Collagen peptide with hematopoietic activity and application thereof in collagen hydrolysate and product

Abstract

The invention discloses an application of two collagen peptides with hematopoietic activity, a collagen hydrolysate containing the collagen peptides and a compound of the collagen peptides. The collagen peptides with hematopoietic activity, the collagen hydrolysate containing the collagen peptides and the compound of the collagen peptides comprise one or a combination of the following polypeptides: 1) a polypeptide of GSAGPpGATGFpGAAGR (p is hydroxyproline), and 2) a polypeptide of which the amino acid sequence is GAAGLpGPK (p is hydroxyproline). The invention also provides a preparation method and application of the collagen peptide with hematopoietic activity and the collagen hydrolysate containing the collagen peptide, the two collagen peptides with hematopoietic activity and the compound thereof have good hematopoietic activity and an effect of relieving hematopoietic inhibition, are non-toxic, can be easily used as functional factors to be added into various health-care foods, and have good health-care effects. Good market prospects are realized in medicine and food industries.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a collagen peptide with hematopoietic activity and its application in collagen hydrolysates and products. Background Art

[0002] Currently, chemotherapy is the main measure for the clinical treatment of malignant tumors. However, about 80% of patients will experience myelosuppression during this process, manifested as adverse reactions such as anemia, bleeding, and reduced immunity, which affect the efficacy of chemotherapy and even lead to the death of patients. Chemotherapy induces the senescence of hematopoietic stem cells (HSCs), resulting in impaired self-renewal ability, which is the most critical mechanism for the occurrence of myelosuppression.

[0003] The application of traditional Chinese medicine diet therapy in myelosuppression after chemotherapy combines "medicine, food, and nourishment", with definite clinical efficacy. At the same time, it has the advantages of being simple, convenient, good palatability, and low toxicity and side effects, and can significantly improve the symptoms of myelosuppression in patients and enhance their quality of life. Collagen peptides have rich biological activities and high nutritional value, and have gradually become a research hotspot. As a traditional product of medicine and food homology, donkey-hide gelatin has attracted much attention due to its good blood-tonifying effect. Exploring the effective components with high hematopoietic efficacy from donkey-hide gelatin helps to develop new medicine and food products. In addition, the research on the combined efficacy of the effective components in donkey-hide gelatin remains the key and difficult point for revealing the high efficacy of products of medicine and food homology in the future. Summary of the Invention

[0004] In order to solve the above technical problems existing in the prior art, the present invention aims at the problems and deficiencies of the existing functional effective components for hematopoiesis and alleviating hematopoietic suppression, and provides an application of a collagen peptide with hematopoietic activity, two polypeptide complexes, and a collagen hydrolysate containing the same. The collagen peptide, polypeptide complexes, and collagen hydrolysate containing the same have hematopoietic activity, no cytotoxicity, the ability to supplement essential amino acids in the human body, and nutritional functions, and can be used to prepare related foods, health products, or drugs for diseases related to hematopoiesis.

[0005] A hematopoietic-active collagen peptide and its collagen hydrolysate have the following amino acid sequences: GSAGPpGATGFpGAAGR (p is hydroxyproline), GAAGLpGPK (p is hydroxyproline). The hematopoietic-active collagen peptide, the complex of the two polypeptides, and their collagen hydrolysate are extracted from donkey-hide gelatin raw materials. An extraction and identification method for the hematopoietic-active collagen peptide and its collagen hydrolysate is provided by combining the techniques of directional protease digestion of donkey-hide gelatin, combined separation and purification methods, theoretical analysis of amino acid composition, and molecular docking prediction of polypeptide biological activity. In the present invention, trypsin is first used to enzymatically hydrolyze donkey-hide gelatin to obtain an enzymatic hydrolysate, and then the enzymatic hydrolysate is analyzed by ultrafiltration, dialysis, and liquid chromatography-tandem mass spectrometry to obtain the polypeptide sequence, and the activities of the polypeptide and its complex in relieving hematopoietic inhibition are verified. Trypsin with specific cleavage sites and the purpose of controlling the molecular weight between 0.5 - 2 kDa are both to obtain amino acids that contribute to the exertion of hematopoietic activity. The molecular docking method is used to predict the hematopoietic activity of the obtained polypeptides, increasing the reliability of the hematopoietic activity of the two polypeptides.

[0006] On the other hand, the present invention also provides a preparation method for a collagen hydrolysate with hematopoietic activity and relieving hematopoietic inhibition activity, including the following steps:

[0007] (1) Using donkey-hide gelatin as the raw material, hydrolyze it with trypsin to obtain a donkey-hide gelatin enzymatic hydrolysate. The enzymatic hydrolysis pH is 9.0 - 11.0, the enzymatic hydrolysis temperature is 35 - 40 °C, the enzymatic hydrolysis time is 10 - 18 h, and the addition ratio of trypsin is 4000 - 8000 U / g. Trypsin with specific cleavage sites helps to obtain arginine and lysine that contribute to the exertion of hematopoietic activity;

[0008] (2) Perform ultrafiltration treatment on the enzymatic hydrolysate. Pass through a filter membrane with a molecular weight of 2 kDa, take the fraction with a molecular weight <2 kDa, and freeze-dry it;

[0009] (3) Further dialyze the fraction retained after ultrafiltration. Select a dialysis bag with a molecular weight cut-off of 500 Da to obtain a dialysis solution containing collagen hydrolysate with a molecular weight >500 Da, freeze-dry it, and verify the activity of relieving hematopoietic inhibition to obtain a collagen hydrolysate with hematopoietic efficacy and relieving hematopoietic inhibition activity. The collagen peptide with a molecular weight between 0.5 - 2 kDa contains amino acids that contribute to the exertion of hematopoietic activity (polypeptides rich in glycine, proline, arginine, and leucine).

[0010] (4) By means of a comprehensive method combining liquid chromatography-tandem mass spectrometry, theoretical analysis of the amino acids in the polypeptide that contribute to hematopoietic activity (polypeptides rich in glycine, proline, arginine, and leucine), and molecular docking (predicting the binding ability to glycogen synthase kinase), the highly hematopoietic active GSAGPpGATGFpGAAGR and / or GAAGLpGPK in the collagen hydrolysate were obtained. (p is hydroxyproline), and the contents of the two peptides and their mass ratio were determined to be between 1:2 - 1:4 (w / w). The polypeptide containing amino acid A and amino acid R is beneficial for binding to glycogen synthase kinase and for the exertion of the hematopoietic activity of the polypeptide. GSAGPpGATGFpGAAGR and GAAGLpGPK were compounded according to the mass ratio in the above-mentioned collagen hydrolysate to increase the content of A and R in the polypeptide, and a highly active hematopoietic active collagen peptide complex was obtained.

[0011] As a preferred technical solution, the products described include foods, health products, and drugs.

[0012] Compared with the prior art, the present invention has the following advantages: The collagen peptide and the collagen hydrolysate containing the same of the present invention are naturally extracted, have hematopoietic activity, can relieve hematopoietic inhibition symptoms, have no cytotoxicity, and have the ability to supplement essential amino acids for the human body and nutritional functions. The related foods, health products, or drugs prepared therefrom have potential application value and broad application prospects for foods or drugs related to hematopoietic diseases. Description of the Drawings

[0013] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only those of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0014] Figure 1 : Schematic diagram of the effects of Peptide 1 (GSAGPpGATGFpGAAGR), Peptide 2 (GAAGLpGPK), and the collagen hydrolysate containing Peptide 1 and Peptide 2 on the proliferation of (A) hematopoietic stem cells (HSCs) and (B) bone marrow mesenchymal stem cells (BMSCs);

[0015] Figure 2 : Schematic diagram of the repair effects of Peptide 1 (GSAGPpGATGFpGAAGR), Peptide 2 (GAAGLpGPK), the complex of Peptide 1 and Peptide 2, and the collagen hydrolysate containing Peptide 1 and Peptide 2 on the bone marrow hematopoietic inhibition and hematopoietic microenvironment damage caused by 5-FU;

[0016] Figure 3: Schematic diagram of the effects of flow cytometry-detected peptide 1 (GSAGPpGATGFpGAAGR), peptide 2 (GAAGLpGPK), the complex of peptide 1 and peptide 2, and the collagen hydrolysate containing peptide 1 and peptide 2 on the cell cycle of HSCs and BMSCs co-cultured under the action of 5-FU;

[0017] Figure 4 : Schematic diagram of the effects of flow cytometry-detected peptide 1 (GSAGPpGATGFpGAAGR), peptide 2 (GAAGLpGPK), the complex of peptide 1 and peptide 2, and the collagen hydrolysate containing peptide 1 and peptide 2 on the apoptosis of HSCs and BMSCs co-cultured under the action of 5-FU;

[0018] Figure 5 : Schematic diagram of the regulation of peptide 1 (GSAGPpGATGFpGAAGR) and peptide 2 (GAAGLpGPK) on the gene pathways related to hematopoiesis and proliferation in cells. Detailed implementation manners

[0019] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. At the same time, it should be noted here that in order to make the embodiments more detailed, the following embodiments are the best and preferred embodiments. For some well-known technologies, those skilled in the art can also adopt other alternative methods for implementation; moreover, the accompanying drawings are only for more specific description of the embodiments and are not intended to specifically limit the present invention.

[0020] It should be pointed out that in the specification, when referring to "an embodiment", "embodiment", "exemplary embodiment", "some embodiments", etc., it indicates that the described embodiment may include specific features, structures or characteristics, but not necessarily every embodiment includes such specific features, structures or characteristics. In addition, when combining an embodiment to describe a specific feature, structure or characteristic, implementing such a feature, structure or characteristic in combination with other embodiments (whether explicitly described or not) should be within the knowledge of those skilled in the relevant art.

[0021] Generally, terms can be understood at least in part from their use in the context. For example, at least in part depending on the context, the term "one or more" used herein can be used to describe any feature, structure or characteristic in a singular sense, or can be used to describe a combination of features, structures or characteristics in a plural sense. In addition, the term "based on" can be understood as not necessarily intended to convey a set of exclusive factors, but rather, at least in part depending on the context, allowing for the existence of other factors that may not be explicitly described.

[0022] Example 1

[0023] Using donkey-hide gelatin as the raw material, it was dissolved with 1% ammonium bicarbonate. 5 mg / mL trypsin and 1% ammonium bicarbonate solution were added, shaken well, and enzymatically hydrolyzed at 37 °C for 12 h. After freeze-drying, donkey-hide gelatin hydrolysate was obtained. It was passed through a 2 kDa ultrafiltration tube to remove the macromolecular peptides with lower biological activity in the donkey-hide gelatin hydrolysate, and the donkey-hide gelatin hydrolysate with a molecular weight below 2 kDa was retained. Then the donkey-hide gelatin hydrolysate was passed through a 500 Da dialysis bag to remove the small molecular peptides, and the donkey-hide gelatin peptides with a molecular weight above 500 Da were retained to preserve their nutrition and biological activity. The donkey-hide gelatin hydrolysate with a molecular weight between 500 Da and 2 kDa was obtained. After freeze-drying, a collagen hydrolysate with hematopoietic efficacy and alleviating hematopoietic inhibition activity was finally obtained. The above collagen hydrolysate was injected into a liquid chromatography-high resolution mass spectrometry instrument for the determination and analysis of the polypeptide sequence. The flow rate was set at 0.3 μL / min, the gradient elution was 60 min, mobile phase A: 0.1% formic acid, mobile phase B: 100% acetonitrile - 0.1% formic acid, and it was directly connected to a Thermo Orbitrap Fusion mass spectrometer. The mass spectrometer used the data-dependent acquisition mode of Xcalibur software, and a single full scan (300 - 1500 m / z, 120000 resolution) was performed in the Orbitrap, and then a 3 s data-dependent mass spectrometry scan was performed in the Ion Routing Multipole. The obtained spectra were analyzed using PEAKS Studio software. The parameters were set as a precursor ion tolerance of 20 ppm and a fragment ion tolerance of 0.02 Da. Theoretical analysis was carried out on the polypeptides rich in glycine, proline, arginine, and leucine that contribute to hematopoietic activity. Subsequently, the potential of the polypeptides for bone marrow hematopoietic activity was predicted by molecular docking of the peptide segments with glycogen synthase kinase GSK3β (ID: 1I09). The molecular docking results are shown in Table 1. The molecular docking binding energies of the polypeptides with glycogen synthase kinase (GSK3β) were -7.12 kcal / mol and -6.893 kcal / mol respectively, indicating that the polypeptides both had strong binding abilities with GSK3β, suggesting that they could prevent the phosphorylation of GSK3β, thereby improving the bone marrow hematopoietic condition. Finally, the peptide segments obtained were: peptide 1 (GSAGPpGATGFpGAAGR, p is hydroxyproline), peptide 2 (GAAGLpGPK, p is hydroxyproline), and it was determined that their mass ratio was between 1:2 - 1:4 (w / w). The polypeptide amino acid sequences and their molecular docking binding energies for bone marrow hematopoietic activity are shown in Table 1.

[0024] Table 1 Peptide sequences of the main collagen peptides in the identified collagen hydrolysate

[0025]

[0026] Impairment of bone marrow hematopoietic function will lead to a decrease in the content of blood cells in the body, and hematopoietic stem cells (HSCs) are mainly responsible for the generation and maintenance of the hematopoietic system. Therefore, by studying the proliferation and growth of bone marrow mesenchymal stem cells (BMSCs) and bone marrow hematopoietic stem cells (HSCs), the bone marrow hematopoietic efficacy of peptide 1 (GSAGPpGATGFpGAAGR) and peptide 2 (GAAGLpGPK) was verified. The CCK-8 method was used to measure the effect of peptide 1 (GSAGPpGATGFpGAAGR), peptide 2 (GAAGLpGPK), and the collagen hydrolysate containing peptide 1 and peptide 2 on the cell proliferation of HSCs. The results are as Figure 1 (A) shown. The survival rates of HSCs under the action of peptide 1 (GSAGPpGATGFpGAAGR), peptide 2 (GAAGLpGPK), and the collagen hydrolysate containing peptide 1 and peptide 2 were all above 95%, and the survival rate increased with the increase in the concentration of the peptide solution. In addition, the effect of the peptide on the cell proliferation of BMSCs was measured, and the results are as Figure 1 (B) shown. The survival rates of BMSCs under the action of peptide 1 (GSAGPpGATGFpGAAGR), peptide 2 (GAAGLpGPK), and the collagen hydrolysate containing peptide 1 and peptide 2 were all above 100%, and the survival rate increased with the increase in the concentration of the peptide solution. It shows that peptide 1 (GSAGPpGATGFpGAAGR), peptide 2 (GAAGLpGPK), and the collagen hydrolysate containing peptide 1 and peptide 2 all have a promoting effect on the proliferation of HSCs and BMSCs, indicating good bone marrow hematopoietic efficacy.

[0027] Example 2

[0028] Peptide 1 (GSAGPpGATGFpGAAGR) protects the hematopoietic microenvironment, promotes the proliferation of hematopoietic stem cells, and reduces the apoptosis rate

[0029] An in vitro co-culture model of bone marrow mesenchymal stem cells (BMSCs) and hematopoietic stem cells (HSCs) damaged by 5-FU in mice was established to observe the effect of the drug-containing serum of peptide 1 (GSAGPpGATGFpGAAGR) on the proliferation of HSCs in the simulated "hematopoietic niche" after chemotherapy, regulate bone marrow hematopoietic function, and protect the hematopoietic microenvironment.

[0030] The CCK-8 method was used to measure the effect of different treatments on the cell proliferation of co-cultured HSCs and BMSCs. The results are as Figure 2 shown. The experimental results showed that compared with the control group, the cell survival rate after treatment with 5-FU (25 μg / mL) decreased from 100% to 59.13%. When peptide 1 (GSAGPpGATGFpGAAGR) was applied to the cells treated with 5-FU, it was found that the cell survival rate increased to above 95%, indicating that it has a relieving effect on the bone marrow hematopoietic inhibition and hematopoietic microenvironment damage caused by 5-FU.

[0031] Flow cytometry was used to detect the cell cycle changes after 24 hours of the action of peptide 1 (GSAGPpGATGFpGAAGR) on co-cultured HSCs and BMSCs treated with 5-FU. The results are as Figure 3 shown. 5-FU significantly inhibited the proliferation of HSCs and BMSCs, significantly increased the proportion of cells in the G0 / G1 phase, while significantly decreased the proportions of cells in the S phase and G2 / M phase, indicating that 5-FU treatment caused G1 phase arrest of cells, thus inhibiting the cell cycle process. Peptide 1 (GSAGPpGATGFpGAAGR) alleviated the inhibition of cell proliferation and G0 / G1 phase arrest by 5-FU, and could restore the G0 / G1 phase of cells to be close to that of the control group, indicating that it could alleviate the damage effect of 5-FU on bone marrow hematopoiesis.

[0032] Flow cytometry was used to detect the apoptosis level of HSCs and BMSCs treated with 5-FU after the addition of peptide 1 (GSAGPpGATGFpGAAGR) ([[]] Figure 4 ). The experimental results showed that peptide 1 (GSAGPpGATGFpGAAGR) significantly alleviated the effect of 5-FU on cell apoptosis, significantly decreased the apoptosis rate, and the apoptosis rate was 25.73% ([[]] Figure 4 ). The above results indicate that peptide 1 (GSAGPpGATGFpGAAGR) can reduce the apoptosis rate caused by 5-FU and alleviate the damage of 5-FU to bone marrow hematopoiesis.

[0033] Example 3

[0034] Peptide 2 (GAAGLpGPK) protects the hematopoietic microenvironment, promotes the proliferation of hematopoietic stem cells and reduces the apoptosis rate

[0035] The CCK-8 method was used to determine the effect of different treatments on the cell proliferation of co-cultured HSCs and BMSCs. The results are as Figure 2 shown. The experimental results showed that compared with the control group, the cell survival rate after treatment with 5-FU (25 μg / mL) decreased from 100% to 59.13%. When peptide 2 (GAAGLpGPK) acted on the cells treated with 5-FU, it was found that the cell survival rate increased to 105.28%, indicating that it had a mitigating effect on the bone marrow hematopoiesis inhibition and hematopoietic microenvironment damage caused by 5-FU.

[0036] Flow cytometry was used to detect the cell cycle changes after 24 hours of the action of peptide 2 (GAAGLpGPK) on co-cultured HSCs and BMSCs treated with 5-FU. The results are as Figure 3As shown, 5-FU significantly inhibited the proliferation of HSCs and BMSCs, significantly increased the proportion of cells in the G0 / G1 phase, while significantly decreasing the proportions of cells in the S phase and G2 / M phase, indicating that 5-FU treatment caused G1 phase arrest in the cells, thereby inhibiting the cell cycle progression. Peptide 2 (GAAGLpGPK) alleviated the inhibition of cell proliferation and G0 / G1 phase arrest by 5-FU, and could restore the G0 / G1 phase of the cells to near the control group, suggesting that it could alleviate the damage to bone marrow hematopoiesis caused by 5-FU.

[0037] Flow cytometry was used to detect the apoptosis level of HSCs and BMSCs treated with 5-FU after the addition of peptide 2 (GAAGLpGPK) Figure 4 ). The experimental results showed that peptide 2 (GAAGLpGPK) significantly alleviated the effect of 5-FU on cell apoptosis, significantly decreasing the apoptosis rate, and the apoptosis rate was 15.65% Figure 4 ). The above results indicate that peptide 2 (GAAGLpGPK) can reduce the apoptosis rate caused by 5-FU and alleviate the damage to bone marrow hematopoiesis by 5-FU.

[0038] Example 4

[0039] Collagen hydrolysate containing peptide 1 (GSAGPpGATGFpGAAGR) and peptide 2 (GAAGLpGPK) protects the hematopoietic microenvironment, promotes the proliferation of hematopoietic stem cells and reduces the apoptosis rate

[0040] An in vitro co-culture model of 5-FU-induced injury to mouse bone marrow mesenchymal stem cells (BMSCs) and hematopoietic stem cells (HSCs) was established to observe the effects of the drug-containing serum of collagen hydrolysate containing peptide 1 and peptide 2 on the proliferation of HSCs in the simulated "hematopoietic niche" after chemotherapy, the regulation of bone marrow hematopoietic function, and the protection of the hematopoietic microenvironment.

[0041] The CCK-8 method was used to measure the effect of different treatments on the cell proliferation of co-cultured HSCs and BMSCs, and the results are as Figure 1 shown. The experimental results showed that when collagen hydrolysate containing peptide 1 and peptide 2 was used for the cells treated with 5-FU, it was found that the survival rate of the cells increased to more than 90%, indicating that it had a mitigating effect on the bone marrow hematopoietic inhibition and hematopoietic microenvironment damage caused by 5-FU (p<0.05).

[0042] Flow cytometry was used to detect the cell cycle changes of co-cultured HSCs and BMSCs treated with 5-FU after the action of collagen hydrolysate containing peptide 1 and peptide 2 for 24 h, and the results are as Figure 3As shown in the figure, the collagen hydrolysate containing Peptide 1 and Peptide 2 alleviated the inhibition of cell proliferation and G0 / G1 phase arrest caused by 5-FU, and could restore the G0 / G1 phase of cells to near the control group, indicating that it could alleviate the damage of 5-FU to bone marrow hematopoiesis.

[0043] Flow cytometry was used to detect the apoptosis level of HSCs and BMSCs treated with 5-FU after the addition of the collagen hydrolysate containing Peptide 1 and Peptide 2 ( Figure 4 ). The experimental results showed that the collagen hydrolysate containing Peptide 1 and Peptide 2 significantly alleviated the effect of 5-FU on cell apoptosis, resulting in a significant decrease in the apoptosis rate, and the apoptosis rate was 9.30% ( Figure 4 ). The above results indicate that the collagen hydrolysate containing Peptide 1 and Peptide 2 can reduce the apoptosis rate caused by 5-FU and alleviate the damage of 5-FU to bone marrow hematopoiesis.

[0044] Example 5

[0045] The complex of Peptide 1 (GSAGPpGATGFpGAAGR) and Peptide 2 (GAAGLpGPK) protects the hematopoietic microenvironment, promotes the proliferation of hematopoietic stem cells and reduces the apoptosis rate

[0046] Peptide 1 (GSAGPpGATGFpGAAGR) and Peptide 2 (GAAGLpGPK) can effectively alleviate the damage of 5-FU to bone marrow hematopoiesis, but their inhibitory ability on 5-FU-induced cell apoptosis is similar to that of the collagen hydrolysate containing Peptide 1 and Peptide 2. The synergistic interaction of protein polypeptides will significantly improve the biological activity of the polypeptide complex. The collagen hydrolysate with hematopoietic activity contains a certain mass ratio of Peptide 1 and Peptide 2. The complex obtained by mixing Peptide 1 and Peptide 2 according to the mass ratio in the collagen hydrolysate may have better bone marrow hematopoietic efficacy.

[0047] An in vitro co-culture model of 5-FU-induced damage to mouse bone marrow mesenchymal stem cells (BMSCs) and hematopoietic stem cells (HSCs) was established to observe the effect of the drug-containing serum of the complex of Peptide 1 and Peptide 2 on the proliferation of HSCs in the simulated "hematopoietic niche" after chemotherapy, the regulation of bone marrow hematopoietic function and the protection of the hematopoietic microenvironment.

[0048] The CCK-8 method was used to measure the effect of different treatments on the cell proliferation of co-cultured HSCs and BMSCs. The results are as Figure 2 shown. The experimental results showed that when the complex of Peptide 1 and Peptide 2 was used for cells treated with 5-FU, the survival rate of the cells was higher than 120%, indicating that it had a relieving effect on the bone marrow hematopoietic inhibition and hematopoietic microenvironment damage caused by 5-FU, and the relieving effect was better than that of Peptide 1 (GSAGPpGATGFpGAAGR), Peptide 2 (GAAGLpGPK) and the collagen hydrolysate containing Peptide 1 and Peptide 2.

[0049] The apoptosis levels of 5-FU-treated HSCs and BMSCs after the addition of the peptide 1 and peptide 2 complex were detected by flow cytometry ( Figure 4 ). The experimental results showed that the collagen hydrolysate containing peptide 1 and peptide 2 significantly alleviated the effect of 5-FU on apoptosis, resulting in a significant decrease in the apoptosis rate, which was 8.70% ( Figure 4 ). The above results indicate that the peptide 1 and peptide 2 complex can reduce the apoptosis rate induced by 5-FU, alleviate the damage of 5-FU to bone marrow hematopoiesis, and the alleviating effect is better than that of peptide 1 (GSAGPpGATGFpGAAGR), peptide 2 (GAAGLpGPK) and the collagen hydrolysate containing peptide 1 and peptide 2. The drug-containing Qing in the Fuzheng Buxue diet therapy formula in the literature can significantly reduce the apoptosis rate, but it is still higher than that of the control group [1]. Compared with the results in the literature, the apoptosis rate of the peptide 1 and peptide 2 complex is lower than that of the control group, indicating that this polypeptide complex can significantly reduce the apoptosis caused by 5-FU and exert higher hematopoietic activity.

[0050] Example 6

[0051] Study on the gene pathways regulated by peptide 1 (GSAGPpGATGFpGAAGR) and peptide 2 (GAAGLpGPK) in cells

[0052] For the gene pathways of peptide 1 (GSAGPpGATGFpGAAGR) and peptide 2 (GAAGLpGPK), the differential genes of each treatment group were analyzed by KEGG enrichment. As Figure 5As shown, both peptide 1 (GSAGPpGATGFpGAAGR) and peptide 2 (GAAGLpGPK) groups were enriched in the hematopoietic pathway: Hematopoietic cell lineage, indicating that both peptide 1 (GSAGPpGATGFpGAAGR) and peptide 2 (GAAGLpGPK) have an impact on genes related to bone marrow hematopoiesis. The amino acid fragment Gly-Ala-Ala exists in both peptide 1 (GSAGPpGATGFpGAAGR) and peptide 2 (GAAGLpGPK), which may be one of the influencing factors for their activity. In addition, the pathways mainly enriched by peptide 1 (GSAGPpGATGFpGAAGR) and peptide 2 (GAAGLpGPK) also include the JAK-STAT signaling pathway. The activation of the JAK-STAT signaling pathway leads to the phosphorylation of STAT family members, which can thus regulate the proliferation, survival, and self-renewal of HSCs. The Rap1 signaling pathway regulates cell signal transduction through various cell receptors. Research has shown that Rap1 can be activated into C3G and Sipa1, and Sipa1 is mainly expressed in hematopoietic tissues such as hematopoietic cells. Therefore, the activation state of Rap1 plays an important role in maintaining long-term hematopoiesis in the bone marrow. The p21ras signal switch molecule family in the Ras signaling pathway is an important part of the proliferative response to many extracellular stimuli (including most hematopoietic growth factors and cytokines). The Ras signaling pathway is triggered by the phosphorylation of tyrosine residues on activated cytokine / growth factor receptors, and the activated Ras protein further activates downstream effectors, while point mutations in the Ras protein often trigger blood diseases, multiple myeloma, etc.

[0053] In summary, both peptide 1 (GSAGPpGATGFpGAAGR) and peptide 2 (GAAGLpGPK) alleviate the bone marrow hematopoietic suppression caused by 5-FU to HSCs and BMSCs by adjusting the above-mentioned physiological processes such as hematopoiesis and HSCs proliferation.

[0054] References:

[0055] [1] Wang Xu, Hao Xiaobei, Yang Min, et al. Mechanism study on the intervention of Notch pathway by the medicated serum of the Fuzheng Buxue diet therapy formula in regulating the damage of bone marrow hematopoietic stem cells after chemotherapy [J]. Information on Traditional Chinese Medicine, 2022, 39(02): 18-24.

[0056] The present invention encompasses any alternatives, modifications, equivalent methods, and solutions made to the essence and scope of the present invention. For the public to have a thorough understanding of the present invention, specific details are described in detail in the following preferred embodiments of the present invention, and those skilled in the art can fully understand the present invention without the description of these details. Additionally, to avoid unnecessary confusion to the essence of the present invention, well-known methods, processes, procedures, components, and circuits are not described in detail.

[0057] The above description is only a preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A hematopoietic active collagen peptide and its compound, characterized in that, The hematopoietic-active collagen peptide includes a combination of one or two of the following polypeptides: 1) A polypeptide with the amino acid sequence GSAGPpGATGFpGAAGR (p is hydroxyproline), 2) A polypeptide with the amino acid sequence GAAGLpGPK (p is hydroxyproline).

2. A composition, characterized in that, The composition contains the hematopoietic-active collagen peptide described in claim 1 and a pharmaceutically, food, or health product-acceptable carrier.

3. Use of the hematopoietic-active collagen peptide described in claim 1 or the composition described in claim 2 in the preparation of a hematopoietic preparation.

4. Use of the hematopoietic-active collagen peptide described in claim 1 or the composition described in claim 2 in the preparation of a preparation for treating diseases related to hematopoiesis.

5. The optimal mass ratio (peptide 1: peptide 2) of the optimized compound of the hematopoietic-active collagen polypeptide described in claim 1 is between 1:2 - 1:4 (w / w).

6. The application according to claim 4, characterized in that, The product includes food, medicine, or health products.

7. A collagen hydrolyzate rich in hematopoietic active collagen peptides, characterized in that: It includes one or two hematopoietic-active peptide segments, and the amino acid sequences of the two hematopoietic-active collagen peptides are GSAGPpGATGFpGAAGR and GAAGLpGPK.

8. The preparation method of the hematopoietic-active collagen peptide described in claim 7 includes the following steps: S1. Using donkey-hide gelatin as the raw material, hydrolyzing it with trypsin to obtain a collagen hydrolysate. The enzymatic hydrolysis pH is 9.0 - 11.0, the enzymatic hydrolysis temperature is 35 - 40 °C, the enzymatic hydrolysis time is 10 - 18 h, and the addition ratio of trypsin is 4000 - 8000 U / g; S2. Ultrafiltrating the enzymatic hydrolysate through a filter membrane with a molecular weight of 2 kDa, and taking the fraction with a molecular weight < 2 kDa; placing the sample between -10 - -70 °C and keeping it for 12 h, and freeze-drying it in a system with a vacuum degree of 10 - 50 Pa; S3. Further dialyzing the fraction retained after ultrafiltration, selecting a dialysis bag with a molecular weight cut-off of 500 Da, and replacing the ultrapure water used for dialysis at intervals of 2 h, 4 h, 8 h, 12 h, and 24 h at 4 °C to obtain a dialysis solution containing collagen hydrolysate with a molecular weight > 500 Da; treating the dialysis solution according to the above freeze-drying conditions to obtain a collagen hydrolysate (with a molecular weight between 0.5 - 2 kDa) that relieves hematopoietic inhibition activity; S4. Obtaining the highly hematopoietic-active GSAGPpGATGFpGAAGR and GAAGLpGPK in the collagen hydrolysate through a comprehensive method of liquid chromatography-tandem mass spectrometry, molecular docking (predicting the binding ability with glycogen synthase kinase), and hematopoietic-related cell experiments; polypeptides containing amino acid A and amino acid R are beneficial for binding with glycogen synthase kinase and for the exertion of the hematopoietic activity of the polypeptide. Recompounding GSAGPpGATGFpGAAGR and GAAGLpGPK according to the mass ratio in the above collagen hydrolysate to increase the content of A and R in the polypeptide, and obtaining a highly active hematopoietic-active collagen peptide complex.

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