An albumin anti-inflammatory peptide for improving muscle loss, its preparation method and application

Abstract

This application relates to the technical field of bioactive peptides, and specifically discloses an albumin anti-inflammatory peptide for improving muscle loss, its preparation method and application. The albumin anti-inflammatory peptide for improving muscle loss provided by this application is composed of a mixture of NKVVR bioactive peptide and FDVFK bioactive peptide; the amino acid sequence of the NKVVR bioactive peptide is: asparagine-lysine-valine-valine-arginine; the amino acid sequence of the FDVFK bioactive peptide is: phenylalanine-aspartic acid-valine-phenylalanine-lysine. The albumin anti-inflammatory peptide prepared by this application can reduce the inflammatory immune response of an individual, has good anti-inflammatory properties, and can be applied to foods or drugs for improving muscle loss.

Description

Technical Field

[0001] The present application relates to the technical field of bioactive peptides, and particularly relates to an albumin anti-inflammatory peptide for improving muscle loss, its preparation method and application. Background Art

[0002] Muscle loss is a problem in the field of health, which refers to the process of decline in muscle mass, volume and function due to reasons such as age, disease or reduced physical activity. Since muscle is one of the main metabolic organs in the human body, muscle loss can also lead to a decrease in basal metabolic rate, which may result in weight gain and obesity.

[0003] Interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) are cytokines that promote the inflammatory response. Human clinical data shows that in the bodies of patients with some special diseases (such as cancer, AIDS, chronic obstructive pulmonary disease, etc.), patients receiving anti-cancer drugs, and the elderly, the concentrations of IL-6 and TNF-α will increase, accompanied by increased muscle breakdown and consumption or increased myocyte death. Research shows that administering IL-6 and TNF-α to experimental animals to increase the concentrations of IL-6 and TNF-α in their bodies will induce myocyte damage (including imbalance of muscle protein metabolism, myocyte apoptosis, etc.), and thus lead to muscle loss or atrophy.

[0004] Anti-inflammatory substances refer to those compounds or elements with the characteristics of reducing or inhibiting the inflammatory response. These substances can reduce inflammation through multiple mechanisms, including but not limited to inhibiting the production of inflammatory mediators, preventing the activation and migration of inflammatory cells, and reducing the oxidative stress response of damaged tissues.

[0005] Therefore, obtaining anti-inflammatory substances for improving muscle loss has good prospects and significance. Summary of the Invention

[0006] In order to solve the above technical problems, the present application provides an albumin anti-inflammatory peptide for improving muscle loss, its preparation method and application.

[0007] In the first aspect, the present application provides an albumin anti-inflammatory peptide for improving muscle loss, which is composed of a mixture of NKVVR bioactive peptide and FDVFK bioactive peptide;

[0008] The amino acid sequence of the NKVVR bioactive peptide is: asparagine-lysine-valine-valine-arginine; the amino acid sequence of the FDVFK bioactive peptide is: phenylalanine-aspartic acid-valine-phenylalanine-lysine.

[0009] Preferably, the albumin anti-inflammatory peptide for improving muscle loss is composed of a mixture of NKVVR bioactive peptide and FDVFK bioactive peptide with a weight ratio of 20-50:0.2-1.

[0010] Preferably, the albumin anti-inflammatory peptide for improving muscle loss is composed of an NKVVR active peptide and an FDVFK active peptide mixed in a weight ratio of 30-40:0.4-0.8.

[0011] In a specific embodiment, the albumin anti-inflammatory peptide for improving muscle loss is composed of an NKVVR active peptide and an FDVFK active peptide mixed in weight ratios of 20:0.2, 20:0.4, 20:0.6, 20:0.8, 20:1, 30:0.2, 30:0.4, 30:0.6, 30:0.8, 30:1, 35:0.2, 35:0.4, 35:0.6, 35:0.8, 35:1, 40:0.2, 40:0.4, 40:0.6, 40:0.8, 40:1, 50:0.2, 50:0.4, 50:0.6, 50:0.8, 50:1.

[0012] In a second aspect, the present application provides a method for preparing the above albumin anti-inflammatory peptide for improving muscle loss, which is characterized by specifically including the following steps in sequence: taking an albumin oligopeptide concentrate, adding physiological saline, adding flavor protease for enzymatic hydrolysis, boiling to inactivate the enzyme activity, and cooling to room temperature; then adding alkaline protease for enzymatic hydrolysis, boiling to inactivate the enzyme activity, and then cooling to room temperature for filtration, ultrafiltration, concentration, vacuum drying, and screening and separation to obtain the albumin anti-inflammatory peptide.

[0013] During the research and test process of the present application, it was found that the NKVVR active peptide and the FDVFK active peptide, as albumin peptides, have significant anti-inflammatory activities, and the activities are closely related to the presence of the anti-inflammatory peptides. However, the preparation of albumin anti-inflammatory peptides is mainly carried out by enzymatic hydrolysis. Due to factors such as the type of enzyme, hydrolysis conditions, and the molecular weight of the peptides, the anti-inflammatory activity of the hydrolysis products is affected, resulting in a low output rate of effective anti-inflammatory peptides. The applicant has greatly improved the output rates of the NKVVR active peptide and the FDVFK active peptide by optimizing and matching the reaction conditions of enzymatic hydrolysis.

[0014] Preferably, the method for preparing the albumin anti-inflammatory peptide for improving muscle loss specifically includes the following steps in sequence: taking 1000 parts by weight of the albumin oligopeptide concentrate, adding 8000-12000 parts by weight of physiological saline, adding 0.01-0.03 parts by weight of flavor protease, carrying out enzymatic hydrolysis at 35-45 °C for 3-5 h, then boiling to inactivate the enzyme activity, and cooling to room temperature; then adding 0.004-0.008 parts by weight of alkaline protease, carrying out enzymatic hydrolysis at 50-60 °C for 2-4 h, boiling to inactivate the enzyme activity, and then cooling to room temperature for filtration, ultrafiltration, concentration, vacuum drying, and screening and separation to obtain the albumin anti-inflammatory peptide.

[0015] Preferably, the dosage of the flavor protease is 0.015-0.025 parts by weight.

[0016] In a specific embodiment, the dosage of the flavor protease is 0.01, 0.015, 0.02, 0.025, 0.03 parts by weight.

[0017] Preferably, before adding the flavor protease, the pH is adjusted to 6.0 - 7.0.

[0018] In a specific embodiment, before adding the flavor protease, the pH is adjusted to 6.0, 6.5, 7.0.

[0019] Preferably, the dosage of the alkaline protease is 0.005 - 0.007 parts by weight.

[0020] In a specific embodiment, the dosage of the alkaline protease is 0.004, 0.005, 0.006, 0.007, 0.008 parts by weight.

[0021] Preferably, before adding the alkaline protease, the pH is adjusted to 7.5 - 9.0.

[0022] In a specific embodiment, before adding the alkaline protease, the pH is adjusted to 7.5, 8.0, 8.5, 9.0.

[0023] In summary, the technical solution of the present application has the following effects:

[0024] The albumin anti-inflammatory peptide composed of a mixture of NKVVR active peptide and FDVFK active peptide prepared by the technical solution of the present application has excellent anti-inflammatory properties.

[0025] The present application optimizes the preparation method of the anti-albumin anti-inflammatory peptide, and obtains NKVVR active peptide and FDVFK active peptide with a relatively high yield. Detailed Description of the Invention

[0026] The present application will be further described in detail below in conjunction with examples, comparative examples and performance detection tests. These examples should not be construed as limiting the scope claimed in the present application.

[0027] Examples

[0028] Example 1

[0029] Example 1 provides an albumin anti-inflammatory peptide for improving muscle loss.

[0030] The preparation method of the albumin anti-inflammatory peptide in Example 1 is as follows:

[0031] Enzymatic hydrolysis: Take 1 kg of albumin oligopeptide concentrate (10% protein content, sourced from Shaoxing Jiayun Biotechnology Co., Ltd.), add 10 kg of physiological saline, adjust the pH to 6.5, add 0.02 kg of flavor protease, and perform enzymatic hydrolysis at 40 °C for 4 h. Then boil to inactivate the enzyme and cool to room temperature. Then adjust the pH to 8.0, add 0.006 kg of alkaline protease, and perform enzymatic hydrolysis at 55 °C for 3 h. Boil to inactivate the enzyme and cool to room temperature.

[0032] Post-treatment of centrifugal filtration, ultrafiltration, concentration, and vacuum drying: Centrifuge the enzyme-hydrolyzed solution cooled to room temperature at 6000 r / min for 15 min to obtain the supernatant, filter it through a 0.45 μm filter membrane, and collect the filtrate for the next step. Separate the filtrate with ultrafiltration membranes of 10 kDa, 5 kDa, and 3 kDa, collect polypeptides with different molecular weights (>10 kDa, 5 - 10 kDa, 3 - 5 kDa, and <3 kDa), and perform vacuum freeze-drying on the 4 polypeptide solutions with different molecular weights and store them at -80 °C.

[0033] Screening and separation: Perform LC-MS / MS liquid chromatography-mass spectrometry analysis on the polypeptide fraction with a molecular weight <3 kDa, draw the 2D structure diagram of the peptide sequence, then draw the 3D structure diagram of the peptide and set it to the structure with the minimum energy (pdkgt format). Download the x-ray crystal structure (PDB format) of inducible nitric oxide synthase (iNOS, PDB I: 3e6t) from the RCSB Protein Data Bank database, and use Autedocktools 1.5.6 software to perform operations such as removing water molecules, adding hydrogen, and calculating charges on the receptor iNOS. Docking all peptides with the receptor protein through BR.0.8 software. Generally, the docking energy is less than 0, and the smaller the value, the closer the docking and the higher the anti-inflammatory potential. Therefore, rank the peptides according to the absolute value of the docking energy, and predict the possible bioactivity values of the peptides with the top rankings by PeptideRanker.

[0034] Inducible nitric oxide synthase (iNOS), a nervous system enzyme closely related to the inflammatory response, is highly expressed in large amounts by LPS or cytokines when stimulated by various inflammatory conditions, and then continuously produces NO, which may cause tissue damage at the inflammatory site. Controlling iNOS is one of the important strategies for controlling the inflammatory response under various pathological conditions, and iNOS has become a key target for treating inflammatory diseases. The PRz software can batch simulate the docking of receptors with different ligands, and judge the strength of their interaction forces through the docking energy to achieve the virtual screening effect. The docking energy score can represent the binding potential between the receptor and the ligand. Generally speaking, a lower score represents a stronger binding ability between the two. The peptides obtained by LC-MSIMS were simulated and scored. The docking energy of the peptides ≤ -8.5 kcal / mol indicates a relatively strong binding ability to iNOS. Two peptides were compared with the BIQPER-UWM database, and among them, the NKVVR active peptide and the FDVFK active peptide were synthesized and verified as anti-inflammatory peptides.

[0035] In this example, the NKVVR active peptide and the FDVFK active peptide were obtained.

[0036] Examples 2-7

[0037] Examples 2-7 respectively provided an albumin anti-inflammatory peptide.

[0038] The differences between the above examples and Example 1 are as follows: The methods for enzymolysis of the albumin oligopeptide concentrate are different, as shown below.

[0039] In Example 2: Take 1 kg of albumin oligopeptide concentrate, add 10 kg of normal saline, adjust the pH to 8.0, add 0.02 kg of flavor protease, enzymolyze at 40 °C for 4 h, then boil to inactivate the enzyme, and cool down to room temperature; then, adjust the pH to 6.5, add 0.006 kg of alkaline protease, enzymolyze at 55 °C for 3 h, boil to inactivate the enzyme, and cool down to room temperature

[0040] In Example 3: Take 1 kg of albumin oligopeptide concentrate, add 10 kg of normal saline, adjust the pH to 6.5, add 0.02 kg of flavor protease, enzymolyze at 55 °C for 4 h, then boil to inactivate the enzyme, and cool down to room temperature; then adjust the pH to 8.0, add 0.006 kg of alkaline protease, enzymolyze at 40 °C for 3 h, boil to inactivate the enzyme, and cool down to room temperature.

[0041] In Example 4: Take 1 kg of albumin oligopeptide concentrate, add 10 kg of normal saline, adjust the pH to 6.5, add 0.01 kg of flavor protease, enzymolyze at 40 °C for 4 h, then boil to inactivate the enzyme, and cool down to room temperature; then adjust the pH to 8.0, add 0.008 kg of alkaline protease, enzymolyze at 55 °C for 3 h, boil to inactivate the enzyme, and cool down to room temperature.

[0042] In Example 5: Take 1 kg of albumin oligopeptide concentrate, add 10 kg of normal saline, adjust the pH to 6.5, add 0.03 kg of flavor protease, enzymatically hydrolyze at 40 °C for 4 h, then boil to inactivate the enzyme, and cool down to room temperature; then adjust the pH to 8.0, add 0.004 kg of alkaline protease, enzymatically hydrolyze at 55 °C for 3 h, boil to inactivate the enzyme, and cool down to room temperature.

[0043] In Example 6: Take 1 kg of albumin oligopeptide concentrate, add 10 kg of normal saline, adjust the pH to 6.0, add 0.015 kg of flavor protease, enzymatically hydrolyze at 40 °C for 4 h, then boil to inactivate the enzyme, and cool down to room temperature; then adjust the pH to 8.0, add 0.007 kg of alkaline protease, enzymatically hydrolyze at 55 °C for 3 h, boil to inactivate the enzyme, and cool down to room temperature.

[0044] In Example 7: Take 1 kg of albumin oligopeptide concentrate, add 10 kg of normal saline, adjust the pH to 7.0, add 0.025 kg of flavor protease, enzymatically hydrolyze at 40 °C for 4 h, then boil to inactivate the enzyme, and cool down to room temperature; then adjust the pH to 8.0, add 0.005 kg of alkaline protease, enzymatically hydrolyze at 55 °C for 3 h, boil to inactivate the enzyme, and cool down to room temperature.

[0045] In the above examples, other process parameters are the same as those in Example 1.

[0046] Comparative Example

[0047] Comparative Examples 1 - 3

[0048] Comparative Examples 1 - 3 respectively provide an albumin anti-inflammatory peptide.

[0049] The differences between the above comparative examples and Example 1 are as follows: The methods for enzymatic hydrolysis of albumin oligopeptide concentrate are different, as specifically shown below.

[0050] In Comparative Example 1: Enzymatic hydrolysis: Take 1 kg of albumin oligopeptide concentrate (10% protein content, sourced from Shaoxing Jiayun Biotechnology Co., Ltd.), add 10 kg of normal saline, adjust the pH to 8.0, add 0.006 kg of alkaline protease, enzymatically hydrolyze at 55 °C for 3 h, then boil to inactivate the enzyme, and cool down to room temperature; then adjust the pH to 6.5, add 0.02 kg of flavor protease, enzymatically hydrolyze at 40 °C for 4 h, boil to inactivate the enzyme, and cool down to room temperature.

[0051] In Comparative Example 2: Enzymatic hydrolysis: Take 1 kg of albumin oligopeptide concentrate (10% protein content, sourced from Shaoxing Jiayun Biotechnology Co., Ltd.), add 10 kg of normal saline, adjust the pH to 7.5, add 0.02 kg of flavor protease and 0.006 kg of alkaline protease, enzymatically hydrolyze at 55 °C for 3 h, boil to inactivate the enzyme, and cool down to room temperature.

[0052] In Comparative Example 3: Enzymatic hydrolysis: Take 1 kg of albumin oligopeptide concentrate (protein content of 10%, sourced from Shaoxing Jiayun Biotechnology Co., Ltd.), add 10 kg of physiological saline, adjust the pH to 6.5, add 0.02 kg of pancreatin, perform enzymatic hydrolysis at 40 °C for 4 h, then boil to inactivate the enzyme activity and cool to room temperature; then adjust the pH to 8.0, add 0.006 kg of alkaline protease, perform enzymatic hydrolysis at 55 °C for 3 h, boil to inactivate the enzyme activity and cool to room temperature.

[0053] For the remaining process parameters in the above comparative examples, they are the same as those in Example 1.

[0054] Performance detection test

[0055] Record the output of albumin anti-inflammatory peptides prepared in Examples 1 - 7 and Comparative Examples 1 - 3. The results are shown in Table 1.

[0056] Table 1 Output of albumin anti-inflammatory peptides in Examples 1 - 7 and Comparative Examples 1 - 3

[0057]

[0058]

[0059] Combined with Table 1, by comparing the detection results of Examples 1 - 7 and Comparative Examples 1 - 3, it can be seen that in the technical solution of this application, after first performing enzymatic hydrolysis on the albumin oligopeptide concentrate using flavor protease, inactivating it, and then performing enzymatic hydrolysis using alkaline protease, the prepared NKVVR active peptide and FDVFK active peptide have higher quality.

[0060] Examples 8 - 15

[0061] Examples 8 - 15 respectively provide an albumin anti-inflammatory peptide.

[0062] The differences in the above examples are as follows: The weight ratio between the NKVVR active peptide and the FDVFK active peptide is specifically as follows. The NKVVR active peptide and the FDVFK active peptide in the above examples are sourced from Example 1.

[0063] In Example 8: The albumin anti-inflammatory peptide is composed of a mixture of NKVVR active peptide and FDVFK active peptide with a weight ratio of 10:3.

[0064] In Example 9: The albumin anti-inflammatory peptide is composed of a mixture of NKVVR active peptide and FDVFK active peptide with a weight ratio of 60:0.1.

[0065] In Example 10: The albumin anti-inflammatory peptide is composed of a mixture of NKVVR active peptide and FDVFK active peptide with a weight ratio of 20:1.

[0066] In Example 11: The albumin anti-inflammatory peptide is composed of an NKVVR active peptide and an FDVFK active peptide mixed in a weight ratio of 50:0.2.

[0067] In Example 12: The albumin anti-inflammatory peptide is composed of an NKVVR active peptide and an FDVFK active peptide mixed in a weight ratio of 30:0.8.

[0068] In Example 13: The albumin anti-inflammatory peptide is composed of an NKVVR active peptide and an FDVFK active peptide mixed in a weight ratio of 40:0.4.

[0069] In Example 14: The albumin anti-inflammatory peptide is the NKVVR active peptide.

[0070] In Example 15: The albumin anti-inflammatory peptide is the FDVFK active peptide.

[0071] Performance detection test

[0072] (1) Animal toxicity test

[0073] Ordinary-grade NIH mice (body weight 20g ± 1g) were used as experimental mice, with an equal number of males and females.

[0074] The albumin anti-inflammatory peptide powder was dissolved in sterile water to prepare an albumin anti-inflammatory peptide sample solution at a concentration of 50mg / mL. The mice were administered at a dose of 1ml / 10g body weight, closely observed for 12h and recorded, and then observed for another 7 days.

[0075] The experimental results showed that: within 7 days, the mice had no deaths, were active, had normal food intake, had bright and non-loose and dirty hair, no drowsiness, no abnormal secretions in the eyes, mouth, nose, ears and perianal area, and no broken tails or rotten toes. Then, 3 mice were selected from each group of experimental mice and sacrificed. Macroscopically, no abnormal changes were found in the important organs such as the heart, liver, lungs, stomach and thymus of the experimental mice, indicating that the albumin anti-inflammatory peptide powder has no acute toxicity.

[0076] (2) Evaluation of the anti-inflammatory performance of the albumin anti-inflammatory peptide

[0077] The albumin anti-inflammatory peptide powder was dissolved in sterile water to prepare an albumin anti-inflammatory peptide sample solution at a concentration of 50mg / mL.

[0078] To confirm whether the albumin anti-inflammatory peptide powder can reduce the inflammatory response of cells, the changes in the gene expression of cytokines in cells during inflammation were measured.

[0079] The mouse macrophage RAW264.7 cell line was used, and lipopolysaccharide (LPS; Sigma-Aldrich, #L2630), the main component of the Gram-negative bacterial cell wall, was used as the substance to induce an inflammatory response. The cells were cultured in DMEM (Lonza, #BE12-604F) containing 10% FBS and 1% penicillin-streptomycin at 37 °C and 5% CO2.

[0080] The cells were seeded in a 96-well plate at a density of 2.5×10 5 cells / well. After 24 h, the prepared cells were treated with the albumin anti-inflammatory peptide sample solution. After incubation at 37 °C for 45 min, the cells were treated with 200 ng / mL LPS to induce inflammation. After incubation at 37 °C for 8 h, the cells in the medium-free state were treated with TRIzolTM (Invitrogen, #15596018) to lyse the cells. Through the given experimental method, RNA was obtained, and cDNA was generated using reverse transcriptase (Promega, #M1705). Subsequently, the cytokine expression levels were measured using SYBR Green (Enzynomics, RT530) and mouse primers for each inflammatory cytokine gene (TNFα, IL-6, and IL-1β). At the same time, the cells not treated with the albumin anti-inflammatory peptide sample solution were used as the blank control group.

[0081] The inhibition rates of cytokines (TNFα, IL-6, and IL-1β) were calculated based on the experimental group and the blank control group.

[0082] Detection results: As shown in Table 2.

[0083] Table 2 Detection results of the application performance of albumin anti-inflammatory peptides in Examples 8-15

[0084]

[0085] Combined with the detection results of Examples 8-15 in Table 2, it can be seen that when the cells were treated with the albumin anti-inflammatory peptide prepared in this application, the inflammatory cytokines (TNFα, IL-6, and IL-1β) were reduced compared with the blank control group; this indicates that the albumin anti-inflammatory peptide prepared in this application can reduce the inflammatory immune response of an individual and has good anti-inflammatory properties.

[0086] Although the present invention has been described in detail with general descriptions and specific embodiments above, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of the present invention claimed.

Claims

1. An albumin anti-inflammatory peptide, characterized in that, Composed of a mixture of NKVVR active peptide and FDVFK active peptide; The amino acid sequence of the NKVVR active peptide is: asparagine - lysine - valine - valine - arginine; the amino acid sequence of the FDVFK active peptide is: phenylalanine - aspartic acid - valine - phenylalanine - lysine.

2. The albumin anti-inflammatory peptide according to claim 1, wherein Composed of a mixture of NKVVR active peptide and FDVFK active peptide with a weight ratio of 20 - 50: 0.2 - 1.

3. The albumin anti-inflammatory peptide according to claim 1, wherein Composed of a mixture of NKVVR active peptide and FDVFK active peptide with a weight ratio of 30 - 40: 0.4 - 0.8.

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