Egg white protein-derived small molecule peptide with the functions of regulating immunity, tonifying qi and blood and uses thereof

Abstract

The application discloses egg white protein source small molecule peptides with the functions of regulating immunity and tonifying qi and blood and application thereof, and belongs to the technical field of small molecule peptides. The application uses alkaline protease, papain and neutral protease to sequentially perform enzymolysis on egg white protein to obtain egg white small molecule peptides containing dipeptide LW. Animal model function verification shows that the dipeptide LW and the egg white small molecule peptides containing the peptide segment have the function of improving qi and blood deficiency, and high-dose (converted into 3.0g / 60kg.d for an adult) egg white small molecule peptides increase red blood cells, hemoglobin, platelets and lymphocytes by 16.6%, 14.7%, 23.4% and 28.75% respectively. Food-derived bioactive peptides have high biological safety, and therefore, the dipeptide LW and the egg white small molecule peptides containing the peptide segment can be applied to the preparation of medicines with the functions of regulating immunity and improving qi and blood, and have good market prospects and application potential.

Description

Technical Field

[0001] This invention relates to the field of small molecule peptide technology, specifically to a small molecule peptide derived from egg white protein that has the effects of regulating immunity and replenishing qi and blood, and its applications. Background Technology

[0002] In Traditional Chinese Medicine (TCM), "Qi and Blood Deficiency" typically refers to various functional or organic problems in Western medicine, such as anemia, endocrine disorders, cardiovascular dysfunction, malnutrition, or insufficient energy metabolism caused by chronic diseases. TCM considers Qi and Blood to be the fundamental substances for maintaining life activities, while Western medicine explains these symptoms through specific indicators or pathological mechanisms. A long-term unbalanced diet can lead to deficiencies in nutrients such as protein, vitamin B12, and folic acid, affecting hematopoiesis and energy metabolism, resulting in symptoms similar to Qi and Blood Deficiency.

[0003] In existing technologies, qi- and blood-tonifying products mainly rely on food-medicine homology. For example, patent document CN120392910A discloses a method for preparing fermented Polygonatum sibiricum with high polysaccharide content for tonifying qi and blood. This method involves mixing low-molecular-weight nutrients extracted from a mixed powder prepared by mixing Polygonatum sibiricum leaves, stems, and roots, and then fermenting the resulting powder with polysaccharides obtained from the filter residue. Patent document CN120381500A discloses a fermented Chinese medicine composition with qi- and blood-tonifying functions. This composition utilizes the synergistic combination of main herbs such as Codonopsis pilosula and Angelica sinensis with Portulaca oleracea and Sinapis alba, combined with a three-stage gradient temperature extraction process and multi-strain synergistic fermentation, to improve the dissolution rate of active ingredients in the herbs and enhance the qi- and blood-tonifying effects of the fermented Chinese medicine composition.

[0004] Bioactive peptides are a class of peptide compounds with physiological regulatory effects. Through natural and green processing methods such as enzymatic hydrolysis, more active peptide components from proteins can be released, offering significant potential application value and market prospects in the food and pharmaceutical industries. For example, patent document CN120021780A discloses a peptide-containing composition that improves blood circulation, including: ginseng oligopeptides, donkey-hide gelatin peptides, bovine blood peptides, wolfberry peptides, fish collagen peptides, and American ginseng peptides. This composition promotes the generation and regulation of blood circulation from multiple levels through various peptide components.

[0005] Eggs are rich in high-quality protein. The protein in egg white is mainly ovalbumin and ovoglobulin, which contain all eight essential amino acids and are very similar in composition to human protein. The human body can absorb up to 98% of egg protein, making the development of high-value-added processed egg products of great significance. Currently, there is no research on the effects of egg white protease hydrolysates on improving nutrition and replenishing qi and blood. Summary of the Invention

[0006] The purpose of this invention is to provide a natural small molecule bioactive peptide with the function of regulating immunity and replenishing qi and blood, and to apply it to the development of products that improve qi and blood function.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] This invention utilizes alkaline protease, papain, and neutral protease to sequentially enzymatically hydrolyze egg white protein powder, extracting the enzymatic hydrolysate with a molecular weight less than 1000 Da to obtain the egg white protease hydrolysate. The peptide sequences in the egg white protease hydrolysate are analyzed using LC-MS / MS peptide mapping, and the interaction between the peptide and the TNF-α receptor is investigated using molecular docking technology. A candidate peptide is screened, and its amino acid sequence is identified as Leu-Trp (LW) by mass spectrometry, with a molecular weight of 317.174 Da. Further functional verification through the artificial synthesis of the dipeptide LW reveals that this peptide has the function of regulating immunity and improving blood circulation.

[0009] Therefore, this invention provides the application of dipeptide LW in the preparation of drugs that regulate immunity and improve blood circulation.

[0010] This invention demonstrates that dipeptide LW has immunomodulatory and blood-tonifying effects. In an anemia model, intervention with dipeptide LW significantly improved blood deficiency and restored red blood cell counts to normal levels. In an immunodeficiency model, intervention with dipeptide LW significantly improved immunodeficiency.

[0011] Furthermore, the drug is used to improve anemia.

[0012] Furthermore, the regulation of immunity and improvement of blood and qi function include increasing the levels of red blood cells, hemoglobin, platelets, and immune cells.

[0013] Furthermore, the dipeptide LW is prepared by solid-phase synthesis; or it is isolated from egg white protease hydrolysis products, wherein the egg white protease hydrolysis products are products obtained by sequentially hydrolyzing egg white protein with alkaline protease, papain, and neutral protease.

[0014] Another objective of this invention is to provide an egg white small molecule peptide with immune-regulating and blood-tonifying effects, wherein the preparation method of the egg white small molecule peptide includes the following steps:

[0015] (1) Mix egg white protein powder with water at a mass ratio of 1:10-20, and then cut to obtain an egg white protein powder solution;

[0016] (2) Heat the egg white protein powder solution to 50℃, adjust the pH value to 8.0±0.2, add 0.5%-1.0% alkaline protease by weight of egg white protein powder, and hydrolyze for 2-2.5 hours; then add 0.4%-0.8% papain by weight of egg white protein powder, and continue hydrolyzing for 4-4.5 hours; then add 0.05%-0.1% neutral protease by weight of egg white protein powder, and continue hydrolyzing for 2-2.5 hours to obtain the enzymatic hydrolysate;

[0017] (3) After centrifuging the enzymatic hydrolysate at a speed of 4000-6000 r / min, the supernatant was collected and filtered through a membrane with a molecular weight cutoff of 1000 Da. The filtrate was then concentrated, sterilized, and dried to obtain the egg white small molecule peptide.

[0018] Furthermore, in step (1), the shearing conditions are: 10000 r / min, 20-30 minutes.

[0019] Further, in step (2), 0.8% of alkaline protease by weight of egg white protein powder is added and enzymatically hydrolyzed for 2 hours; then 0.6% of papain by weight of egg white protein powder is added and enzymatically hydrolyzed for 4 hours; then 0.1% of neutral protease by weight of egg white protein powder is added and enzymatically hydrolyzed for 2 hours to obtain the enzymatic hydrolysate.

[0020] The egg white small molecule peptide contains a dipeptide LW with the amino acid sequence Leu-Trp. This invention demonstrates that the egg white small molecule peptide has immunomodulatory and blood-tonifying effects. In an anemia model, intervention with the egg white small molecule peptide significantly improves blood deficiency and increases the levels of red blood cells, hemoglobin, platelets, and lymphocytes.

[0021] Therefore, the present invention provides the application of the egg white small molecule peptide in the preparation of drugs that regulate immunity and improve blood circulation.

[0022] Furthermore, the improvement of blood and qi function includes improving anemia.

[0023] Specifically, the present invention provides a drug for improving qi and blood deficiency, the drug comprising an effective dose of egg white small molecule peptide or dipeptide LW and a pharmaceutically acceptable carrier.

[0024] In this invention, the pharmaceutically acceptable carrier is any formulation or carrier medium capable of delivering an effective dose of the active substance of this invention, without interfering with the biological activity of the active substance, and without toxic side effects on the host or subject.

[0025] Furthermore, the pharmaceutically acceptable carrier includes one or more of the following: fillers, wetting agents, disintegrants, binders, or lubricants.

[0026] The beneficial effects of this invention are as follows:

[0027] This invention provides a dipeptide LW with immunomodulatory and blood-nourishing effects, as well as an egg white small molecule peptide containing this peptide. The peptide can be obtained through artificial synthesis or targeted enzymatic hydrolysis of egg white protein. Animal model functional validation shows that the dipeptide LW and the egg white small molecule peptide containing this peptide can improve blood deficiency. High doses (equivalent to 3.0g / 60 kg / day for adults) of the egg white small molecule peptide increased erythrocytes, hemoglobin, platelets, and lymphocytes by 16.6%, 14.7%, 23.4%, and 28.75%, respectively. Furthermore, food-derived bioactive peptides have high biosafety. Therefore, the dipeptide LW and the egg white small molecule peptide containing this peptide can be used to prepare drugs that modulate immunity and improve blood function, showing good market prospects and application potential. Attached Figure Description

[0028] Figure 1 The effect of small molecule peptides from egg white on erythrocytes.

[0029] Figure 2 The effect of small molecule peptides from egg white on hemoglobin.

[0030] Figure 3 The effect of small molecule peptides from egg white on platelets.

[0031] Figure 4 The effects of small molecule peptides from egg white on lymphocytes.

[0032] Figure 5 This is the primary mass spectrum of the dipeptide LW.

[0033] Figure 6 This is the secondary mass spectrum of the dipeptide LW. In the figure, y1-NH3 represents the first deamination fragment ion generated by the C-terminus cleavage of the peptide, and y1 represents the first fragment ion generated by the C-terminus cleavage of the peptide.

[0034] Figure 7 This is a schematic diagram illustrating the binding interaction between dipeptide LW and the TNF-α receptor.

[0035] Figure 8 The relative fluorescence area of ​​cardiac erythrocytes after intervention with dipeptide LW in a zebrafish model of blood and qi deficiency.

[0036] Figure 9 The relative fluorescence intensity of cardiac erythrocytes after intervention with dipeptide LW in a zebrafish model of blood and qi deficiency.

[0037] Figure 10 The figure shows the change in macrophage number after intervention with dipeptide LW in a zebrafish immunodeficiency model. The # symbol indicates a significant difference compared to the control group (CON), and #### indicates... P<0.0001; * indicates a significant difference compared to the model group (MOD), **** indicates P <0.0001; *** indicates P <0.001; ** indicates P <0.01. Detailed Implementation

[0038] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0039] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0040] The egg white protein powder used in the following examples was purchased from Zhejiang Aige Egg Products Co., Ltd.; the alkaline protease (derived from Bacillus subtilis) was purchased from Nanning Pangbo Biological Products Co., Ltd.; the papain was purchased from Guangxi Dongheng Huadao Biological Products Co., Ltd.; and the neutral protease (derived from Aspergillus oryzae) was purchased from Nanning Pangbo Biological Products Co., Ltd.

[0041] Example 1: Preparation of small molecule peptides from egg white

[0042] 1. This embodiment provides a method for protein hydrolysis of egg white, the specific steps of which are as follows:

[0043] (1) Add 1500 kg of water to the reactor, and add 100 kg of egg white protein powder while stirring. Stir and mix evenly, then start the shearing process. The shearing conditions are: 10000 r / min for 30 minutes. The high-speed shearing destroys the globulin structure in the egg white protein powder, exposes the enzymatic hydrolysis sites, and makes the next enzymatic hydrolysis more complete and improves the thermal stability.

[0044] (2) After shearing, the temperature of the liquid is raised to 50°C. After the temperature is reached, the pH of the material is adjusted to 8.0 with alkaline solution. 800 g of alkaline protease (from Bacillus subtilis) is added to the egg white protein powder solution and enzymatically hydrolyzed for 2 hours. Then 600 g of papain is added and enzymatically hydrolyzed for another 4 hours. Then 100 g of neutral protease (from Aspergillus oryzae) is added and enzymatically hydrolyzed for another 2 hours to obtain the enzymatic hydrolysate.

[0045] (3) The enzymatic hydrolysate was separated using a disc separator (6000 r / min) to obtain the supernatant. The supernatant was then passed through a membrane with a molecular weight cutoff of 1000 Da, and the filtrate was collected.

[0046] (4) Then the liquid is evaporated and concentrated in a vacuum low-temperature thin film evaporator until the sugar content of the liquid is 25° to obtain a concentrated liquid;

[0047] (5) The concentrated liquid is sterilized at high temperature and spray-dried to obtain egg white protease hydrolysate (egg white small molecule peptides or albumin peptides).

[0048] 2. The relative molecular weight distribution of small molecule peptides in egg white was determined using the method described in Appendix A of the national standard GB / T 22729-2008, "Marine Fish Oligopeptide Powder". The test results are shown in Table 1.

[0049] Table 1. Relative molecular weight distribution of small peptides in egg white

[0050] molecular weight Test results, % ＞5000 0.36 1000-5000 8.92 ＜1000 90.73

[0051] The results in Table 1 show that after enzymatic hydrolysis and a series of treatments, the proportion of peptides with a molecular weight of less than 1000 Daltons in egg white protein was as high as 90.73%, indicating that the preparation process successfully enriched most of the small molecular weight peptides, providing an effective material basis for subsequent functional verification.

[0052] Example 2: Effects of egg white small molecule peptides on immune regulation and qi and blood replenishment in a mouse model

[0053] In this embodiment, the egg white small molecule peptide prepared in Example 1 was applied to a dexamethasone-induced mouse model to study its effect on replenishing qi and blood.

[0054] 1. Experimental Principle

[0055] Dexamethasone is a long-acting glucocorticoid drug with anti-inflammatory, immunosuppressive, antitoxin, anti-shock, hematopoietic system-affecting, and central nervous system-stimulating effects.

[0056] Red blood cells are the most numerous type of blood cell and are the primary means of oxygen transport in the blood of vertebrates and some invertebrates. They also serve as acid-base buffers and play an immune role. Erythrocyte production originates from pluripotent hematopoietic stem cells and undergoes three key differentiation processes: the hematopoietic stem cell stage, the directed progenitor cell stage, and the precursor cell stage. Erythrocytes require iron, vitamin B12, and folic acid, and their lifespan is influenced by factors such as metabolism and body temperature. A decrease in the number or quality of red blood cells can cause red blood cell diseases, the most common being anemia and sickle cell disease.

[0057] Hemoglobin (HGB or Hb) is a special protein in red blood cells that transports oxygen. It is the protein that gives blood its red color and is composed of globin and heme. The globin portion is a tetramer composed of two pairs of different globin chains (α chain and β chain). Hemoglobin and red blood cells are highly synergistic "transport partners," and the clinical significance of elevated or decreased hemoglobin levels can be referenced from that of elevated or decreased red blood cell levels.

[0058] Platelets are the smallest anucleated cell fragments in mammalian blood, formed from the shedding of megakaryocytes from the bone marrow. Platelets are small pieces of cytoplasm detached from the cytoplasm of mature megakaryocytes in the bone marrow. Although megakaryocytes are the fewest number of hematopoietic cells in the bone marrow, accounting for only 0.05% of the total number of nucleated cells, the platelets they produce are extremely important for the body's hemostatic function.

[0059] Lymphocytes are a type of white blood cell, and are the smallest white blood cells. Produced by lymphatic organs, they are mainly found in the circulating lymphatic fluid within lymphatic vessels. They are crucial cellular components of the body's immune response, serving as the primary executors of almost all immune functions in the lymphatic system, and acting as the frontline "soldiers" against external infections and monitoring cellular mutations within the body. Lymphocytes are a cell lineage with immune recognition functions. Based on their origin, migration, surface molecules, and functions, they can be divided into T lymphocytes (also known as T cells), B lymphocytes (also known as B cells), and natural killer (NK) cells. Both T cells and B cells are antigen-specific lymphocytes, and they share the same initial origin: hematopoietic tissue.

[0060] 2. Experimental Methods

[0061] Six- to eight-week-old male C57BL / 6J mice (weighing 20-25 g) were selected and acclimatized for one week. Dexamethasone was administered to establish the model. After 35 days of intervention, the mice were sacrificed, and whole blood erythrocyte, hemoglobin, platelet, and lymphocyte levels were measured using a blood analyzer. This experiment was approved by the Animal Ethics Center of Zhejiang University of Traditional Chinese Medicine, ethics approval number IACUC-20250331-15.

[0062] Administration method: Mice were administered the appropriate dose of the test substance daily according to their body weight, at a gavage dose of 0.01 mL / g bw, once daily. Specific groupings are shown in Table 2.

[0063] Table 2. Experimental Groups and Doses

[0064] Group Gavage Dosage (mg / g.bw / d) Blank group (CON) distilled water Equal volume Model Group (DEX) DEX (solvent is water) 5 mg / kg / day.ih Low-dose group of small molecule peptides in egg white (OP-L+DEX) Egg white small molecule peptides + DEX (solvent is water) 0.25 g / kg / day bw + 5 mg / kg / day ih High-dose group of small molecule peptides in egg white (OP-H+DEX) Egg white small molecule peptides + DEX (solvent is water) 0.5 g / kg / day bw + 5 mg / kg / day ih

[0065] 3. Experimental Results

[0066] The results are as follows Figures 1-3As shown, compared with the blank group, the platelet count in the model group showed a significant decrease, while the red blood cell count did not show a significant difference. This suggests that the modeling intervention has a selective inhibitory effect on the hematopoietic system, mainly targeting the platelet lineage, which may be related to the imbalance of bone marrow microenvironment regulation.

[0067] Compared with the control group, the low-dose (equivalent to 1.5 g / 60 kg.d for adults) of egg white small molecule peptides increased the number of red blood cells and hemoglobin by 15.1% and 15.3%, respectively; the high-dose (equivalent to 3.0 g / 60 kg.d for adults) of egg white small molecule peptides increased the number of red blood cells and hemoglobin by 16.5% and 15%, respectively.

[0068] Compared with the model group, low-dose (equivalent to 1.5 g / 60 kg / day for adults) of egg white small molecule peptides increased the number of red blood cells, hemoglobin, and platelets by 15.2%, 15%, and 10.8%, respectively; high-dose (equivalent to 3.0 g / 60 kg / day for adults) of egg white small molecule peptides increased the number of red blood cells, hemoglobin, and platelets by 16.6%, 14.7%, and 23.4%, respectively.

[0069] The results are shown in Table 3 and Figure 4 As shown, compared with the blank group, the lymphocyte count in the model group did not change significantly, but it decreased by 0.32%. Intervention with egg white small molecule peptides can increase the serum lymphocyte count. Among them, high dose (equivalent to 3.0 g / 60 kg / day for adults) of egg white small molecule peptides can significantly increase the lymphocyte count. Compared with the model group, high dose (equivalent to 3.0 g / 60 kg / day for adults) of egg white small molecule peptides increased the serum lymphocyte count by 28.75%.

[0070] Table 3. Serum lymphocyte count

[0071] Group <![CDATA[Lymphocyte count, 10 9 cells / L]]> Blank group 0.9093±0.1928 Model group 0.9064±0.2346 low-dose group 0.9996±0.3375 High-dose group 1.1670±0.2469*

[0072] The above results indicate that egg white small molecule peptides significantly improve the decrease in platelet count induced by dexamethasone in mice, and the improvement effect is positively correlated with the dose, with higher doses showing a more significant effect on platelet count recovery. Intervention with egg white small molecule peptides significantly improved the decrease in lymphocytes caused by dexamethasone. Low doses of egg white small molecule peptides effectively increased erythrocyte and hemoglobin levels. These results validate the mechanism by which egg white small molecule peptides replenish qi and blood by regulating hematopoietic system function, providing important experimental evidence for subsequent clinical applications.

[0073] The experimental data also showed that egg white small molecule peptides exhibited a good dose-response relationship in improving blood and qi indicators. In particular, the high-dose group increased platelet count by 23.4%, suggesting that it may exert its blood-replenishing effect through multiple synergistic pathways.

[0074] Example 3: Screening of active ingredients in egg white small molecule peptides

[0075] In this embodiment, LC-MS / MS analysis was used to perform peptide mapping on the egg white small molecule peptides prepared in Example 1, and then molecular docking technology was used to screen for functional peptides. Details are as follows:

[0076] 1. LC-MS / MS analysis

[0077] 2. Molecular docking

[0078] Qi and blood complement each other. Sufficient qi ensures smooth blood circulation, which in turn nourishes the internal organs and strengthens the immune system. Conversely, deficient qi leads to weak blood, weakening the body's resistance and making it more susceptible to external pathogens, resulting in various illnesses. Common immune targets include IL-6, TNF-α, and PD-1. When screening for active peptides, the TNF-α receptor is used as the docking target for molecular docking.

[0079] The PeptdieRanker score (≥0.95) and relative peak area (≥10) were used to determine the performance of the peaks. 6 The selected peptides were molecularly docked with the TNF-α receptor. The docking score (≤-7.0), number of hydrogen bonds (≥4), docking frequency of amino acid residues, and amino acid residues related to immune regulation and blood replenishment were used for sequence screening and identification. Finally, the dipeptide LW was determined to have the potential to improve blood deficiency, as shown in Table 4.

[0080] Table 4. Potentially bioactive peptides that bind to TNF-α receptors

[0081] peptide sequence Peptide score Combined Mass-to-charge ratio (m / z) Number of charges relative peak area Number of hydrogen bonds LW 0.985 -8.2 318.182 1 <![CDATA[2.36×10 8 ]]> 5

[0082] The primary and secondary structures of the dipeptide LW, as analyzed by mass spectrometry, are as follows: Figure 5 and Figure 6 As shown, the [M+H]+ ion signal of dipeptide LW in the primary structure is 318.182 m / z, which is basically consistent with the theoretical molecular weight of 317.174 Da, and the amino acid sequence of dipeptide LW in the secondary structure is Leu-Trp.

[0083] Molecular docking analysis results are as follows Figure 7This indicates that the interaction between LW and TNF-α involves amino acid residues GLU116, SER99, GLN102, and PRO100, and is achieved through five hydrogen bonds. This multi-point hydrogen bond interaction mode enhances the binding stability of LW to the TNF-α receptor, suggesting that it may exert an immunomodulatory effect by blocking the TNF-α-mediated inflammatory signaling pathway, thereby achieving the effect of replenishing qi and blood.

[0084] Example 4: Synthesis of active peptides

[0085] This embodiment utilizes the artificially synthesized peptide LW. Specifically, it was synthesized by Shenzhen Borunsida Biotechnology Co., Ltd., with a purity ≥98%. The synthesis method is as follows:

[0086] S1. Weigh Fmoc-Trp-Wang Resin and place it in a glass reaction column. Add DCM to swell the solution for 30 min, then remove the DCM under reduced pressure.

[0087] S2. Wash the resin three times with DMF, add 20% piperidine / DMF solution and react for 20 min to remove the protecting group fmoc, remove the solution under reduced pressure, and wash with DMF six times.

[0088] S3. Weigh out the second amino acid Fmoc-Leu(otbu)-oh, then wash it three times each with DMF, DCM and methanol, and dry the resin.

[0089] S4. Add lysis buffer to remove resin and amino acid side chain protecting groups, filter with sand core, add ether to the filtrate to precipitate, centrifuge and wash the solid 3 times, dry and detect by MS to obtain dipeptide LW.

[0090] Example 5: Verification of the Qi-tonifying and Blood-nourishing Efficacy of Active Peptides

[0091] This embodiment utilizes a zebrafish anemia model induced by phenylhydrazine (PHZ) to verify the efficacy of the active peptides. Wild-type zebrafish (AB) were bred at the zebrafish platform of Zhejiang University School of Medicine, and this experiment was approved by the Zhejiang University Center for Experimental Animal Ethics (ethics number ZJU20250649).

[0092] 1. Experimental intervention treatment

[0093] Four dpf zebrafish were randomly divided into four groups, with 10 fish per well per group and three parallel wells per group, and placed in a 6-well plate. Specific treatments are shown in Table 5.

[0094] Table 5. Zebrafish treatment with peptide fragments

[0095] Group Intervention normal group 3 mL system water Modeling group Phenylenol pH (final concentration 8 μg / mL) + 3 mL system water LW Phenylehydrazine PHZ (final concentration 8 μg / mL) + 3 mL of LW (final concentration 50 μg / mL)

[0096] LW is the peptide LW synthesized in Example 4, dissolved in the system water. The system water formula is as follows: 35 g of sodium chloride (NaCl), 2 g of sodium bicarbonate (NaHCO3), 1 g of calcium chloride (CaCl2) and 0.5 g of potassium chloride (KCl) are dissolved in 1 liter of pure water.

[0097] 2. Staining analysis

[0098] Weigh 10 mg of 3,3'-dimethylbenzidine, add 10 mL of PBS, and add 500 μL of 10% H2O2 to prepare a 1 g / L DAB staining solution. 3,3'-dimethylbenzidine itself has fluorescent properties; therefore, the stained cardiac region will show a fluorescent signal under a fluorescence microscope, and the fluorescence intensity is positively correlated with the erythrocyte staining intensity.

[0099] After culturing in a constant temperature incubator at 28℃ for 18 h, zebrafish were anesthetized with tricaine for approximately 1 min. They were then washed three times with phosphate-buffered saline (PBS), each time for 5-10 minutes. 1.5 mL of 3,3-dimethylbenzidine staining solution was added, and staining was performed at 28℃ for 40 min. The staining was then repeated three times with butylene terephthalate-butylene succinate (PBST). The samples were observed and photographed using a stereomicroscope, and the relative fluorescence area and relative fluorescence intensity of cardiac erythrocytes were analyzed using normalization.

[0100] from Figure 8 and Figure 9 As can be seen, compared with the normal group, the model group (phenylhydrazine) significantly reduced the fluorescence area and fluorescence intensity of zebrafish cardiac erythrocytes, indicating that the phenylhydrazine-induced anemia model in zebrafish was successful. Compared with the model group, the dipeptide significantly increased the fluorescence area and fluorescence intensity of cardiac erythrocytes, indicating that the dipeptide significantly improved the effects of phenylhydrazine-induced anemia in zebrafish. Specifically, the fluorescence area and fluorescence intensity of cardiac erythrocytes increased by 71.4% and 99.8% respectively compared with the model group, essentially returning to normal levels.

[0101] Example 6: Verification of the immunomodulatory efficacy of active peptides (synthesized peptides in Example 4)

[0102] This embodiment utilizes a zebrafish immune-depressed model induced by cyclophosphamide (CTX) to verify the efficacy of the active peptide. Wild-type zebrafish (AB) were housed at the zebrafish platform of Zhejiang University School of Medicine, and this experiment was approved by the Zhejiang University Center for Experimental Animal Ethics (ethics number ZJU20250649).

[0103] Wild-caught AB strain zebrafish embryos (48 hpf) were demembranous and placed in six-well plates, with two parallel wells per group and 10-15 embryos per well. After 48 h of treatment with the drug, 2.5 μg / mL neutral red dye and PTU were added to each well, and the plates were stained in the dark for 6 h. The embryos were washed with systemic water in the dark, anesthetized, and fixed with 6% methylcellulose. The embryos were photographed under a stereomicroscope, and the number of macrophages in the head was counted. Data were analyzed using GraphPad Prism software.

[0104] The groups are as follows:

[0105] (1) Blank control group (CON): 0.5% DMSO + system water + PTU;

[0106] (2) Model group (MOD): 200 μg / mL CTX (dissolved in DMSO) + system water + PTU, DMSO volume fraction is 0.5%;

[0107] (3) LW group (LW): LW 0.5, 1, 5, 10, 25, 50 μg / mL + 200 μg / mL CTX (dissolved in DMSO) + system water + PTU, with DMSO volume fraction of 0.5%.

[0108] from Figure 10 As can be seen, compared with the blank control group, the model group (cyclophosphamide) significantly reduced the number of head macrophages in zebrafish, indicating that the cyclophosphamide-induced immune decline model in zebrafish was successful. Compared with the model group, the dipeptide LW significantly increased the number of head macrophages in zebrafish, indicating that the dipeptide can significantly improve the immune decline effect of cyclophosphamide in zebrafish. Among them, 0.5 μg / mL of LW dipeptide can significantly increase the number of head macrophages in zebrafish, increasing it by 18.55% compared with the model group.

[0109] The above description is merely a specific embodiment of the present invention, intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly, and should not be construed as limiting the scope of protection of the present invention. All equivalent modifications or substitutions made based on the essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. The application of dipeptide LW in the preparation of products that improve blood and qi function, characterized in that, The amino acid sequence of the dipeptide LW is Leu-Trp.

2. The application as described in claim 1, characterized in that, The product is used to improve anemia.

3. The application as described in claim 1, characterized in that, The improvement in blood and qi function is manifested by increasing the levels of red blood cells, hemoglobin, platelets, and immune cells.

4. The application as described in claim 1, characterized in that, The dipeptide LW was prepared by solid-phase synthesis or isolated from egg white protein hydrolysate.

5. A small molecule peptide from egg white with qi- and blood-tonifying effects, characterized in that, The egg white small molecule peptide contains a dipeptide LW with the amino acid sequence Leu-Trp; the preparation method of the egg white small molecule peptide includes the following steps: (1) Mix egg white protein powder with water at a mass ratio of 1:10-20, and then cut to obtain an egg white protein powder solution; (2) Heat the egg white protein powder solution to 50℃, adjust the pH value to 8.0±0.2, add 0.5%-1.0% alkaline protease by weight of egg white protein powder, and hydrolyze for 2-2.5 hours; then add 0.4%-0.8% papain by weight of egg white protein powder, and continue hydrolyzing for 4-4.5 hours; then add 0.05%-0.1% neutral protease by weight of egg white protein powder, and continue hydrolyzing for 2-2.5 hours to obtain the enzymatic hydrolysate; (3) After centrifuging the enzymatic hydrolysate at a speed of 4000-6000 r / min, the supernatant was collected and filtered through a membrane with a molecular weight cutoff of 1000 Da. The filtrate was then concentrated, sterilized, and dried to obtain the egg white small molecule peptide.

6. The application of the egg white small molecule peptide as described in claim 5 in the preparation of products that improve blood and qi function.

7. The application as described in claim 6, characterized in that, The improvement of blood and qi function includes improving anemia.

8. The application as described in claim 6, characterized in that, The product is available in tablet, hard capsule, soft capsule, oral solution, granules, or powder form.

9. The application as described in claim 6, characterized in that, The product comprises an effective dose of small molecule peptides from egg white and a pharmaceutically acceptable carrier.

Images