Active peptide for promoting osteoblast differentiation and its application

Abstract

The present invention discloses an active peptide that promotes osteocyte differentiation and its application, and relates to the technical field of biological peptides. The amino acid sequence of the active peptide that promotes osteocyte differentiation of the present invention is: Phe-Asp-Phe-Ser-Phe. The active peptide of the present invention has osteogenic activity and strong bone density regulating activity, and the active peptide of the present invention is a single purified active peptide with clear and safe ingredients, which is of great significance for the development of osteoporosis drugs, and solves the problem that the ingredients of the existing enzymatic hydrolysis products of collagen are unclear, which is not conducive to the later drug development and application.

Description

Technical Field

[0001] The present invention relates to the technical field of biological peptides, and in particular to an active peptide for promoting osteocyte differentiation and application thereof. Background Art

[0002] Osteoporosis is a systemic bone disease caused by a variety of reasons, resulting in decreased bone density and bone quality, destruction of bone microstructure, increased bone brittleness, and easy fracture. With the increase of age, the prevalence of osteoporosis in men and women increases significantly, and the treatment cost of patients is high and the compliance is low.

[0003] At present, Western medicine generally treats osteoporosis with drugs, and commonly used drugs include calcium, vitamin D, calcitonin, bisphosphonates, and estrogens. Although these drugs can relieve pain for patients, they are often accompanied by side effects such as allergic reactions, gastrointestinal side effects, and liver and kidney damage. In addition to the traditional Chinese medicine compound prescriptions of Liuwei Dihuang Pills, Zuogui Pills, and Jinkui Shenqi Pills, single Chinese medicines are also used in clinical practice for the treatment of osteoporosis. Generally, Chinese medicines with the effects of invigorating qi, strengthening tendons and bones, and replenishing kidney yang are selected, such as epimedium, eucommia, psoralea, astragalus, radix dipsaci, bone-setting bud, wolfberry, tortoise shell glue, and deer antler glue. Traditional Chinese medicine has unique advantages in preventing and treating postmenopausal osteoporosis. Its treatment focuses on the whole body, and can simultaneously regulate multiple systems such as endocrine and immune systems to improve patients' osteoporosis. A large number of studies have shown that the rational intake of some Chinese patent medicines that have the function of regulating bone growth can effectively prevent the occurrence of osteoporosis and is an effective auxiliary method for medical treatment.

[0004] More and more studies have shown that bioactive peptides have multiple biological activities, can regulate the body's metabolism, play multiple functions, and are easy to digest and absorb, playing an increasingly important role in the prevention or treatment of diseases. In recent years, research on finding peptides with bone density regulating activity from different food-derived proteins has gradually attracted attention. For example, tilapia collagen peptides, bovine bone collagen peptides, pig bone collagen peptides, Antarctic krill peptides, haddock skin collagen peptides, cucumber seed peptides and other substances have been reported to have certain bone density regulating activity. The above-mentioned collagen-derived active peptides are mostly enzymatic hydrolysis products of collagen, and there is no single purified active peptide product. The product composition is unclear, and various problems will be encountered in the later drug development process. Therefore, it is very necessary to obtain efficient and safe anti-osteoporosis active peptides. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to propose an active peptide that promotes osteocyte differentiation, the amino acid sequence of which is Phe-Asp-Phe-Ser-Phe. The active peptide of the present invention has osteogenic activity and strong bone density regulating activity. Moreover, the active peptide of the present invention is a single purified active peptide with clear ingredients and safety, which is of great significance for the development of osteoporosis drugs and solves the problem that the ingredients of the existing collagen enzymatic hydrolysis products are unclear, which is not conducive to the later drug development and application.

[0006] Another object of the present invention is to propose an active peptide (Phe-Asp-Phe-Ser-Phe) for use in preparing a drug for promoting osteoblast differentiation.

[0007] Another object of the present invention is to provide an active peptide (Phe-Asp-Phe-Ser-Phe) for use in preparing a drug for preventing and / or treating osteoporosis.

[0008] Another object of the present invention is to provide a drug comprising the active peptide (Phe-Asp-Phe-Ser-Phe).

[0009] To achieve this object, the present invention adopts the following technical solutions:

[0010] An active peptide for promoting osteocyte differentiation, wherein the amino acid sequence of the active peptide is: Phe-Asp-Phe-Ser-Phe.

[0011] Preferably, the active peptide is obtained from tortoise shell glue enzymatic hydrolysis products after separation and purification, or synthesized by solid phase synthesis.

[0012] The invention discloses an application of an active peptide in preparing a drug for promoting osteocyte differentiation. The amino acid sequence of the active peptide is: Phe-Asp-Phe-Ser-Phe.

[0013] The invention discloses an application of an active peptide in preparing a medicine for preventing and / or treating osteoporosis. The amino acid sequence of the active peptide is: Phe-Asp-Phe-Ser-Phe.

[0014] The technical solution provided in the embodiment of the present application may include the following beneficial effects: the active peptide of the amino acid sequence of Phe-Asp-Phe-Ser-Phe (named: FDFSF) in the technical solution has osteogenic activity and strong bone density regulating activity, and has a good differentiation-promoting effect on osteoblasts (MC3T3-E1 cells), which is of great significance for the development of osteoporosis drugs. Moreover, the active peptide of the technical solution is a single purified active peptide product, the product composition is clear and safe, and can avoid various problems encountered in the later drug development process. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is a high performance liquid chromatogram of the active peptide Phe-Asp-Phe-Ser-Phe of the present invention;

[0016] Figure 2 is a mass spectrum (MS) of the active peptide Phe-Asp-Phe-Ser-Phe of the present invention;

[0017] Figure 3 The figure is a staining effect diagram of alkaline phosphatase in the experimental group (FDFSF), the control group and the blank group in Example 1;

[0018] Figure 4 Statistical graph of alkaline phosphatase content in MC3T3-E1 cells in the experimental group (FDFSF), control group and blank group in Example 1. DETAILED DESCRIPTION

[0019] In order to facilitate the understanding of the present invention, the present invention is described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0020] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. Raw materials used without specifying the manufacturer are all conventional products that can be obtained through commercial purchase.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0022] The technical solution of the present invention is further explained below in conjunction with specific implementation methods.

[0023] An active peptide for promoting osteocyte differentiation, wherein the amino acid sequence of the active peptide is: Phe-Asp-Phe-Ser-Phe.

[0024] It is worth noting that the active peptide of the amino acid sequence of Phe-Asp-Phe-Ser-Phe (named: FDFSF) in the technical solution has osteogenic activity and strong bone density regulating activity, and has a good effect on promoting differentiation of osteoblasts (MC3T3-E1 cells), which is of great significance for the development of osteoporosis drugs. Moreover, the active peptide of the technical solution is a single purified active peptide product, the product composition is clear and safe, and can avoid various problems encountered in the later drug development process.

[0025] To further illustrate, the active peptide is obtained from the tortoise shell glue enzymatic hydrolysis product after separation and purification, or synthesized by solid phase synthesis method.

[0026] The method of the present technical scheme for obtaining active peptides from the enzymatic hydrolysis product of tortoise shell glue by separation and purification is as follows: after treating the tortoise shell glue with pepsin and trypsin respectively, the supernatant is taken out by centrifugation for dialysis purification and then freeze-dried to obtain a low-molecular peptide extract, the low-molecular peptide extract is subjected to LC-MSMS detection, and the active peptide sequence of the tortoise shell glue is obtained to obtain the active peptide (FDFSF) of the present scheme.

[0027] In a most preferred embodiment of the present invention, the active peptide (FDFSF) of the present technical solution is obtained by separation and purification from the tortoise shell glue enzymatic hydrolysis product, comprising the following steps:

[0028] (1) After the tortoise shell glue is crushed and ground, it is passed through a 200-mesh sieve to obtain fine powder, and then 50 mL of artificial gastric juice containing 0.1 g of pepsin is added, and the pH value is adjusted to 1.3. After being shaken on a shaker at 37°C and 100 rpm for 3 hours, it is taken out and neutralized with NaHCO3 to neutrality. 0.5 g of trypsin (2500 U / mg) and 0.34 g of potassium dihydrogen phosphate are added, and after being shaken on a shaker at 37°C and 100 rpm for 6 hours, it is taken out and quenched with 10% TFA, and then centrifuged to obtain the supernatant. The supernatant is dialyzed with a 100DA dialysis bag for 6 hours (to remove the inorganic salt components in the tortoise shell glue), and then freeze-dried to obtain the low-molecular peptide segment of the tortoise shell glue.

[0029] (2) The low molecular peptides of the tortoise shell glue were analyzed by LC-MS / MS, and compared with the proteome of the turtle species in the NCBI database to detect the sequence of the low molecular peptides. After desalting, the peptide sample was centrifuged and dried, then redissolved in 100uL Nano-LC mobile phase A (0.1% formic acid / water), bottled and loaded for online LCMS analysis. The dissolved sample was loaded onto a nanoViper C18 pre-column (3μm, ), and then 20uL volume was used for washing and desalting. The liquid phase was Easy nLC 1200 nanoliter liquid phase system (ThermoFisher, USA). The sample was desalted and retained on the pre-column and then separated by the analytical column. The analytical column specification was C18 reverse phase chromatography column (Acclaim PepMap RSLC, 75μm×25cm C18-2μm ), the gradient used in the experiment was that the mobile phase B (80% acetonitrile, 0.1% formic acid) increased from 5% to 38% in 60 min. The mass spectrometer used the ThermoFisher Q Exactive system (ThermoFisher, USA) combined with the nanospray Nano Flex ion source (ThermoFisher, USA), the spray voltage was 1.9 kV, and the ion transfer tube heating temperature was 275 ° C. The mass spectrometer scanning mode was in the information-dependent acquisition working mode (DDA, Data Dependent Analysis), the primary mass spectrometer scanning resolution was 70000, the scanning range was 100-1500 m / z, and the maximum injection time was 100 ms. In each DDA cycle, up to 20 secondary spectra with charges from 1+ to 3+ were collected, and the maximum injection time of the secondary mass spectrometer ions was 50 ms. The collision cell energy (high-energy collision-induced dissociation, HCD) was set to 28 eV, applicable to all precursor ions, and the dynamic exclusion was set to 6 seconds.

[0030] (3) The original raw spectrum files collected by mass spectrometry were processed and searched using PEAKS Studio 8.5 (Bioinformatics Solutions Inc. Waterloo, Canada) software. The database was the protein database of Mauremys reevesii species (turtle species) downloaded from NCBI. The search parameters were set as follows: the mass tolerance of the primary mass spectrometry was 10ppm, and the secondary mass spectrometry was 0.05Da. The variable modifications were Oxidation (M), Acetylation (Protein N-term), Deamidation (NQ), Pyro-glu from E, Pyro-glu from Q, and hydroxyproline. According to the length of the measured peptides, the amino acids of the obtained peptides ranged from 5 to 36 amino acids. The fewer the number of amino acids, the easier it is for the peptide to be absorbed and function. Since type I collagen is related to osteoporosis and bone development, peptides from type I collagen are more likely to have a pro-osteoblastic effect. Finally, based on the abundance of the peptides measured by LC-MSMS, the higher the abundance, the higher the possibility of exerting an effect. Based on the protein source of the peptides, the length of the peptides and the abundance of the peptides, the active peptide Phe-Asp-Phe-Ser-Phe that promotes osteocyte differentiation in this scheme was screened. The information of the active peptide Phe-Asp-Phe-Ser-Phe is shown in Table 1 below.

[0031] Table 1 Information of active peptide Phe-Asp-Phe-Ser-Phe

[0032]

[0033] Further explanation, the present technical scheme can also use the solid phase synthesis method to synthesize the active peptide Phe-Asp-Phe-Ser-Phe. The solid phase synthesis method can avoid the problem that the application of the active peptide of this scheme is limited by factors such as the source and cost of tortoise shell glue.

[0034] The technical solution of obtaining the active peptide Phe-Asp-Phe-Ser-Phe by solid phase synthesis method comprises the following steps:

[0035] (1) Fmoc-Phe-Wang Resin (Fmoc-Phe-Wang Resin) was placed in a glass reaction column and DCM (dichloromethane) was added to swell for a specified time, and then the DCM was removed under reduced pressure;

[0036] (2) After washing the resin with DMF (dimethylformamide), add piperidine / DM solution and react for 10 to 30 minutes to remove the protective group fmoc, remove the solution under reduced pressure, and wash with DMF several times;

[0037] (3) Add the second amino acid FMOC-Ser(tbu)-OH (fluorenylmethoxycarbonyl-tert-butyloxy-serine) and tbtu (O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate) to the resin, dissolve it in DMF and add DIEA (N,N-diisopropylethylamine). After reacting for 10 to 50 minutes, take the resin for color test reaction and observe the color of the solution and the resin. If the solution is bright yellow and the resin is yellow, it means that the reaction is complete, and remove the solvent under reduced pressure;

[0038] (4) Repeat steps (2) and (3) until the last amino acid FMOC-Phe-OH (fluorenylmethoxycarbonyl-L-phenylalanine) is connected, and then wash with DMF, DCM, and methanol for multiple times, and then drain the resin;

[0039] (5) adding lysis solution to remove the resin and amino acid side chain protecting groups, filtering, adding ether to the filtrate for precipitation, centrifuging and washing in sequence, and draining to obtain 9Phe-Asp-Phe-Ser-Phe.

[0040] Specifically, in a preferred embodiment of the present invention, obtaining the active peptide Phe-Asp-Phe-Ser-Phe by solid phase synthesis method comprises the following steps:

[0041] (1) Weigh 3.35 g of Fmoc-Phe-Wang Resin (fluorenylmethoxycarbonyl-phenylalanine-polybenzyloxybenzyl alcohol), place it in a glass reaction column, add 100 mL of DCM (dichloromethane) to swell for 30 min, and remove the DCM under reduced pressure.

[0042] (2) The resin was washed three times with 100 mL of DMF (dimethylformamide), and 100 mL of 20% piperidine / DM solution was added. The reaction was allowed to proceed for 20 min to remove the protective group fmoc. The solution was removed under reduced pressure and the resin was washed six times with 100 mL of DMF.

[0043] (3) Weigh 1.15 g of the second amino acid FMOC-Ser(tbu)-OH (fluorenylmethoxycarbonyl-tert-butyloxy-serine) and 0.96 g of tbtu (O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate), add them to the resin, dissolve them in DMF and add 2 mL of DIEA (N,N-diisopropylethylamine); react for 30 min, take the resin for color test reaction, observe the color of the solution and the resin, the solution is bright yellow and the resin is yellow, indicating that the reaction is complete. Remove the solvent under reduced pressure.

[0044] (4) Repeat steps (2) and (3) until the last amino acid FMOC-Phe-OH (fluorenylmethoxycarbonyl-L-phenylalanine) 1.16 g is received, and then the resin is washed three times with DMF, DCM, and methanol (each solvent, 150 mL each time), and the resin is drained.

[0045] (5) Add lysis buffer (83% TFA: thioanisole: phenol: DODT: water = 83:5:3:7:2 in lysis buffer) to remove the resin and amino acid side chain protecting groups, filter with sand core, add ether to the filtrate to precipitate, centrifuge, wash the solid three times, and drain to obtain 95% pure Phe-Asp-Phe-Ser-Phe. Use liquid chromatography and liquid chromatography-mass spectrometry to identify the solid phase synthesized peptide fragments. The results are shown in the attached figure. Figure 1 and attached Figure 2 shown.

[0046] The invention discloses an application of an active peptide in preparing a drug for promoting osteocyte differentiation. The amino acid sequence of the active peptide is: Phe-Asp-Phe-Ser-Phe.

[0047] It is worth noting that the active peptide (Phe-Asp-Phe-Ser-Phe) of the present technical solution has the potential to regulate the osteogenic differentiation of MC3T3-E1 cells, has a promoting effect on osteoblast differentiation, and is expected to be used in drugs that promote osteoblast differentiation.

[0048] The invention discloses an application of an active peptide in preparing a medicine for preventing and / or treating osteoporosis. The amino acid sequence of the active peptide is: Phe-Asp-Phe-Ser-Phe.

[0049] It is worth noting that the active peptide (Phe-Asp-Phe-Ser-Phe) of the present technical solution has the potential to regulate the osteogenic differentiation of MC3T3-E1 cells and has a differentiation-promoting effect on osteoblasts. It is expected to be used in the preparation of drugs for preventing and / or treating osteoporosis, thereby preparing drugs for preventing and / or treating osteoporosis.

[0050] A drug comprising an active peptide having the following amino acid sequence: Phe-Asp-Phe-Ser-Phe.

[0051] Specifically, the drug of the present invention can be made into various forms commonly used in the art, for example, it can be made into powders, tablets (including various coated tablets, sustained-release or controlled-release tablets), lozenges, capsules (including soft capsules and hard capsules), granules, pills, dispersible powders, aqueous or oily suspensions, aqueous or oily solutions, emulsions, elixirs, syrups, etc. suitable for oral administration; it can also be made into creams, ointments, gels, aqueous or oily solutions, aqueous or oily suspensions, etc. suitable for local use; it can also be made into powders or liquid aerosols suitable for inhalation; and sterile aqueous or oily injections or lyophilized powder injections, suppositories, etc. suitable for intravenous, subcutaneous or intramuscular injections for parenteral administration.

[0052] Example 1

[0053] The amino acid sequence of the active peptide of this embodiment is Phe-Asp-Phe-Ser-Phe. The active peptide of this technical solution is obtained by separating and purifying the enzymatic hydrolysis product of tortoise shell glue.

[0054] The effect of the active peptide (FDFSF) of this example on osteoblast differentiation was tested below. The testing method comprises the following steps:

[0055] (1) Detection of osteoblast differentiation: Osteoblasts can secrete alkaline phosphatase (ALP), which is an enzyme necessary for bone formation and an early marker of osteoblast differentiation and functional maturation. First, 2×10 4 MC3T3-E1 mice were inoculated in a 24-well plate, and experimental groups, control groups and blank groups were set up respectively, with three replicates in each group; after 24 hours of cell attachment, the culture medium was removed, wherein, in the experimental group, a polypeptide induction solution A containing 100 μg / mL of active peptide (FDFSF) was added (polypeptide induction solution A is a complete culture medium containing 100 μg / mL of active peptide (FDFSF), 10 nmol of dexamethasone, 10 mmol of sodium β-glycerophosphate and 5 μg / ml of ascorbic acid); the control group was added with a control induction solution (the control induction solution is a complete culture medium containing 10 nmol of dexamethasone, 10 mmol of sodium β-glycerophosphate and 5 μg / ml of ascorbic acid); the blank group was added with a complete culture medium (10% FBS+90% DMEM); and then placed in a cell culture incubator at a constant temperature and humidity of 37°C and 5% CO2 concentration for 7 days (the solution was changed every three days). After 7 days, the culture medium was discarded and washed twice with PBS. Then add paraformaldehyde fixative for 30 minutes, rinse once with deionized water, add 500μL BCIP / NBT alkaline phosphatase colorimetric kit, and stain for 30 minutes. Then rinse twice with deionized water to stop staining, and take pictures with an inverted microscope. The results are as follows: Figure 3 shown.

[0056] (2) Quantification of ALP: 2×10 per well 4 MC3T3-E1 mice were inoculated into 24-well plates, and experimental groups, control groups and blank groups were set up respectively, with three duplicate wells in each group. After 24 hours of cell attachment, the culture medium was aspirated, wherein, polypeptide induction solution B containing 100 μg / mL of active peptide (FDFSF) was added to the experimental group (polypeptide induction solution B is a complete culture medium containing 100 μg / mL of active peptide (FDFSF), 10 nmol of dexamethasone, 10 mmol of sodium β-glycerophosphate and 5 μg / ml of ascorbic acid); control induction solution was added to the control group (control induction solution is a complete culture medium containing 10 nmol of dexamethasone, 10 mmol of sodium β-glycerophosphate and 5 μg / ml of ascorbic acid); complete culture medium (10% FBS+90% DMEM) was added to the blank group, and then the 24-well plates were placed in a CO2 constant temperature incubator and cultured for 7 days (replacement of the medium every three days). The culture medium was discarded and washed twice with PBS. 200 μL of cell lysis solution was added to each well, and the cells were placed in an ice bath for 5 min, and then centrifuged at 6000 rpm and 4°C. The supernatant was taken and ALP was detected using an alkaline phosphatase (ALP) kit. The results were as follows: Figure 4 shown.

[0057] Specifically, Figure 3 From left to right, they are the alkaline phosphatase staining results of MC3T3-E1 cells treated with the blank group, control group and experimental group (i.e., FDFSF). Figure 3 It can be seen that the staining range of MC3T3-E1 cells treated with the active peptide FDFSF was significantly increased, which shows that the active peptide FDFS in this scheme can effectively promote osteoblast differentiation.

[0058] Figure 4 The following is a statistical chart obtained after the quantitative test of alkaline phosphatase in MC3T3-E1 cells treated with the blank group, control group and experimental group (i.e., FDFSF). Figure 4 The results showed that the alkaline phosphatase content in MC3T3-E1 cells treated with the active peptide FDFSF was significantly different from that in the control group (P < 0.01), and was much better than that in the blank group. It can be seen that the active peptide FDFSF has the potential to regulate the osteogenic differentiation of MC3T3-E1 cells and is expected to be used in drugs that promote osteoblast differentiation, and is also expected to be used in the preparation of drugs for the prevention and / or treatment of osteoporosis.

[0059] The technical principle of the present invention is described above in conjunction with specific embodiments. These descriptions are only for explaining the principle of the present invention and cannot be interpreted as limiting the scope of protection of the present invention in any way. Based on the explanations herein, those skilled in the art can associate other specific implementations of the present invention without paying creative labor, and these methods will fall within the scope of protection of the present invention.

Claims

1. Use of an active peptide in the preparation of a drug for promoting osteoblast differentiation, characterized in that: The amino acid sequence of the active peptide is: Phe-Asp-Phe-Ser-Phe.

2. Use of an active peptide in the preparation of a drug for preventing and / or treating osteoporosis, characterized in that: The amino acid sequence of the active peptide is: Phe-Asp-Phe-Ser-Phe.

3. Use of an active peptide according to claim 1 in the preparation of a drug for promoting osteoblast differentiation, characterized in that: The active peptide is obtained from tortoise shell glue enzymatic hydrolysis products after separation and purification, or synthesized by a solid phase synthesis method.

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