating system water at 28 ± 0.5 ℃ and pH 7.0-7.5, with a photoperiod of 14 h light / 10 h dark, and were fed brine shrimp twice a day. Healthy adult zebrafish were placed in a special fish tank in the evening of the previous day at a ratio of 1:1 between male and female, with the water level at about 2 / 3, and the male and female fish were separated by a partition. At about 8 am the next day, the partition was removed to allow the zebrafish to fertilize freely and obtain zebrafish embryos, which were 0 hpf at this time. After removing impurities from the embryos, they were placed in a culture dish filled with methylene blue system water, and the culture dish was placed in a constant temperature incubator at 28 ± 0.5 ℃ for culture.

[0071] (2) Zebrafish grouping and drug administration 4 dpf zebrafish were selected and placed in a six-well plate and divided into 7 groups, with 30 fish in each well. The specific groups were blank (NC), model (MC), SR7 (5 μg / mL, SR7) and QR6 (5 μg / mL, QR6). The blank group was added with the same volume of system water, the model group was added with 2.5 μmol / L dexamethasone, and the sample group was added with the corresponding dose of sample and dexamethasone. The culture was continued in a constant temperature incubator at 28 ± 0.5 ℃ for 4 days, and the medium was changed every day. The zebrafish larvae were not fed during the culture process.

[0072] (3) Exercise capacity measurement After the intervention, the zebrafish were transferred to a 96-well cell culture plate, with one fish per well, and then placed in a soundproof box (temperature set at 28 °C) for 5 min of adaptation before the experiment officially began. The DanioVisionTracking System was used to collect the movement of zebrafish within 20 min (5 min of light and 5 min of darkness). The total movement distance, average speed, activity frequency, and cumulative duration of activity of zebrafish were calculated using Noldus behavioral analysis software.

[0073] (4) Data analysis This study used Graphpad Prism 8.0 software for statistical analysis and plotting, and all data were expressed as mean ± standard error. One-way analysis of variance was used to compare the differences between the groups (compared with the NC group, * P <0.05, ** P <0.01, *** P <0.001; compared with the MC group, # P <0.05, ## P <0.01, ### P <0.001).

[0074] (5) Results analysis Comparison of the effects of heptapeptide SR7 and hexapeptide QR6 on the motor ability of zebrafish Figure 13-Figure 16 Compared with the NC group, the total movement distance, movement speed and activity frequency of the MC group were significantly reduced ( P <0.001, P <0.001, P <0.01), indicating that the sarcopenia model was successfully established. Compared with the MC group, the total exercise distance, movement speed and activity frequency of the QR6 group increased by 9.41%, 7.01% and 4.50%, respectively, but none of them reached a significant level (P >0.05); the total movement distance, movement speed and activity frequency of the SR7 group increased significantly by 31.19% ( P <0.001), 22.08% ( P <0.001) and 32.69% ( P <0.05), indicating that SR7 has a muscle-building effect. Analysis of zebrafish movement trajectories further proved that the activity and range of activity of zebrafish increased significantly after SR7 intervention.

[0075] Based on the above data, it can be seen that the heptapeptide SR7 has a muscle-building effect from the perspective of zebrafish's athletic ability.

[0076] Example 6: Comparison of the muscle-building effects of heptapeptide SR7 and wheat peptide on zebrafish sarcopenia The experimental method was the same as that of "Comparison of the muscle-building effects of heptapeptide SR7 and hexapeptide QR6 on zebrafish sarcopenia", and was divided into 7 groups, with 30 fish in each well. The specific groups were blank group (NC), model group (MC), wheat peptide low-dose (10 μg / mL, WP-10) and high-dose (100 μg / mL, WP-100) groups, and SR7 low-dose (1 μg / mL, SR7-1), medium-dose (5 μg / mL, SR7-5) and high-dose (10 μg / mL, SR7-10) groups.

[0077] Comparison of the effects of heptapeptide SR7 and wheat peptide on the motor ability of zebrafish Figure 17-Figure 20 Compared with the NC group, the total movement distance, movement speed and activity frequency of the MC group were significantly reduced by 26.12%, 23.92%, 39.68% and 40.88%, respectively (all P <0.001), indicating that the sarcopenia model was successfully established. Compared with the MC group, the total movement distance, movement speed and activity frequency of the 10 μg / mL wheat peptide group increased by 14.77%, -0.82% and 23.64%, respectively, but none of them reached a significant level ( P >0.05); the above indicators in the 100 μg / mL wheat peptide group increased significantly by 24.54% ( P <0.01), 24.11% ( P <0.05) and 36.78% ( P <0.01), indicating that 100 μg / mL wheat peptide has a muscle-building effect. The above indicators in the 1 μg / mL SR7 group increased by 11.52%, 16.32% and 25.45%, respectively, but none of them reached a significant level ( P >0.05); the above indicators in the 5 μg / mL SR7 group increased by 21.23% ( P <0.01), 23.59% ( P<0.05) and 41.22% ( P <0.01), and the above indicators in the 10 μg / mL SR7 group increased by 33.86% ( P <0.001), 28.22% ( P <0.01) and 47.71% ( P <0.05), indicating that 5 μg / mL and 10 μg / mL SR7 have a muscle-building effect. Analysis of zebrafish movement trajectories further proved that zebrafish activity and range of activity increased significantly after intervention with 100 μg / mL wheat peptide, 5 μg / mL SR7, and 10 μg / mL SR7.

[0078] Based on the above data, it can be seen that from the perspective of zebrafish's athletic ability, including total movement distance, movement speed and activity frequency, the muscle-enhancing effect of the heptapeptide SR7 is about 20 times that of wheat peptide.

[0079] In summary, the wheat peptide provided by the present invention has a good effect on improving muscle strength and muscle quality, and can regulate lipid metabolism at the same time. Its effect of improving muscle strength is significantly better than that of wheat oligopeptides. The heptapeptide SSGKFLR (SR7) was further screened from the wheat peptide by mass spectrometry identification and molecular docking of the peptide segment. The peptide segment showed an efficient muscle-enhancing effect in the sarcopenia model, which was mainly reflected in enhancing athletic ability, including increasing total exercise distance, movement speed and activity frequency. The present invention provides a theoretical basis for the development of new active substances with muscle-enhancing functions, and can be applied to the preparation of drugs or muscle-enhancing foods for improving sarcopenia. The heptapeptide SSGKFLR can be used alone to prepare drugs or foods with muscle-enhancing functions, or it can be compounded with other active substances with muscle-enhancing functions. The active components with muscle-enhancing functions may include plant extracts, probiotics and other protein peptides. The food or drug can be prepared into microcapsules to improve the gastrointestinal digestion stability, bioavailability and shelf life of the polypeptide, so that it can be better applied to the food industry and health care fields.

Claims

1. A biologically active heptapeptide SR7, characterized in that: The amino acid sequence of the heptapeptide SR7 is Ser-Ser-Gly-Lys-Phe-Leu-Arg.

2. The method for preparing the heptapeptide SR7 according to claim 1, characterized in that: The heptapeptide SR7 is prepared by solid phase synthesis; or obtained by enzymatic hydrolysis of wheat gluten protein.

3. Use of the heptapeptide SR7 according to claim 1 in the preparation of a drug for treating sarcopenia or a muscle-building food, characterized in that: The heptapeptide SR7 has a muscle-building effect.

4. The use according to claim 3, characterized in that The manifestations of sarcopenia include decreased athletic ability, decreased muscle mass and muscle strength.

5. A wheat peptide with muscle-building function, characterized in that: The wheat peptide contains the heptapeptide SR7 as claimed in claim 1. The preparation method of the wheat peptide comprises: mixing water and wheat gluten at a mass ratio of 15-20:1, adjusting the pH of the slurry to 8.0±0.2, adding 1.0% of alkaline protease accounting for the total weight of the gluten for enzymolysis for 60 minutes, then adding 1.5% of neutral protease accounting for the total weight of the gluten for enzymolysis for 30 minutes, then adding 0.5% of flavor protease accounting for the total weight of the gluten for 30 minutes, and finally inactivating the enzyme, concentrating and drying to obtain the wheat peptide.

6. The use of the wheat peptide according to claim 5 in the preparation of a drug for treating sarcopenia or a muscle-building food, characterized in that: The wheat peptide has a muscle-building effect.

7. A pharmaceutical composition for treating sarcopenia, characterized in that: The pharmaceutical composition comprises an effective dose of heptapeptide SR7 or wheat peptide containing heptapeptide SR7, and the amino acid sequence of the heptapeptide SR7 is Ser-Ser-Gly-Lys-Phe-Leu-Arg.

8. The pharmaceutical composition according to claim 7, characterized in that Also included are pharmaceutically acceptable carriers.

9. The pharmaceutical composition according to claim 7, characterized in that The pharmaceutical composition is in the form of an oral preparation.

10. A health food for muscle growth, characterized in that: The invention comprises heptapeptide SR7 or wheat peptide containing heptapeptide SR7 as an active ingredient, and excipients acceptable in food science, wherein the amino acid sequence of the heptapeptide SR7 is Ser-Ser-Gly-Lys-Phe-Leu-Arg.

Images