A heptapeptide with muscle-building function, its preparation and application

Abstract

The present invention discloses a heptapeptide with muscle-building function, its preparation and application, belonging to the technical field of biomedicine. The amino acid sequence of the heptapeptide SR7 is Ser-Ser-Gly-Lys-Phe-Leu-Arg. This heptapeptide can be obtained by artificial synthesis or directional enzymatic hydrolysis of wheat protein. Functional verification shows that the heptapeptide SR7 and the wheat peptides obtained by directional enzymatic hydrolysis have muscle-building functions, which are mainly reflected in enhancing exercise ability, including increasing the total exercise distance, movement speed and activity frequency; and increasing muscle strength and muscle mass. Moreover, food-derived bioactive peptides have high biosafety. Therefore, it can be applied to the preparation of drugs for improving sarcopenia. The present invention provides a new active substance with muscle-building function, having good market prospects and application potential.

Description

Technical Field

[0001] The present invention relates to the field of biological medicine technology, and particularly relates to a heptapeptide with muscle-building function, its preparation method and application. Background Art

[0002] Sarcopenia (also known as muscle loss, muscle attenuation syndrome), refers to age-related reduction in muscle mass, and there must also be a decrease in muscle strength and / or decline in physical function. According to the 2019 Asian Sarcopenia Working Group guidelines, the assessment of sarcopenia mainly includes three aspects: ① muscle mass, ② muscle strength, and ③ muscle function performance. When meeting ①+② or ①+③ or ①+②+③, it can be determined that sarcopenia has occurred. Sarcopenia is an important inducement for falls and disability and an inflection point for the decline of end-stage function. The occurrence of sarcopenia increases with aging. Therefore, strengthening the prevention and treatment of sarcopenia may be a very important task in the field of geriatrics.

[0003] Dietary protein has the most direct impact on muscle tissue protein synthesis, can provide the amino acids required for protein anabolic metabolism, and can stimulate protein anabolic metabolism. 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 activities, and the characteristics of being safe and non-toxic.

[0004] For example, patent document CN118852406A discloses 5 ovalbumin peptides with muscle-building effects isolated from ovalbumin protease 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 processes such as biological enzymatic hydrolysis, refinement, and spray drying. Due to the rich biological functions of wheat peptides, they have important research value in improving age-related muscle loss. Currently, most of the research on the functions of wheat peptides is for mixed peptides. Deeply exploring highly effective peptide segments with muscle-building functions 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 effects and apply it to the development of drugs for improving sarcopenia.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] The present invention uses LC-MS / MS peptide mapping analysis technology to analyze the peptide sequences in wheat peptides, and then uses molecular docking technology to explore the interaction between peptide segments and fibroblast growth factor receptor 1 (FGFR1). A candidate peptide segment is screened out. 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. It is named SR7. Further, the heptapeptide SR7 is synthesized artificially, and functional verification shows that this peptide segment has the function of increasing muscle mass.

[0009] Therefore, the present invention provides a biologically active heptapeptide SR7, and the amino acid sequence of the heptapeptide SR7 is Ser-Ser-Gly-Lys-Phe-Leu-Arg.

[0010] 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 the Fmoc solid-phase synthesis strategy, using Fmoc-protected amino acids as raw materials, selecting Wang resin as the solid-phase carrier, and introducing arginine, leucine, phenylalanine, lysine, glycine, serine, and serine residues in sequence to extend the peptide chain from the C-terminus to the N-terminus, and solid-phase synthesizing the heptapeptide SR7.

[0011] The heptapeptide SR7 can also be obtained by enzymatic hydrolysis of wheat gluten. Specifically, according to the mass ratio of water to wheat protein (gluten powder) of 15:1, it is put into a reaction kettle. After the feeding is completed, the pH of the feed liquid is adjusted to 8.0 ± 0.2, and then alkaline protease (accounting for 1.0% of the total weight of gluten powder) is added for enzymatic hydrolysis for 60 min. After that, neutral protease (accounting for 1.5% of the total weight of gluten powder) is added for enzymatic hydrolysis for 30 min. After the enzymatic hydrolysis is completed, flavor protease (accounting for 0.5% of the total weight of gluten powder) is used for 30 min, and then it is placed at 100 °C for 30 min to inactivate the enzyme. Subsequently, it is concentrated and dried to obtain wheat peptide powder, which contains the heptapeptide SR7.

[0012] The present invention also provides the application of the heptapeptide SR7 in the preparation of drugs for treating sarcopenia. The research of the present invention shows that the heptapeptide SR7 has the effect of increasing muscle mass. In the sarcopenia model, after the intervention of the heptapeptide SR7, the exercise ability can be significantly improved, which is mainly reflected in the significant increase in the total exercise distance, moving speed, and activity frequency, as well as the significant increase in activity and the obvious increase in the activity range. Therefore, it can be applied to the development of related products for improving sarcopenia or increasing muscle mass and muscle strength.

[0013] Furthermore, the manifestations of sarcopenia include: decreased motor ability, decreased muscle mass, and decreased muscle strength.

[0014] Another object of the present invention is to provide a wheat peptide having a muscle-building function. The preparation method of the wheat peptide includes: mixing water and wheat gluten in a mass ratio of 15-20:1, adjusting the pH of the material liquid to 8.0±0.2, adding alkaline protease accounting for 1.0% of the total weight of gluten and enzymolyzing for 60 min, then adding neutral protease accounting for 1.5% of the total weight of gluten and enzymolyzing for 30 min, then adding flavor protease accounting for 0.5% of the total weight of gluten and acting for 30 min, and finally inactivating the enzyme, concentrating, and drying to obtain the wheat peptide.

[0015] The wheat peptide contains the heptapeptide SR7 described above. The research of the present invention shows that the wheat peptide has a significant muscle-building effect. In the sarcopenia model, after the wheat peptide intervention, it can significantly improve muscle strength and muscle mass, and significantly improve blood lipid levels and then regulate lipid metabolism.

[0016] Therefore, the present invention provides the application of the wheat peptide described above in the preparation of a drug for treating sarcopenia.

[0017] The present invention provides a pharmaceutical composition for treating sarcopenia. The pharmaceutical composition includes an effective dose of heptapeptide SR7 or the wheat peptide. The amino acid sequence of the heptapeptide SR7 is Ser-Ser-Gly-Lys-Phe-Leu-Arg.

[0018] In the pharmaceutical composition provided by the present invention, the heptapeptide SR7 or the wheat peptide can be used as the only active ingredient for exerting the muscle-building effect, or can be compounded with other active ingredients having a muscle-building function. The active components having a muscle-building function can be, but are not limited to, plant extracts, probiotics, and other protein peptides.

[0019] Furthermore, the pharmaceutical composition further includes a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is any preparation or carrier medium that can deliver the 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 or side effects on the host or subject.

[0020] Furthermore, the pharmaceutically acceptable carrier includes one or more of fillers, wetting agents, disintegrants, binders, or lubricants.

[0021] The present invention uses the heptapeptide SR7 or the wheat peptide as the main active ingredient, adds a pharmaceutically acceptable carrier, and prepares a preparation according to the preparation method of the preparation recorded in pharmacy.

[0022] Furthermore, the pharmaceutical composition can be in a dosage form of, but not limited to, an oral preparation. Specifically, the dosage form can be, but not limited to, oral liquid, capsule, microcapsule powder, tablet, granule or emulsion.

[0023] Advantages of the present invention:

[0024] The present invention provides a heptapeptide SR7 with muscle-building function and a wheat peptide containing this peptide segment, which can be obtained by artificial synthesis or directional enzymatic hydrolysis of wheat protein. Functional verification on animal models shows that the heptapeptide SR7 and the wheat peptide containing this peptide segment have muscle-building function, mainly reflected in enhancing exercise ability, including increasing total movement distance, movement speed and activity frequency; and increasing muscle strength and muscle mass. Moreover, food-derived bioactive peptides have high biological safety. Therefore, the heptapeptide SR7 and the wheat peptide containing this peptide segment can be applied to the preparation of drugs for improving sarcopenia. The present invention provides a new active substance with muscle-building function, having good market prospects and application potential. Description of the Drawings

[0025] Figure 1 Effect of wheat peptide containing SR7 on the grasping force of sarcopenia mice.

[0026] Figure 2 Effect of wheat peptide containing SR7 on the liver index of sarcopenia mice.

[0027] Figure 3 Effect of wheat peptide containing SR7 on the kidney index of sarcopenia mice.

[0028] Figure 4 Effect of wheat peptide containing SR7 on the gastrocnemius index of sarcopenia mice.

[0029] Figure 5 Effect of wheat peptide containing SR7 on the soleus index of sarcopenia mice.

[0030] Figure 6 Effect of wheat peptide containing SR7 on the total cholesterol in the serum of sarcopenia mice.

[0031] Figure 7 Effect of wheat peptide containing SR7 on the triglyceride in the serum of sarcopenia mice.

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

[0033] Figure 9 Effect of wheat peptide containing SR7 on the high-density lipoprotein cholesterol in the serum of sarcopenia mice.

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

[0035] Figure 11 It is the secondary mass spectrometry diagram of heptapeptide SR7.

[0036] Figure 12 It is the schematic diagram of the binding of heptapeptide SR7 and FGFR1.

[0037] Figure 13 To compare the effects of heptapeptide SR7 and hexapeptide QR6 on the moving distance in the locomotor ability of zebrafish.

[0038] Figure 14 To compare the effects of heptapeptide SR7 and hexapeptide QR6 on the moving speed in the locomotor ability of zebrafish.

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

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

[0041] Figure 17 To compare the effects of heptapeptide SR7 and wheat peptides on the moving distance in the locomotor ability of zebrafish.

[0042] Figure 18 To compare the effects of heptapeptide SR7 and wheat peptides on the moving speed in the locomotor ability of zebrafish.

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

[0044] Figure 20 To compare the effects of heptapeptide SR7 and wheat peptides on the locomotor trajectory of zebrafish. Detailed implementation manners

[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 used to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, any modification or replacement made to the methods, steps or conditions of the present invention shall fall within the scope of the present invention.

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

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

[0048] Example 1: Preparation of wheat peptide

[0049] According to the mass ratio of water to wheat protein (gluten) of 15:1, it was put into the reaction kettle. After the feeding was completed, the pH of the feed liquid was adjusted to 8.0 ± 0.2, and then alkaline protease (accounting for 1.0% of the total weight of gluten) was added for enzymatic hydrolysis for 60 min. After that, 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 it was placed at 100 °C for 30 min to inactivate the enzyme. Subsequently, the feed liquid was concentrated and then spray-dried to obtain wheat peptide powder.

[0050] Example 2: Effect of wheat peptide on sarcopenia in mice

[0051] (1) Mouse experiment design

[0052] SPF-grade male C57BL / 6 mice at 2 months old (about 20 g in weight) were selected for the experiment. All procedures were carried out in accordance with the Guidelines for the Care and Use of Laboratory Animals of Zhejiang Chinese Medical University and were approved by the Animal Ethics Committee of Zhejiang Chinese Medical University (No. 20231204-05). After one week of adaptive feeding of 36 mice, they were randomly divided into 3 groups (12 mice in each group): ① blank control (NC) group, ② model (MC) group, ③ wheat peptide (0.70 mg / g, WP) group. The experiment lasted for 6 weeks. In the first 3 weeks, mice in the MC group and the WP group were subcutaneously injected with dexamethasone solution (5 mg / kg) at a fixed time every day, and the NC group was given sterile normal saline, and the injection was continuous for 3 weeks (volume 0.1 mL / 10g). After 3 weeks, the WP group was intragastrically administered with wheat peptide (0.70 mg / g), and the NC group and the MC group were intragastrically administered with the same volume of sterile normal saline (volume 0.1 mL / 10g), and the intragastric administration was continuous for 3 weeks. During the experiment, the body weight and food intake of the mice were recorded at a fixed time every week.

[0053] (2) Determination of mouse grip strength

[0054] After the experiment, a mouse grip strength tester was used to evaluate the forelimb grip strength of the mice. During the test, the tail of the mouse was lifted and it was induced to grasp a rigid grid attached to a digital dynamometer. The tail of each mouse was gently pulled backward, and the tension reading of the digital dynamometer was defined as the grip strength before the mouse released the grid. Three consecutive tests were performed on each mouse, and the average grip strength value was calculated.

[0055] (3)Serum and tissue sample collection

[0056] After the experiment, the mice were fasted but allowed free access to water for 12 h. Blood was collected by eye puncture, allowed to stand for 1 - 2 h until it stratified, and then centrifuged at 4℃ and 3000 rpm for 15 min using a low-temperature high-speed centrifuge to separate the serum, which was stored at -80℃ for later use. After blood collection, the mice were sacrificed by cervical dislocation. The liver, kidney, gastrocnemius, and soleus muscle tissues were accurately removed, weighed, and the organ indices were calculated. Subsequently, the samples were stored in a -80℃ ultra-low temperature freezer.

[0057] (4)Biochemical index determination

[0058] The total cholesterol (T-CHO), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) in the serum were measured according to the kit instructions (kits from Nanjing Jiancheng Bioengineering Institute).

[0059] (5)Result analysis

[0060] Muscle atrophy often leads to a decrease in 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 disrupted, muscle mass loss occurs and may lead to muscle atrophy. Muscle mass and muscle strength play very important core roles in the evaluation of sarcopenia. Therefore, the effect of wheat peptides on the grip strength of sarcopenic mice was measured, and the results are as Figure 1 shown. Compared with the NC group, the grip strength of the mice in the MC group was significantly reduced by 18.00% ( P <0.001). Compared with the MC group, the grip strength of the mice in the WP group was significantly increased by 20.49% ( P <0.001), indicating that WP intervention may have a good effect on improving muscle strength.

[0061] The results of the effect of wheat peptides on the organ indices of sarcopenic mice are as Figures 2 - 5 shown. For the liver index and kidney index, there were no significant differences among the NC group, MC group, and WP group ( P >0.05), indicating that dexamethasone had no significant effect on the liver and kidney, and WP intervention also had no significant effect on the liver and kidney. 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 on improving muscle mass.

[0062] Metabolic disorders and metabolites of lipids in muscle may be able to exacerbate the reduction of skeletal muscle mass. Therefore, the effects of wheat peptides on blood lipid levels in sarcopenia mice were measured, and the results are as Figures 6 - 9 shown. There were no significant differences in the levels of TCHO, TG, and LDL-C among the NC group, MC group, and WP group ( P > 0.05). Compared with the NC group, the HDL-C level in the MC group was significantly decreased by 15.47% ( P <0.05); compared with the MC group, the HDL-C level in the WP group was significantly increased by 21.14% ( P <0.01). The results indicate that wheat peptide intervention can significantly improve the blood lipid levels in sarcopenia mice, and thus can regulate lipid metabolism.

[0063] Example 3: Comparison of wheat peptides and wheat oligopeptides on the arm muscle strength of mice

[0064] (1) Mouse experiment design

[0065] Forty-eight ICR male mice with a body weight of 20 ± 2 g were selected for the experiment. They were fed standard pellet feed and water ad libitum, and were adaptively raised for one week at an environmental temperature of 25 ± 2 °C and a light cycle of 12 h:12 h before use. 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 the blank group (CON), the wheat peptide group (WP), and the wheat oligopeptide (WOP) group, with 16 mice in each group. Four mice were placed in one cage, and there were 4 cages in each group. According to the publicly reported concentration, the wheat peptide group was intragastrically administered the wheat peptide prepared in Example 1 at a dose of 0.1 mg / g per day, the wheat oligopeptide group was given wheat oligopeptide at a dose of 0.1 mg / g, and the blank group was intragastrically administered the same volume of sterile normal saline (the intragastric administration volume was 0.01 mL / g·bw) for 30 consecutive days.

[0066] (2) Grip strength measurement

[0067] On the 29th day of the experiment, the forelimb grip strength of the mice was measured using a grip strength testing system (Model-RX-5, Aikoh Engineering, Nagoya, Japan). When the front paws of the mice grasped the available grid in the instrument, the tails of the mice were lifted and gently pulled upward, and the instrument automatically displayed the maximum force value. The forelimb grip strength of each mouse was measured three times repeatedly, and the average value was taken.

[0068] Grip strength mainly depends on the strength of the forearm flexor and extensor muscle groups. The force - generating parts of the grip strength mainly include the forearm flexor and extensor muscle groups, and in addition, the wrist flexor, superficial flexor digitorum, and deep flexor digitorum, etc. To a certain extent, the arm muscle strength can be evaluated by the size of the grip strength. The results of comparing the effects of wheat peptide and wheat oligopeptide on the arm muscle strength of mice are as Figure 10 shown. Compared with the blank group (CON) and wheat oligopeptide (WOP), wheat peptide (WP) showed a significant increase in grip strength ( P <0.001). It shows that the effect of wheat peptide in enhancing arm muscle strength is significantly better than that of wheat oligopeptide.

[0069] Example 4: Screening of bioactive peptide segments

[0070] In this example, LC - MS / MS peptide mapping analysis was performed on wheat peptide. The obtained peptide segments of wheat peptide were sorted according to the screening conditions, and then further molecular docking was used to determine the final theoretical effective peptide segments according to the scores. The specific analysis process is as follows:

[0071] (1) Identification of wheat peptide sequence

[0072] Dissolve the wheat peptide sample in NH4HCO3 solution, add dithiothreitol solution, and place it in a 56 °C water bath for reduction for 1 h. Then add iodoacetamide solution and react in the dark for 40 min. After desalting, evaporate the solvent to dryness, and then dissolve the sample in 10 μL of mobile phase A (0.1% formic acid) into a liquid phase injection vial. Subsequently, LC - MS / MS analysis was carried out.

[0073] 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 - 66min, 95%B.

[0074] Mass spectrometry conditions: Full - scan MS was performed using Orbitrap for the first - stage scan, scanning range (100~1500 m / z), resolution (70,000), maximum ion injection time (100 ms), automatic gain control (3×10 6 ); High - energy collision dissociation was used to fragment the top 20 precursor ions that met the tandem (MS / MS) fragmentation conditions and scan with Orbitrap, resolution (17,500), maximum ion injection time (50 ms), automatic gain control (1×10 5). The raw data obtained by mass spectrometry was analyzed for polypeptide sequence using the De novo software of PEAKSStudio.

[0075] (2) Screening of active peptides with potential muscle-building function

[0076] Fibroblast growth factor receptor 1 (FGFR1), a transmembrane protein, consists 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 growth and differentiation) and muscle regeneration. Therefore, molecular docking of wheat peptide fragments with FGFR1 was performed to screen for small peptide fragments that may have muscle-building functions.

[0077] Peptide fragments meeting the criteria were screened according to the conditions of average local confidence (ALC) greater than 95%, peak area greater than 2×10 6 , and PeptideRanker score greater than 0.8. Subsequently, the peptide fragments were molecularly docked with fibroblast growth factor receptor 1 (5AM6).

[0078] First, the crystal structure of FGFR1 (5AM6) was downloaded from the PDB protein database. After removing water molecules and adding hydrogen atoms to the receptor target through Discovery Studio software, its active center was defined. The structures of the selected wheat peptide fragments were constructed by Discovery Studio, and their energies were minimized by the CHARMm force field. These peptides were defined as ligands. The constructed peptides were docked with FGFR1 using CDOCKER to simulate the binding mode, site, and amino acid residues with the lowest binding energy and 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 content of the target peptide fragments in the wheat peptides prepared in Example 1 was measured, and the content of SSGKFLR (on a dry basis) was 8 mg / 100 g.

[0079] Table 1 Peptide fragments in wheat peptides with potential muscle-building functions

[0080] 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 QWLQPR 96.9 6 414.228 2 2.34E+06 826.445 0.77 -68.82

[0081] Among them, the secondary mass spectrometry diagram of the heptapeptide SR7 is as Figure 11As shown, the cleavage fragment ions of peptides include: N-terminal fragment ions (types a, b, c) and C-terminal fragment ions (types x, y, z). The side-chain cleavage of a, y, and z type ions forms d, v, w type ions respectively. In addition, there are internal ions formed by the cleavage at both ends, etc. The b and y series ions are the most common. The primary structure of the peptide can be deduced based on the b or y series fragment ions of the peptide. 397.728 m / z is the [M+H] of the heptapeptide + ion signal, the charge number (z) is 2, and the molecular weight is 793.445 Da. Further, the heptapeptide was subjected to MS / MS analysis by in-source collision-induced dissociation technology (as Figure 10 shown), and the primary structure of the heptapeptide was determined to be Ser-Ser-Gly-Lys-Phe-Leu-Arg.

[0082] The 2D and 3D maps of the molecular docking of SR7 and FGFR1 are as Figure 12 shown. By analyzing the chemical bonds, it was found that SR7 binds to FGFR1 mainly through van der Waals forces, hydrogen bonds (including conventional hydrogen bonds and carbon-hydrogen bonds), and electrostatic interactions (salt bridges and Attractivecharge), and the docking energy is -99.04 kcal / mol. SR7 forms 4 van der Waals forces with the amino acid residues ASN568, GLY567, LEU494, and ALA564, forms 12 hydrogen bonds with GLU571, LYS566, LYS482, SER565, GLU633, LYS510, TYR563, ASP634, and THR632, and forms 5 hydrophobic interactions with GLU571, LYS482, GLU633, and ASP634.

[0083] From the results of molecular docking, it can be seen that at the molecular docking level, SR7 can bind to FGFR1 and has the potential function of increasing muscle mass.

[0084] The peptide segments SSGKFLR and QWLQPR were synthesized by Shenzhen Borun Sida Biotechnology Co., Ltd. with a purity ≥98% for subsequent functional verification.

[0085] Example 5: Comparison of the muscle mass-increasing effects of the heptapeptide SR7 and the hexapeptide QR6 on zebrafish sarcopenia

[0086] 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 indicators for measuring muscle ability.

[0087] (1) Rearing and embryo collection of zebrafish

[0088] In this experiment, wild-type AB strain zebrafish were used for research. AB zebrafish (4 months old) were reared in a circulating water system at 28 ± 0.5 °C and pH 7.0 - 7.5. The photoperiod was 14 h light / 10 h dark, and brine shrimp were fed regularly twice a day. Healthy adult zebrafish were placed in a special mating tank at a ratio of 1:1 male to female the evening before, with the water level 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 zebrafish embryos were obtained. At this time, the embryos were at 0 hpf. After removing impurities from the embryos, they were placed in a Petri dish containing methylene blue system water, and the Petri dish was placed in an incubator at a constant temperature of 28 ± 0.5 °C for cultivation.

[0089] (2)Grouping and administration of zebrafish

[0090] Zebrafish at 4 dpf were selected and placed in a six-well plate, divided into 7 groups, with 30 fish in each well. The specific grouping was blank (NC) group, model (MC) group, SR7 (5 μg / mL, SR7) group, and QR6 (5 μg / mL, QR6) group. 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 groups were added with the corresponding dose of the sample and dexamethasone at the same time. They were continued to be cultured in an incubator at 28 ± 0.5 °C for 4 days, and the liquid was changed every day. The zebrafish larvae were not fed during the culture process.

[0091] (3)Measurement of motor ability

[0092] The zebrafish after the intervention were transferred to a 96-well cell culture plate, with 1 fish in each well, and then placed in a soundproof box (temperature set at 28 °C) to adapt for 5 min, and then the experiment was officially started. The DanioVision Tracking System was used to collect the movement of zebrafish within 20 min (alternating 5 min of light and 5 min of darkness). The Noldus behavior analysis software was used to statistically analyze the total movement distance, average speed, activity frequency, and cumulative activity duration of zebrafish.

[0093] (4)Data analysis

[0094] In this study, Graphpad Prism 8.0 software was used for statistical difference analysis and drawing, and each data was expressed as mean ± standard error. One-way ANOVA was used for inter-group difference comparison (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).

[0095] (5) Result analysis

[0096] The results of comparing the effects of heptapeptide SR7 and hexapeptide QR6 on the locomotor ability of zebrafish are as Figures 13 - 16 shown. Compared with the NC group, the total locomotor distance, movement speed and activity frequency of the MC group were significantly decreased ( P < 0.001, P < 0.001, P < 0.01), indicating that the sarcopenia model was successfully established. Compared with the MC group, the total locomotor distance, movement speed and activity frequency of the QR6 group were increased by 9.41%, 7.01% and 4.50% respectively, but not significantly ( P > 0.05); the total locomotor distance, movement speed and activity frequency of the SR7 group were significantly increased 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. The analysis of the locomotor trajectories of zebrafish further demonstrated that the activity and activity range of zebrafish were significantly increased after SR7 intervention.

[0097] Based on the above data, it can be seen that heptapeptide SR7 has a muscle-building effect from the perspective of the locomotor ability of zebrafish.

[0098] Example 6: Comparison of the muscle-building effects of heptapeptide SR7 and wheat peptide on zebrafish sarcopenia

[0099] The test method was the same as that of "Comparison of the muscle-building effects of heptapeptide SR7 and hexapeptide QR6 on zebrafish sarcopenia", and it was divided into 7 groups with 30 tails in each well. The specific grouping was blank group (NC), model group (MC), low-dose wheat peptide (10 μg / mL, WP-10) and high-dose (100 μg / mL, WP-100) groups, low-dose SR7 (1 μg / mL, SR7-1), medium-dose (5 μg / mL, SR7-5) and high-dose (10 μg / mL, SR7-10) groups.

[0100] The results of comparing the effects of heptapeptide SR7 and wheat peptide on the locomotor ability of zebrafish are as Figures 17 - 20 shown. Compared with the NC group, the total locomotor distance, movement speed and activity frequency of the MC group were significantly decreased 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 significance ( P >0.05); the above indicators of 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 of the 1 μg / mL SR7 group increased by 11.52%, 16.32%, and 25.45%, respectively, but none of them reached significance ( P >0.05); the above indicators of 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 of 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. The analysis of zebrafish movement trajectories further demonstrated that the activity of zebrafish increased significantly and the activity range increased significantly after intervention with 100 μg / mL wheat peptide, 5 μg / mL SR7, and 10 μg / mL SR7.

[0101] Based on the above data, it can be seen that from the perspective of zebrafish movement ability, including total movement distance, movement speed, and activity frequency, the muscle-building effect of heptapeptide SR7 is about 20 times that of wheat peptide.

[0102] In summary, the wheat peptides provided by the present invention have good effects in improving muscle strength and muscle mass, and can also regulate lipid metabolism. The effect of enhancing muscle strength is significantly better than that of wheat oligopeptides. Further, through mass spectrometry identification of peptide segments and molecular docking, the heptapeptide SSGKFLR (SR7) was screened from wheat peptides. This peptide segment showed an efficient muscle-building effect in the sarcopenia model, mainly reflected in enhancing exercise ability, including increasing the total movement distance, movement speed, and activity frequency. The present invention provides a theoretical basis for the development of novel active substances with muscle-building functions, which can be applied to the preparation of drugs for improving sarcopenia. The heptapeptide SSGKFLR can be used alone in the preparation of drugs with muscle-building functions, or can be compounded with other active substances with muscle-building functions. The active components with muscle-building functions may include plant extracts, probiotics, and other protein peptides. The drug can be prepared into microcapsules to improve the gastrointestinal digestion stability, bioavailability, and shelf life of the polypeptide, so as to better apply it to the food industry and health care field.

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, wherein the enzymatic hydrolysis conditions are as follows: water and wheat protein 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 then 1.0% of alkaline protease accounting for the total weight of gluten powder is added for enzymatic hydrolysis for 60 minutes, and after the end, 1.5% of neutral protease accounting for the total weight of gluten powder is added for enzymatic hydrolysis for 30 minutes; after the enzymatic hydrolysis is completed, 0.5% of flavor protease accounting for the total weight of gluten powder is used for 30 minutes, and then the enzyme is inactivated at 100°C for 30 minutes, and then concentrated and dried to obtain wheat peptide powder, which contains the heptapeptide SR7.

3. Use of the heptapeptide SR7 according to claim 1 in the preparation of a drug for treating sarcopenia, 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. Use of the wheat peptide according to claim 5 in the preparation of a medicament for treating sarcopenia, 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.

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