A tetrapeptide that promotes bone growth

Abstract

The present invention provides a tetrapeptide for promoting bone growth, whose amino acid sequence is Glu-Pro-Gly-Phe (EPGF) and molecular weight is 448.48Da. The tetrapeptide can be prepared from egg yolk protein by protease hydrolysis, separation and purification, or by chemical solid phase synthesis, and belongs to the technical field of functional bioactive peptides. The tetrapeptide prepared by the present invention can promote the growth of osteoblasts in the body, increase the secretion levels of insulin-like growth factor-1 (IGF-1), transforming growth factor-β1 (TGF-β1) and type I procollagen amino-terminal propeptide (PINP) in the cells, thereby achieving the effect of promoting bone growth, and providing new functional ingredients for products that improve bone growth.

Description

Technical Field

[0001] The invention belongs to the technical field of functional bioactive peptides, and in particular relates to a tetrapeptide for promoting bone growth. Background Art

[0002] Eggs are a source of high-quality protein. Their amino acid composition is very close to the needs of the human body. They can provide the body with sufficient protein to promote muscle growth and repair.

[0003] Studies have shown that the protein bioactive peptides in egg yolk can promote the growth and development of bones in the body, participate in the synthesis of substances necessary for the construction and repair of bone tissue, support the growth and differentiation of bone cells, and thus help promote the healthy development of bones and the stability of their structure. In addition, they work synergistically with other nutrients to provide comprehensive nutritional support for bone strength and growth. Therefore, the protein bioactive peptides contained in egg yolk powder have the same function and can be used as raw materials for the preparation of functional products.

[0004] Bone is a strong connective tissue that supports movement, protects important organs, produces blood, and stores minerals. Bone health has become a fundamental condition for achieving a quality and healthy life. The current focus on bone health issues involves bone growth and development in children and osteoporosis in the middle-aged and elderly population. Bone growth disorders not only seriously affect the physical health of children and the middle-aged and elderly population, but also cause great harm to the mental health of these groups. Therefore, it is very urgent to solve the problem of bone growth disorders.

[0005] Existing methods of improving bone health mainly involve supplementing calcium, vitamin D or taking drugs. For example, growth hormones are prone to dependence and affect the function of the body's endocrine system. Alendronate sodium tablets, for example, are bisphosphonates that can inhibit osteoclast activity and reduce the absorption of bone matrix to promote bone growth. However, long-term use can lead to drug resistance, affecting its related effects and making bones fragile and prone to breakage again.

[0006] There have been studies on peptides that promote bone growth in the prior art. For example, patent CN117143189A discloses that a bone growth promoting peptide VPLWMAR is extracted from enzymatically defatted egg yolk powder. VPLWMAR can bind to epidermal growth factor to secrete Wnt glycoprotein, bind to LRP5 receptor protein to upregulate β-catenin protein expression, thereby expressing RUNX2 gene, secreting human type I collagen, alkaline phosphatase and human osteocalcin, and regulating the proliferation and differentiation of osteoblasts. For example, patent CN117859870A discloses that a bone growth promoting peptide TVVEPADR is isolated from hydrolyzed egg yolk powder, which can promote the secretion of osteocalcin and bone alkaline phosphatase, activate the Wnt3a / β-catenin signaling pathway to promote bone formation, and inhibit osteoclast differentiation. There are also studies on extracting water-soluble proteins from fresh egg yolks, and then separating and purifying them after enzymatic hydrolysis to obtain active peptides that promote bone growth. The molecular weight of the peptide segment that effectively promotes bone growth is concentrated in 10-30kDa. However, the large molecular weight and complex amino acid structure of polypeptides can increase the difficulty of absorption and utilization, resulting in low bioavailability of active peptides, thus failing to achieve the corresponding effect. Therefore, these methods cannot effectively solve the problem of bone growth. It is urgent to find ingredients that are effective for bone cell growth to provide new methods for improving bone growth problems in children, adolescents, and middle-aged and elderly people. Summary of the invention

[0007] Technical problems to be solved: In view of the above technical problems, the purpose of the present invention is to provide a tetrapeptide for promoting bone growth, whose amino acid sequence is Glu-Pro-Gly-Phe (EPGF) and molecular weight is 448.48Da. The tetrapeptide can be prepared from egg yolk protein by protease hydrolysis, separation and purification, or by chemical solid phase synthesis, and belongs to the technical field of functional polypeptides. The tetrapeptide prepared by the present invention can promote the growth of osteoblasts in the body, increase the secretion level of insulin-like growth factor-1 (IGF-1), transforming growth factor-β1 (TGF-β1) and type I procollagen amino-terminal propeptide (PINP) in the cells, thereby achieving the effect of promoting bone growth, and providing new functional ingredients for products that improve bone growth.

[0008] Technical solution: A tetrapeptide for promoting bone growth, wherein the tetrapeptide is EPGF, and its amino acid sequence is Glu-Pro-Gly-Phe.

[0009] Furthermore, the OPG protein receptor binding sites of the tetrapeptide are Lys243, Ser245, Ser56, Glu58, Cys59, and Tyr61.

[0010] Furthermore, the preparation method of the tetrapeptide includes an egg yolk protein hydrolysis extraction method and a chemical solid phase synthesis method.

[0011] Use of any of the above tetrapeptides for promoting bone growth in the preparation of products for improving bone growth.

[0012] Furthermore, the tetrapeptide can significantly promote osteoblast proliferation.

[0013] Furthermore, the tetrapeptide can increase the secretion levels of insulin-like growth factor-1, transforming growth factor-β1 and type I procollagen amino-terminal propeptide.

[0014] Furthermore, the tetrapeptide can be used in combination with a component having sleep-promoting activity.

[0015] Furthermore, the product for improving bone growth is in the form of oral liquid, capsule, tablet, and granule. Beneficial Effects

[0016] 1. The amino acid sequence of the tetrapeptide EPGF provided by the present invention is Glu-Pro-Gly-Phe, and the molecular weight is 448.48Da. It can increase the secretion levels of IGF-1, TGF-β1 and PINP, among which IGF-1 can promote bone growth by stimulating the production of extracellular matrix and increasing bone mineral density, and plays an important role in the proliferation and differentiation of chondrocytes in the growth plate; at the same time, TGF-β1 promotes osteoblast proliferation and differentiation, inhibits osteoclast differentiation and its bone absorption function, and helps to regulate bone growth disorders, and PINP can reflect osteoblast activity and promote bone matrix formation.

[0017] 2. The tetrapeptide EPGF provided by the present invention can significantly promote osteoblast proliferation and can be applied to promote bone growth. The tetrapeptide EPGF can not only be chemically solid-phase synthesized, but also can be directionally extracted from egg yolk protein hydrolysates, which will help promote the further development of the preparation of bone growth promoting products. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the total ion map of mass spectrometry analysis of hydrolyzed egg yolk powder;

[0019] Figure 2 This is the molecular docking result diagram of the tetrapeptide EPGF in Example 1;

[0020] Figure 3 is the primary mass spectrum of the tetrapeptide EPGF in Example 1;

[0021] Figure 4 is the secondary mass spectrum of the tetrapeptide EPGF in Example 1;

[0022] Figure 5 The effect of the tetrapeptide EPGF in Example 2 on the proliferation rate of MC3T3-E1 osteoblast precursor cells;

[0023] Figure 6 The effect of the octapeptide TVVEPADR in comparative example 2 on the proliferation rate of MC3T3-E1 osteoblast precursor cells;

[0024] Figure 7 The effect of the hydrolyzed egg yolk powder on the proliferation rate of MC3T3-E1 osteoblast precursor cells in Comparative Example 3;

[0025] Figure 8 The effect of the tetrapeptide EPGF on the secretion level of alkaline phosphatase in Example 4;

[0026] Fig. 9 The effects of the tetrapeptide EPGF in Example 4 on insulin-like growth factor-1 (IGF-1), transforming growth factor-β1 (TGF-β1), CTX-1 (C-terminal peptide of type I collagen) and PINP (amino-terminal propeptide of type I procollagen). DETAILED DESCRIPTION

[0027] The present invention will be further described below in conjunction with embodiments, which are explanations of the present invention and are not limited to the following embodiments: Example 1

[0028] A method for preparing a tetrapeptide for promoting bone growth comprises the following steps:

[0029] S1. Weigh 150kg defatted egg yolk powder, add 1000kg water and mix well, then add 7.5% NaOH solution to adjust the pH to 8.2, homogenize to obtain egg yolk powder solution, raise the temperature to 118 ° C in a high temperature cooking reactor, cook at high temperature for 5h, and continue to keep warm for 5h;

[0030] S2. After the insulation, 450 g of composite protease (128.6 g of papain, 192.8 g of alkaline protease, 64.3 g of bromelain, 64.3 g of flavor enzyme) was added to the egg yolk powder solution, and the enzyme was inactivated at 55 ° C for 8 h to obtain an enzymatic solution;

[0031] S3. The enzymatic hydrolyzate was separated using a disc separator to obtain a supernatant, 20 kg of activated carbon was added to the supernatant, and 40 kg of diatomaceous earth was adsorbed and filtered to obtain a supernatant;

[0032] S4. The concentrate was then evaporated and concentrated by a vacuum low-temperature thin film evaporator to a sugar content of 25 ° to obtain a concentrate;

[0033] S5. The concentrated solution is sterilized at high temperature and spray-dried to obtain hydrolyzed egg yolk powder;

[0034] S6. Perform LC-MS / MS peptide spectrum analysis on the hydrolyzed egg yolk powder to obtain the peptides contained in the hydrolyzed egg yolk powder, sort and classify the peptides, and finally use bioinformatics methods to further perform molecular docking on the initially screened peptides, and determine the final theoretical effective peptides based on the scores. The specific analysis process is as follows:

[0035] (1) Sample pretreatment:

[0036] ① Take an appropriate amount of sample and dissolve it in 50mM NH4HCO3, add DTT solution to the dissolved sample to make the final concentration 10mmol / L, and reduce it in a 56℃ water bath for 1h;

[0037] ② Add IAM solution to make the final concentration 50mmol / L, and react for 40 minutes in the dark;

[0038] ③ Desalt using a self-filled desalting column and evaporate the solvent in a vacuum centrifugal concentrator at 45°C;

[0039] ④ Take 2 μL of blank control buffer and desalted peptide solution for sample loading respectively, and use NanoDrop software to save the A280nm test data.

[0040] (2) Analysis of hydrolyzed egg yolk powder samples by LC-MS / MS

[0041] Capillary liquid chromatography conditions:

[0042] ①Analytical column: 150μm id×150mm, Acclaim PepMap RPLC C18 column, 1.9μm, 100Å;

[0043] ②Mobile phase A: 0.1% formic acid, mobile phase B: 0.1% formic acid, 80% ACN;

[0044] ③Flow rate: 600mL / min;

[0045] ④Analysis time for each component: 66min;

[0046] ⑤Mobile phase method: 0min, 4%B; 2min, 8%B; 45min, 28%B; 55min, 40%B; 56min, 95%B; 66min, 95%B;

[0047] Mass spectrometry parameters:

[0048] ① Primary mass spectrometry parameters:

[0049] Resolution: 70000;

[0050] AGCtarget: 3e6;

[0051] MaximumIT: 100ms;

[0052] Scan range: 300-1800 m / z;

[0053] ② Secondary mass spectrometry parameters:

[0054] Resolution: 17500;

[0055] AGCtarget: 1e5;

[0056] MaximumIT: 50ms;

[0057] TopN: 20;

[0058] NCE / steppedNCE:28.

[0059] The raw file generated by LC-MS / MS data acquisition of hydrolyzed egg yolk powder sample is opened with Xcalibur, and you can see Figure 1 Total ion current chromatogram shown.

[0060] (3) Analysis and docking

[0061] PeptdieRanker scoring was used for preliminary screening, and then molecular docking with the OPG protein receptor was performed to screen out the tetrapeptide EPGF.

[0062] Depend on Figure 1 It can be seen that based on the analysis of the spectrum by Xcalibur software and matching the database by PEAKS Studio10.6 software, 1724 peptides were separated and identified from the hydrolyzed egg yolk powder, and the relative molecular weight of the peptides was less than 1000Da, and the peptide length was between 3-10 peptides.

[0063] 138 peptides were screened out from 1724 peptides by PeptdieRanker score (>0.8) and relative peak area (>106), and molecular docking was performed with the OPG protein receptor. A total of 50 peptides were successfully docked, and the sequences were screened and identified by docking score (<-7.0), number of hydrogen bonds (>6), number of docked amino acid residues and docked amino acids related to bone growth.

[0064] Table 1 Potential bioactive peptides that bind to OPG protein receptors

[0065] Peptide sequence PeptideRanks docking score intensity Amino acid sequence Number of hydrogen bonds EPGF 0.842858 -7.4 63483000 lys243, ser245, ser56, glu58, cys59, tyr61 8

[0066] From Table 1 and Figure 2It can be seen that the docking score of the tetrapeptide EPGF is -7.4, the number of hydrogen bonds formed is 8, and it can bind to the OPG protein receptor at the amino acid residues of Lys243, Ser245, Ser56, Glu58, Cys59, and Tyr61, indicating that the tetrapeptide EPGF can bind to the OPG protein receptor and exert its effect.

[0067] Depend on Figure 3 and Figure 4 It can be seen that the primary and secondary structures of the tetrapeptide EPGF were analyzed by mass spectrometry. The [M+H] + The ion signal is m / z 448.21, which is basically consistent with the molecular weight of the tetrapeptide 448.48 Da, and the amino acid sequence of the tetrapeptide EPGF in the secondary structure is Glu-Pro-Gly-Phe. Example 2

[0068] A method for preparing a tetrapeptide for promoting bone growth comprises the following steps:

[0069] S1. Weigh Fmoc-Phe-Wang Resin and place it in a glass reaction column. Add DCM and swell for 30 minutes. Remove the DCM under reduced pressure.

[0070] S2. The resin was washed with DMF 3 times, 20% piperidine / DMF solution was added and reacted for 20 min to remove the protecting group fmoc, the solution was removed under reduced pressure, and washed with DMF 6 times;

[0071] S3. Weigh the second amino acid Fmoc-Gly-OH and TBTU separately and add them to the resin, dissolve them in DMF and add DIEA, react for 30 min, take the resin for color reaction, observe the color of the solution and the color of the resin, bright yellow solution and yellow resin indicate that the reaction is complete, and remove the solvent under reduced pressure;

[0072] S4. Repeat steps S2 and S3 until the last amino acid Fmoc-Glu(otbu)-oh is connected, then wash with DMF, DCM, and methanol three times each, and drain the resin;

[0073] S5. Add lysis solution to remove the 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, drain and send to MS to obtain the tetrapeptide. Comparative Example 1

[0074] A method for preparing an octapeptide (TVVEPADR) for promoting bone growth comprises the following steps:

[0075] S1. Weigh Fmoc-Arg-Wang Resin and place it in a glass reaction column. Add DCM and swell for 30 minutes. Remove the DCM under reduced pressure.

[0076] S2. The resin was washed with DMF 3 times, 20% piperidine / DMF solution was added and reacted for 20 min to remove the protecting group fmoc, the solution was removed under reduced pressure, and washed with DMF 6 times;

[0077] S3. Weigh the second amino acid Fmoc-Asp-OH and TBTU respectively, add them to the resin, dissolve them in DMF and add DIEA, react for 30 min, take the resin for color reaction, observe the color of the solution and the color of the resin, bright yellow solution and yellow resin indicate complete reaction, remove the solvent under reduced pressure;

[0078] S4. Until the last amino acid Fmoc-Thr-oh is received, then the resin is washed three times with DMF, DCM and methanol, and then dried;

[0079] S5. Add lysis solution to remove the 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, drain and send to MS to obtain the octapeptide TVVEPADR. Example 3

[0080] In vitro proliferation study of EPGF tetrapeptide on MC3T3-E1 osteoblast precursor cells

[0081] The proliferation effect of tetrapeptide EPGF on cell growth was analyzed using MC3T3-E1 osteoblast precursor cell test, as follows: a certain amount of sample was weighed and placed in a 5 mL centrifuge tube, 1 mL of cell culture medium was added and vortexed to prepare a sample mother solution, and then the sample mother solution was gradiently diluted with cell culture medium to prepare a sample working solution, and 100 μL of different sample working solutions were added to a 96-well plate, and the plate was placed in an incubator for 24 h. The next day, the original sample working solution was removed, and the plate was washed once with PBS buffer, and 100 μL of cell proliferation detection solution containing CCK8 was added. After being placed in an incubator for 1 h, the absorbance OD value was measured at 450 nm and the corresponding calculation was performed.

[0082] Depend on Figure 5 It can be seen that when the concentration of tetrapeptide EPGF is in the range of 1-50 μg / mL, tetrapeptide EPGF can significantly promote the proliferation of MC3T3-E1 osteoblast precursor cells, especially when the concentration is 5-10 μg / mL, it has a significant proliferation effect on MC3T3-E1 osteoblast precursor cells, indicating that tetrapeptide EPGF will not affect MC3T3-E1 osteoblast precursor cells under the condition of concentration range of 1-50 μg / mL, and at the same time has a proliferation promoting effect. Comparative Example 2

[0083] Effect of octapeptide TVVEPADR on proliferation of osteoblasts (MC3T3-E1 osteoblast precursor cells)

[0084] The proliferation effect of octapeptide TVVEPADR on cell growth was analyzed using MC3T3-E1 osteoblast precursor cells, as follows:

[0085] Weigh a certain amount of sample, place it in a 5mL centrifuge tube, add 1mL of cell culture medium and vortex to mix, to prepare the sample mother solution, then use the cell culture medium to dilute the sample mother solution in sequence to prepare the sample working solution, add 100μL of different sample working solutions to the 96-well plate, place it in an incubator and culture it for 24h; the next day, remove the original sample working solution, wash it once with PBS buffer, add 100μL of cell proliferation detection solution containing CCK8, place it in an incubator and culture it for 1h, then measure the absorbance OD value at 450nm and perform corresponding calculations.

[0086] Depend on Figure 6 It can be seen that the octapeptide TVVEPADR did not improve the growth and proliferation of osteoblasts at a concentration of 1-500 μg / mL. Comparative Example 3

[0087] Effect of hydrolyzed egg yolk powder on the proliferation of osteoblasts (MC3T3-E1 osteoblast precursor cells)

[0088] The hydrolyzed egg yolk powder prepared in steps S1-S5 of Example 1 was used to analyze the proliferation effect of the hydrolyzed egg yolk powder on cell growth using MC3T3-E1 osteoblast precursor cells, as follows:

[0089] Weigh a certain amount of sample, place it in a 5mL centrifuge tube, add 1mL of cell culture medium and vortex to mix, to prepare the sample mother solution, then use the cell culture medium to dilute the sample mother solution in sequence to prepare the sample working solution, add 100μL of different sample working solutions to the 96-well plate, place it in an incubator and culture it for 24h; the next day, remove the original sample working solution, wash it once with PBS buffer, add 100μL of cell proliferation detection solution containing CCK8, place it in an incubator and culture it for 1h, then measure the absorbance OD value at 450nm and perform corresponding calculations.

[0090] Depend on Figure 7 It can be seen that hydrolyzed egg yolk powder can promote the growth and proliferation of osteoblasts at a concentration of 0.05-1 mg / mL (i.e. 50-1000 μg / mL), especially at a concentration of 0.5 mg / mL (i.e. 500 μg / mL). p <0.05). Example 4

[0091] Effects of Tetrapeptide EPFG on Bone-Related Growth Factors

[0092] The concentration determined in Example 3 was selected to prepare a differentiated culture medium, and the induction intervention was carried out when the MC3T3-E1 cell density grew to 70%. The medium was changed every 2-3 days, and the induced cells on the 7th day were taken for alkaline phosphatase staining and quantitative analysis was performed using image j software; and the cell supernatant was taken on days 0, 7, and 14, and TGF-β1 (transforming growth factor-β1), IGF-1 (insulin-like growth factor-1), CTX-1 (type I collagen C-terminal peptide) and PINP (type I procollagen amino-terminal propeptide) were measured using a kit, and the determination was performed on day 7 of induction.

[0093] Alkaline phosphatase plays an important role in bone maturation. It mainly reflects the activity and function of osteoblasts and is a sign of osteoblast maturation and activity. It usually begins to express on the 4th day and is highly expressed on the 7th to 14th day. Therefore, the induced cells on the 7th day were taken for detection. Figure 8 It can be seen that compared with the blank group, after 7 days of culture, the alkaline phosphatase secretion in the tetrapeptide EPFG group reached an extremely significant level, indicating that the tetrapeptide EPFG can significantly promote the secretion of alkaline phosphatase.

[0094] Transforming growth factor-β1 (TGF-β1) is the most widely studied member of the TGF-β family. It is ubiquitously expressed and can affect the growth of most tissues. Studies have shown that TGF-β1 can promote the growth of osteoblasts, inhibit the production of factors that promote osteoclasts (such as interferon IFN-γ), promote osteoblast proliferation and differentiation, and inhibit bone resorption. Type I procollagen amino-terminal propeptide (PINP) is a bone formation marker secreted by osteoblasts. An increase in its content indicates a rapid synthesis rate of type I collagen. Type I collagen C-terminal peptide (CTX-Ⅰ) is a bone resorption marker and a decomposition product of type I collagen. An increase in its expression indicates increased bone resorption. Insulin-like growth factor-1 (IGF-1) is a broad-spectrum growth factor synthesized mainly in the liver. It is a polypeptide substance that can promote the growth of bones, muscles, and internal organs.

[0095] Depend on Fig. 9 It can be seen that compared with the blank group, after 7 days of culture, TGF-β1 increased by 10.87%, IGF-1 increased by 10.74%, PINP increased by 7.44%, and CTX-Ⅰ increased by 3.98%; after 14 days of culture, TGF-β1 increased by 15.07%, IGF-1 increased by 16.52%, PINP increased by 6.63%, and CTX-Ⅰ increased by 5.70%. The results show that the tetrapeptide EPFG can significantly promote the secretion levels of TGF-β1, IGF-1, and PINP, among which TGF-β1 and IGF-1 have extremely significant promoting effects, proving that the tetrapeptide EPFG can significantly increase the content of TGF-β1 and IGF-1, thereby achieving the effect of promoting bone growth.

[0096] The above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the art can make many possible changes and modifications to the technical solution of the present invention by using the above disclosed methods and technical contents without departing from the spirit and technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the scope of protection of the technical solution of the present invention.

Claims

1. Use of a tetrapeptide for promoting bone growth in the preparation of a product for improving bone growth, characterized in that: The tetrapeptide is EPGF, and its amino acid sequence is Glu-Pro-Gly-Phe.

2. The use according to claim 1, characterized in that: The tetrapeptide can significantly promote the proliferation of osteoblasts.

3. The use according to claim 1, characterized in that: The tetrapeptide can increase the secretion levels of insulin-like growth factor-1, transforming growth factor-β1 and type I procollagen amino terminal propeptide.

4. The use according to claim 1, characterized in that: The product for improving bone growth includes oral liquid, capsule, tablet and granule.

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