A tripeptide that promotes bone growth

Abstract

The present invention provides a tripeptide for promoting bone growth, wherein the tripeptide is VPF, and its amino acid sequence is Val‑Pro‑Phe, and its molecular weight is 361.44Da, and it belongs to the technical field of functional bioactive peptides. The tripeptide in the present invention can not only be obtained by separation and purification from egg yolk protein hydrolysis, but also can be obtained by chemical solid phase synthesis, and has a good effect on promoting bone growth, can significantly improve the growth and proliferation level of osteoblasts in the body, promote the synthesis and secretion of bone matrix, and at the same time efficiently promote the secretion of insulin-like growth factor (IGF‑1), transforming growth factor (TGF‑β1) and type I procollagen amino-terminal propeptide (PINP) in the body, and has a good market prospect.

Description

Technical Field

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

[0002] The protein small molecule peptides in egg yolk can increase bone bud cells and promote their growth into bone, thereby supporting and promoting bone elongation, activating cells in the growth area of ​​bones during growth, supporting the growth and differentiation of bone cells, and promoting bone growth and development. The protein small molecule peptides in egg yolk can be used in combination with other nutrients to synergistically stabilize the healthy development and growth of bones, which not only helps the formation of new bones, but also reduces bone damage and makes bone growth healthier.

[0003] Bones are an important supporting structure of the human body, composed of hard bone tissue and elastic tissues such as cartilage, and are an indispensable part of life activities. Bone health is a necessary condition for people to achieve a quality and healthy life, but most of the current attention to bone health issues is focused on bone growth and development during the growth period of children and adolescents and osteoporosis in the middle-aged and elderly population. Poor bone growth and development can cause serious health and psychological problems for children and adolescents, and to a large extent have a serious impact on the lives of the middle-aged and elderly population. Therefore, it is of great significance to ensure the healthy growth and development of bones.

[0004] In the prior art, improving bone disorders and promoting bone growth are mainly achieved through calcium supplementation, vitamin D or drug treatment, but the relevant therapeutic drugs currently on the market all have certain side effects. For example, bisphosphonates reduce bone absorption and increase bone mineralization by inhibiting the activity of osteoclasts, but long-term use may lead to excessive inhibition of bone reconstruction, resulting in accumulation of bone damage, making bones brittle and easy to break; for example, denosumab is a fully human monoclonal antibody against RANKL that can effectively increase bone density and reduce the risk of fractures, but denosumab cannot be stopped at will during use, otherwise it will cause increased bone absorption and a rapid return of bone density to pre-treatment levels.

[0005] Although there have been corresponding studies on active peptides that promote bone growth, such as the invention patent CN117143189A, which discloses a bone growth promoting peptide VPLWMAR, which can be obtained by enzymatic purification of defatted egg yolk powder, and can promote the proliferation, differentiation and mineralization of osteoblasts through the Wnt signaling pathway. For example, the invention patent CN117859870A discloses that the functional peptide extracted from defatted egg yolk powder is TVVEPADR, which can promote bone growth, enhance bone density and reduce osteoporosis; as related studies report, after the egg yolk water-soluble protein is enzymatically hydrolyzed by trypsin and flavor protease, its peptide segment with a molecular weight of 10-30kDa can significantly promote osteoblast proliferation and achieve the purpose of promoting bone formation. However, the large molecular weight of the polypeptide makes its oral bioavailability low, and it usually needs to be delivered by injection, which increases the patient's pain and the risk of treatment. At the same time, the large molecular weight also increases the difficulty of the peptide to pass through the cell membrane, affecting its distribution and metabolic process in the body.

[0006] Therefore, neither oral related drugs nor peptides (such as heptapeptides and octapeptides) have been able to effectively solve the problem of directly targeting bone cell growth. In addition, the peptide sequences in the existing technology are relatively long and cannot be directly absorbed and utilized when entering the human body orally. They need to be further decomposed before utilization. It is urgent to find ingredients that have efficacy against bone cell production and small molecule ingredients that can be directly absorbed, so as to provide a new method 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 tripeptide for promoting bone growth, the tripeptide is VPF, its amino acid sequence is Val-Pro-Phe, and its molecular weight is 361.44Da, belonging to the technical field of functional bioactive peptides. The tripeptide in the present invention can not only be obtained by separation and purification from egg yolk protein hydrolysis, but also by chemical solid phase synthesis. It has a good effect on promoting bone growth, can significantly improve the growth and proliferation level of osteoblasts in the body, promote the synthesis and secretion of bone matrix, and at the same time efficiently promote the secretion of insulin-like growth factor (IGF-1), transforming growth factor (TGF-β1) and type I procollagen amino-terminal propeptide (PINP) in the body, and has a good market prospect.

[0008] Technical solution: A tripeptide for promoting bone growth, wherein the tripeptide is VPF, and its amino acid sequence is Val-Pro-Phe.

[0009] Furthermore, the OPG protein receptor binding sites of the tripeptide are Asp57, Ser245, and Lys243.

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

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

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

[0013] Furthermore, the tripeptide 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 tripeptide can be used in combination with a component having sleep-promoting activity.

[0015] Furthermore, the product for improving bone growth may be in the form of, but not limited to, oral liquid, capsule, microcapsule powder, tablet, granule, and emulsion tablet. Beneficial Effects

[0016] 1. The amino acid sequence of the tripeptide synthesized by the present invention is Val-Pro-Phe (VPF), and the molecular weight is 361.44Da. It can 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). Insulin-like growth factor-1 (IGF-1) promotes the proliferation and differentiation of osteoblasts, enhances the synthesis and mineralization of bone matrix, thereby increasing bone density and bone strength. At the same time, transforming growth factor-β1 (TGF-β1) promotes the proliferation and differentiation of osteoblast precursor cells, increases the number and activity of osteoblasts, and inhibits bone resorption. In addition, the increased secretion of type I procollagen amino-terminal propeptide (PINP) can reflect the activity of osteoblasts and promote the formation of bone matrix, so that the tripeptide VPF has a good effect of promoting osteoblast proliferation and growth.

[0017] 2. The tripeptide VPF prepared by the present invention can be used to prepare products that promote bone growth. It is natural and non-toxic, highly safe, and can be taken for a long time. The synthetic method of the tripeptide VPF does not require harsh operating conditions. It can not only be chemically solid-phase synthesized, but also can be directionally extracted from egg yolk protein hydrolysates. It is also suitable for industrial production. It not only provides a new research direction for improving bone growth disorders, but also can realize the high-value application of defatted egg yolk powder. 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 is the molecular docking diagram of the tripeptide VPF in Example 1;

[0020] Figure 3is the primary mass spectrum of the tripeptide VPF in Example 1;

[0021] Figure 4 is the secondary mass spectrum of the tripeptide VPF in Example 1;

[0022] Figure 5 The effect of the tripeptide VPF in Example 3 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 tripeptide VPF on the secretion content of alkaline phosphatase in Example 4;

[0026] Fig. 9 The effects of the tripeptide VPF in Example 4 on insulin-like growth factor-1 (IGF-1), transforming growth factor-β1 (TGF-β1), CTX-1 (type I collagen C-terminal peptide) and PINP (type I procollagen amino-terminal propeptide). 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 tripeptide for promoting bone growth comprises the following steps:

[0029] S1. Weigh 150kg egg yolk powder (skim), add 1000kg water and mix well to obtain an egg yolk powder mixture;

[0030] S2. Add 7.5% sodium hydroxide aqueous solution to the egg yolk powder mixture, adjust the pH to 8.0, homogenize to obtain a homogenous solution, raise the temperature to 118 ° C in a high-temperature cooking reactor, cook at high temperature for 3h, and continue to keep warm for 5h;

[0031] S3. After the insulation is completed, 450 g of composite protease (150 g of papain, 150 g of alkaline protease, 75 g of bromelain, and 75 g of flavor enzyme) is added to the homogenized liquid, and the enzyme is inactivated after enzymolysis at 55 ° C for 8 h to obtain an enzymolysis solution;

[0032] S4. The enzymatic hydrolysate 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;

[0033] S5. The concentrated solution was evaporated and concentrated by vacuum low-temperature thin film evaporator to a sugar content of 25 °, sterilized by UHT instantaneous 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 of 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, 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] The PeptdieRanker score was used for preliminary screening, and then molecular docking was performed with the OPG protein (osteoprotegerin) receptor to screen out the tripeptide VPF.

[0062] Depend on Figure 1 It can be seen that the total ion chromatogram of the hydrolyzed egg yolk powder was analyzed using Xcalibur software, and the database was matched using PEAKSStudio10.6 software. A total of 1724 peptides were identified, and the relative molecular weights were all <1000Da, and the peptide lengths were distributed between 3-10 peptides.

[0063] The results of PeptdieRanker score (>0.8) and relative peak area (>10 6 ) A total of 138 peptides were screened and screened based on the docking score (<-7.0), the number of docking times of amino acid residues and the docking amino acids related to bone growth. Molecular docking and mass spectrometry identification were also performed with the OPG protein receptor.

[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 VPF 0.873193 -8.2 66140000 asp57,ser245,lys243 3

[0066] From Table 1 and Figure 2 It can be seen that the docking score of the tripeptide VPF is -8.2, and it can bind to the OPG protein receptor at the amino acid residue sites of Asp57, Ser245, and Lys243 to form hydrogen bonds, indicating that the tripeptide VPF can better bind to the OPG protein receptor and exert its effect.

[0067] Depend on Figure 3 and Figure 4 It can be seen that the fragmentation ions of peptides include: N-terminal fragment ions (a, b, c types) and C-terminal fragment ions (x, y, z types). The side chains of a, y, and z-type ions are broken to form d, v, and w-type ions, respectively. In addition, there are internal ions formed by the breakage of both ends. The b and y series ions are the most common. The primary structure of the tripeptide VPF can be analyzed and inferred based on the b or y series fragment ions of the peptide. The [M+H] + The ion signal was m / z 366.20, which was consistent with its molecular weight of 366.44. At the same time, secondary mass spectrometry analysis determined that the primary structure of the tripeptide was Val-Pro-Phe. Example 2

[0068] A method for preparing a tripeptide 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 3 times with DMF, 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 6 times with DMF;

[0071] S3. Weigh the second amino acid Fmoc-Pro-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 the reaction is complete, and remove the solvent under reduced pressure;

[0072] S4. Until the last amino acid Fmoc-Val-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 tripeptide. 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 3 times with DMF, 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 6 times with DMF;

[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] Effect of tripeptide VPF on proliferation of osteoblasts (MC3T3-E1 osteoblast precursor cells)

[0081] The MC3T3-E1 osteoblast precursor cell experiment was used to analyze the proliferation effect of the tripeptide VPF on cell growth, as follows:

[0082] 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.

[0083] Depend on Figure 5It can be seen that the tripeptide VPF prepared in Example 1 can promote the growth and proliferation of osteoblasts at a concentration of 1-100 μg / mL, especially at a concentration of 5-10 μg / mL. p <0.05), but when the concentration of tripeptide VPF was greater than 200 μg / mL, the cell proliferation rate was significantly lower than that of the control group, indicating that tripeptide VPF can survive and rapidly proliferate at a concentration of 1-100 μg / mL for osteoblasts, and will inhibit osteoblasts once the concentration is greater than 200 μg / mL. Comparative Example 2

[0084] Effect of octapeptide TVVEPADR on proliferation of osteoblasts (MC3T3-E1 osteoblast precursor cells)

[0085] The proliferation effect of octapeptide TVVEPADR on cell growth was analyzed using MC3T3-E1 osteoblast precursor cells, as follows:

[0086] 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 stock solution, then use the cell culture medium to dilute the sample stock 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.

[0087] Depend on Figure 6 It can be seen that the octapeptide TVVEPADR prepared in Comparative Example 2 did not achieve improvement effect on the growth and proliferation of osteoblasts at a concentration of 1-500 μg / mL. Comparative Example 3

[0088] Effect of hydrolyzed egg yolk powder on the proliferation of osteoblasts (MC3T3-E1 osteoblast precursor cells)

[0089] The hydrolyzed egg yolk powder was prepared according to steps S1-S5 of Example 1, and the proliferation effect of the hydrolyzed egg yolk powder on cell growth was analyzed using MC3T3-E1 osteoblast precursor cells, as follows:

[0090] 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 stock solution, then use the cell culture medium to dilute the sample stock 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.

[0091] 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 1-1000μg / mL, especially at a concentration of 500μg / mL. p <0.05), but when the concentration of hydrolyzed egg yolk powder was greater than 1000 μg / mL, the cell proliferation rate was significantly lower than that of the control group, indicating that hydrolyzed egg yolk powder can survive and rapidly proliferate at a concentration of 1-1000 μg / mL, and will inhibit osteoblasts once the concentration is greater than 1000 μg / mL. Example 4

[0092] Effects of tripeptide VPF on bone-related growth factors

[0093] The concentration determined in Example 3 was selected to prepare a differentiated culture medium. When the density of MC3T3-E1 cells grew to 70%, induction intervention was performed. The medium was changed every 2-3 days. The induced cells on the 7th day were taken for alkaline phosphatase staining and quantitative analysis was performed using image j software. The cell supernatant was then 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 measurement was performed on day 7 of induction.

[0094] 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, the secretion of alkaline phosphatase in the tripeptide VPF group reached a significant level after 7 days of culture, indicating that the tripeptide VPF promotes the secretion of alkaline phosphatase.

[0095] 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.

[0096] Depend on Fig. 9 It can be seen that compared with the blank group, after 7 days of culture, TGF-β1 increased by 3.99%, IGF-1 increased by 5.79%, PINP increased by 6.41%, and CTX-Ⅰ increased by 0.21%; after 14 days of culture, TGF-β1 increased by 4.08%, IGF-1 increased by 6.28%, PINP increased by 5.55%, and CTX-Ⅰ increased by 1.49%. The results show that the tripeptide VPF can significantly promote the secretion levels of TGF-β1, IGF-1, and PINP at both 7 and 14 days, among which IGF-1 reached a very significant level. Therefore, the tripeptide VPF can promote bone growth by increasing the content of TGF-β1, IGF-1 and PINP.

[0097] 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 tripeptide for promoting bone growth in the preparation of a product for improving bone growth, characterized in that: The tripeptide is VPF, and its amino acid sequence is Val-Pro-Phe.

2. The use according to claim 1, characterized in that: The tripeptide can significantly promote osteoblast proliferation.

3. The use according to claim 1, characterized in that: The tripeptide 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 tripeptide can be used in combination with a component having sleep-aiding activity.

5. 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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