CN115990239A - Application of small molecular peptide and derivatives thereof in preparation of bone health products - Google Patents

Abstract

The invention discloses application of a small molecular peptide and a derivative thereof in preparing bone health products, wherein the small molecular peptide comprises dipeptide Cyclo (L-Phe-L-Phe). The dipeptide Cyclo (L-Phe-L-Phe) has remarkable effect of promoting osteoblast proliferation at the concentration lower than 20 mu M, can effectively increase the bone density of the spine vertebrae of the zebra fish, reverse the decrease of the bone density of the spine vertebrae of the zebra fish induced by dexamethasone and remarkably promote the bone formation of transgenic zebra fish (osx:cy 25), and in addition, the dipeptide Cyclo (L-Phe-L-Phe) is derived from natural protein, has low toxicity and high safety, and has good industrial application prospect.

Description

Application of small molecular peptide and derivatives thereof in preparation of bone health products

Technical Field

The invention relates to the technical field of food and medical health, in particular to application of a small molecular peptide and a derivative thereof in preparing bone health products.

Background

Bones are important components of human body structures and are also support brackets for various functions of the human body. Bone health plays a central role in human health, and abnormal bone metabolism can affect metabolism and health of other systems such as cardiovascular system, wherein osteoporosis is the most common systemic abnormal bone metabolism disease and is characterized by decreased bone density value (Bone mineral density, BMD), decreased bone strength, impaired bone mineralization and increased fracture risk. Brittle fracture of multiple parts such as spine, hip, wrist and the like caused by osteoporosis seriously affects the exercise function and life quality of patients, and brings great economic and medical burden to society. Current treatments for osteoporosis are based mainly on two strategies, one is to increase osteoblast differentiation and mineralization, promote bone formation, aim to supplement missing bone tissue by increasing osteoblast and bone cells, increase bone mass, related commercial drugs are mainly parathyroid hormone (PTH); another strategy is to reduce bone resorption by promoting osteoclast apoptosis and inhibiting osteoclast activation, relieve continuous loss of bone mass, and improve osteoporosis and related symptoms, wherein related clinical drugs mainly comprise bisphosphates. However, these drugs have a lot of side effects after long-term use, such as toxic and side effects on kidney, liver, gastrointestinal tract and other tissues and organs caused by long-term use of bisphosphate drugs, and the risk of osteosarcoma is increased after long-term use of parathyroid hormone drugs.

In the related art, research and industrialization of bioactive peptides are of great interest, and bioactive peptides generally have 2-20 amino acid residues and have a molecular weight of less than 6000Da. The food-borne bioactive peptide has the characteristics of high safety, good absorption, rich nutrition and the like, and has wide application prospect in the fields of foods, health-care foods and biological medicines. Related researches show that chicken peptides have wide biological activities such as antioxidation, anti-inflammation, immunoregulation and the like, but the market mainly uses oligomeric mixed peptides, and the action mechanism and the material basis of the chicken peptides in bone diseases are not clear.

Therefore, development of a small-molecule peptide which has high safety and low side effects and is suitable for long-term administration is very necessary and urgent for preparing bone health products.

Disclosure of Invention

The present invention aims to solve at least one of the technical problems existing in the prior art. Therefore, the invention provides application of the small molecular peptide and the derivative thereof in preparing bone health products, which can effectively increase the bone density of zebra fish spine vertebrae, reverse the decrease of the bone density of the zebra fish spine vertebrae induced by dexamethasone and obviously promote the bone formation of transgenic zebra fish (osx:cy 25), and has obvious effects on preventing and improving osteoporosis symptoms.

The invention also provides application of the small molecular peptide or the derivative thereof in preparing a product for promoting proliferation of osteoblast precursor cells.

The invention also provides application of the small molecular peptide or the derivative thereof in preparing a product for promoting animal bone formation.

The invention also provides application of the small molecular peptide or the derivative thereof in preparing a product for increasing animal bone density.

The invention also provides application of the small molecular peptide or the derivative thereof in preparing a product for preventing and/or improving osteoporosis symptoms.

In a first aspect, the invention provides an application of a small molecular peptide and a derivative thereof in preparing bone health products, wherein the small molecular peptide is dipeptide Cyclo (L-Phe-L-Phe).

The application according to the embodiment of the invention has at least the following beneficial effects: the oligopeptide CPP (namely dipeptide Cyclo (L-Phe-L-Phe)) provided by the invention has excellent effects on promoting osteogenesis, increasing bone density and the like, and specifically comprises the following aspects:

(1) The oligopeptide CPP provided by the invention can promote the proliferation of osteoblast precursor cells MC3T3-E1 at a low dosage, and has no obvious toxicity at a higher concentration;

(2) The oligopeptide CPP provided by the invention has no obvious toxic effect on juvenile zebra fish;

(3) The oligopeptide CPP provided by the invention can effectively increase the bone density of the zebra fish spine vertebrae;

(4) The oligopeptide CPP provided by the invention can effectively reverse dexamethasone-induced reduction of the spine bone density of the zebra fish;

(5) The oligopeptide CPP provided by the invention significantly promotes the bone formation of transgenic zebra fish (osx:cy 25).

In a second aspect of the invention, there is provided the use of a small molecule peptide, which is the dipeptide Cyclo (L-Phe-L-Phe), or a derivative thereof, in the manufacture of a product for promoting proliferation of osteoblast precursor cells.

In a third aspect of the invention, there is provided the use of a small molecule peptide, which is the dipeptide Cyclo (L-Phe-L-Phe), or a derivative thereof, in the manufacture of a product for promoting osteogenesis in an animal.

In a fourth aspect of the invention, there is provided the use of a small molecule peptide, which is the dipeptide Cyclo (L-Phe-L-Phe), or a derivative thereof, in the manufacture of a product for increasing bone mineral density in an animal.

In a fifth aspect, the invention provides the use of a small molecule peptide or derivative thereof for the preparation of a product for preventing and/or ameliorating the symptoms of osteoporosis.

In some embodiments of the invention, the dipeptide Cyclo (L-Phe-L-Phe) has the structural formula:

in some embodiments of the invention, the products include pharmaceuticals and functional foods

In some embodiments of the invention, the dosage form of the pharmaceutical product is in solid, semi-solid or liquid form;

preferably, the dosage form of the drug is an aqueous solution, a non-aqueous solution, a suspension or a paste;

more preferably, the medicament is in the form of tablets, capsules, granules, pills, oral liquid, emulsion, dry suspension, dry extract or injection.

In some embodiments of the present invention, the preparation raw materials of the medicine may further include pharmaceutical excipients.

In some preferred embodiments of the present invention, the pharmaceutical excipients are conventional pharmaceutical carriers in the art, and may be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipients;

preferably, the pharmaceutical excipients are selected from at least one of disintegrants, diluents, lubricants, binders, wetting agents, flavoring agents, suspending agents, surfactants, preservatives;

more preferably, the disintegrating agent is at least one selected from corn starch, potato starch, crosslinked polyvinylpyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, crosslinked sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, alginic acid;

more preferably, the diluent is selected from at least one of lactose, sucrose, mannitol, corn starch, potato starch, calcium phosphate, calcium citrate, crystalline cellulose;

more preferably, the lubricant is at least one selected from the group consisting of silica gel micropowder, magnesium stearate, calcium stearate, stearic acid, talc, and anhydrous silica gel;

more preferably, the binder is at least one selected from acacia, gelatin, dextrin, hydroxypropyl cellulose, methylcellulose, polyvinylpyrrolidone;

more preferably, the wetting agent is selected from sodium dodecyl sulfate;

more preferably, the flavoring agent is at least one selected from aspartame, stevioside, sucrose, maltitol, citric acid;

more preferably, the suspending agent is at least one selected from acacia, gelatin, methylcellulose, sodium carboxymethylcellulose, hydroxymethyl cellulose, and aluminum stearate gel;

more preferably, the surfactant is at least one selected from lecithin, sorbitan monooleate, and glyceryl monostearate;

more preferably, the preservative is selected from at least one of methylparaben or propylparaben.

In some embodiments of the invention, the functional food comprises at least one of a decoction, a beverage, a candy, an oral liquid, a capsule, a tablet, and a powder.

In some embodiments of the invention, the dipeptide Cyclo (L-Phe-L-Phe) includes natural products of animal, plant or microbial origin.

In some embodiments of the invention, the dipeptide Cyclo (L-Phe-L-Phe) includes compounds having the same structure or similar structures synthesized by fermentation synthesis, eukaryotic expression systems, or synthesized artificially.

In some embodiments of the invention, the dipeptide Cyclo (L-Phe-L-Phe) is extracted from chicken with an organic reagent that is one or more of acetic acid, diethyl ether, citric acid, formic acid, tartaric acid, malic acid, acetone, chloroform, ethyl acetate, dichloromethane, benzene, and petroleum ether.

In some embodiments of the invention, the dipeptide Cyclo (L-Phe-L-Phe) is extracted from chicken using acetic acid and diethyl ether as organic reagents.

In some embodiments of the invention, the dipeptide Cyclo (L-Phe-L-Phe) is extracted by:

step 1, drying and crushing chicken to obtain chicken powder;

step 2, adding an organic reagent into chicken powder to extract the oligopeptide and the derivative thereof, collecting the organic solvent and drying to obtain crude oligopeptide and the derivative thereof;

step 3, dissolving the powder in a buffer solution, eluting through a Sephadex hh-20 chromatographic column, collecting fractions and drying to obtain oligopeptide and derivative semi-finished product powder thereof;

and 4, dissolving the semi-finished product in a buffer solution, eluting through an ODS-HG-5 liquid chromatographic column, collecting fractions and drying to obtain dipeptide Cyclo (L-Phe-L-Phe) powder.

In some embodiments of the invention, the buffer solution is an aqueous solution of 80wt% acetonitrile.

Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Drawings

The invention is further described with reference to the accompanying drawings and examples, in which:

FIG. 1 is a graph showing the effect of the oligopeptide CPP of the present invention on the viability of osteoblast precursor cells MC3T 3-E1;

FIG. 2 is a graph showing the effect of the oligopeptide CPP of the present invention on increasing zebra fish bone density;

FIG. 3 is a fluorescence microscopy image of a zebra fish spine vertebra in accordance with example 4 of the present invention;

FIG. 4 is a graph showing the statistics of fluorescence areas of the vertebrae of the zebra fish spine of example 4 of the present invention;

FIG. 5 is a microscopic view of the hard bones of zebra fish of example 5 of the present invention;

FIG. 6 is a graph showing the fluorescence intensity statistics of hard bones of zebra fish in example 5 of the present invention.

Detailed Description

The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention.

In the description of the present invention, the descriptions of the terms "one embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

In the embodiment of the invention, the oligopeptide CPP is extracted from chicken, and the specific acquisition method is as follows:

step S1, preparing chicken powder: cutting fresh chicken, drying, pulverizing at 10000rpm/min with pulverizer, suspending for one minute every 2 minutes, pulverizing for 5 minutes to obtain dry powder, sieving with 40 mesh sieve, and sieving to obtain chicken powder.

Step S2, extracting small molecule peptides: 135g of the chicken powder prepared above is weighed, then acetic acid solution with the concentration of 1 mol/L (mol/L) is added to prepare chicken acetic acid solution with the concentration of 200mg/mL, then an equal volume of diethyl ether solution is added, and immediately shaking is carried out to fully extract, after standing for 10 minutes, diethyl ether layer solution is collected and dried by a rotary evaporator, and the obtained dry powder is small molecular peptide crude product powder.

Step S3, preparation of an active site containing CPP: the dry powder obtained above was dissolved in 80% acetonitrile/water solution and equilibrated with 80% acetonitrile/water solution as buffer, then the sample was loaded onto Sephadex hh-20 column (6×18 cm), and isothermal elution was performed, the eluted fraction was collected every 80mL volume and fractions containing CPP were identified by mass spectrometry, and the fractions containing CPP were dried to prepare a small molecular peptide semi-finished powder.

Step S4, preparation of CPP: dissolving the dry powder prepared in the step S3 in 8% acetonitrile/water, balancing by taking 8% acetonitrile/water solution as a buffer solution, then loading a sample on an ODS-HG-5 liquid chromatographic column, balancing by using the same buffer solution, comparing CPP standard substances, collecting elution fractions at corresponding retention time of the CPP, and then drying the CPP to prepare the CPP powder.

Step S5, CPP powder identification: dissolving the CPP powder prepared in the step S4 in dimethyl sulfoxide (DMSO), and then detecting the purity of the CPP powder by using a high performance liquid chromatography tandem ultraviolet detector under the following chromatographic conditions: the liquid phase is separated by a reversed phase system, 0.1% formic acid water is used as an eluting A phase, and acetonitrile is used as an eluting B phase. The column model was (HSS T31.8 μm, C18, 100X 2.1mm,Waters Acquity). The elution procedure was as follows: 0min:25% B; 25% B for 0.25 min; 1.5min:55%B3min:65%B;5.5min 99% B;9min 99% B;9.5min 25% B;15min 25% B. The flow rate was 0.25mL/min. The column temperature was 40 ℃. The sample injection amount was 2. Mu.L. The detection wavelength of the ultraviolet detector was 254nm. Next, the characterization verification is performed on the CPP powder by using high performance liquid chromatography tandem mass spectrometry, and the chromatographic and mass spectrometry conditions are as follows: the Siemens flight (Thermo Fisher Scientific) dual ternary high performance liquid chromatography (DGLC) is connected with a quadrupole orbitrap high-resolution mass spectrometer (Q-exact) for LC-MS/MS analysis. The liquid phase is separated by a reversed phase system, 0.1% formic acid water is used as an eluting A phase, and acetonitrile is used as an eluting B phase. The column model was (HSS T31.8 μm, C18, 100X 2.1mm,Waters Acquity). The elution procedure was as follows: 0min:25% B; 25% B for 0.25 min; 1.5min:55%B;3min 65% B;5.5min 99% B;9min 99% B;9.5min 25% B;15min 25% B. The flow rate was 0.25mL/min. The column temperature was 40 ℃. The sample injection amount was 2. Mu.L. The mass spectrum scanning mode is positive and negative ion switching full scanning. The scanning mass range of the primary mass spectrum is 100-1000m/z, the resolution is 35000, and the maximum injection time is 35ms; the MS2 scan resolution was 17,500, the maximum injection time was 50MS, the collision energy was 35eV, and the scan isolation window was set to 0.8m/z. The spray voltage of the positive ion capillary and the negative ion capillary is set to 3.0kV, the capillary temperature is 350 ℃, the heating temperature of the auxiliary device is 320 ℃, and the S-lens Rf is set to 60. The mass spectrum identification result shows that the product obtained in the embodiment is CPP, and the chemical structure is as follows:

the purity is more than 95 percent.

Example 1: oligopeptide CPP promotes osteoblast precursor cell MC3T3-E1 proliferation experiment

The cell viability was tested in this embodiment using a classical MTT (3- (4, 5) -dimethylhiahizo (-z-y 1) -3,5-di-phenytetrazoliumromi de) assay. MTT is a yellow powder, which is fully called thiazole blue, and the detection principle of the MTT experiment is that MT can penetrate through cell membranes into cells, is reduced into blue-violet needle crystal formazan which is difficult to dissolve in water by amber dehydrogenase in mitochondria of living cells and is deposited in the cells, and dead cells have no function. Crystals formed by living cells can be dissolved by dimethyl sulfoxide (DMSO), and the detected light absorption value can indirectly reflect the survival and growth conditions of the cells.

The experimental procedure for the influence of oligopeptide CPP on the activity of osteoblast precursor cells MC3T3-E1 is as follows:

(1) The method comprises the steps of placing mouse osteoblast precursor cells MC3T3-E1 (cells are purchased from Jiangsu Kaiki Biotechnology Co., ltd.) with good selection state and logarithmic growth phase and growth density of about 80% in a culture dish in 96-well plates, placing 8000 cells per well in an incubator for culture (culture medium: alpha-MEM culture medium+10% fetal bovine serum, 5% CO) ₂ Cell drug experiments were performed after overnight at 37 ℃.

(2) The cell culture solution in the 96-well plate is discarded, 100. Mu.L of prepared CPP solution with serial concentration of 0.06. Mu.M, 0.6. Mu.M, 6. Mu.M, 10. Mu.M, 20. Mu.M, 40. Mu.M, 60. Mu.M, 80. Mu.M and 100. Mu.M is added to each well, 5-6 compound wells are arranged for each dose group, and the culture is continued for 24 hours in an incubator.

(3) After the drug action was completed, 10. Mu.L of the prepared 5mg/ml MTT solution was added to each well, the mixture was placed in an incubator for further culture for 3 hours, the stock solution was discarded, 200. Mu.L of DMSO was added to each well, and after shaking at room temperature for 10 minutes, the absorbance value (A) of each well at 570nm was measured by an enzyme-labeled instrument. Cell viability (%) = (a) was calculated according to the following formula _{Test set} -A _{Blank spaceGroup of} )/(A _{Control group} -A _{Blank group} ) X 100%, wherein the control group was treated without drug addition relative to the test group, and the blank group contained medium only.

As shown in FIG. 1, when the concentration of the oligopeptide CPP is low (less than or equal to 20 mu M), the CPP has a certain promotion effect on MC3T3-E1 cell proliferation. When the CPP concentration is more than 20 mu M and less than 100 mu M, the activity of the osteoblast precursor cells MC3T3-E1 is still maintained above 80%. The CPP has the effect of promoting the proliferation of osteoblasts at a lower concentration, and the MC3T3-E1 cell which is the precursor of the osteoblasts still maintains higher activity at a higher concentration, so that the CPP has better safety.

Example 2: toxicity influence experiment of oligopeptide CPP on juvenile zebra fish

The toxicity of oligopeptide CPP to young zebra fish is detected in the embodiment, and the specific detection method is as follows:

(1) Feeding zebra fish into 28 deg.C water (200 mg instant sea salt is added into 1L reverse osmosis water, the conductivity is 450-550 mu S/cm, pH is 6.5-8.5, and hardness is 50-100 mg/L CaCO) ₃ ). Transgenic hard bone green fluorescent zebra fish (cy 25) is carried out in a natural pairing mating propagation mode. Zebra fish aged 3 days after fertilization (3 dpf) were selected for use in detecting the effect of oligopeptide CPP on toxicity of young zebra fish.

(2) Randomly selecting 3dpf transgenic hard bone green fluorescent zebra fish (cy 25) in 6-well plates, and treating 30 zebra fish in each well (experimental group). The concentrations of the oligopeptides CPP were set to 3.75. Mu.M, 7.50. Mu.M, 15.0. Mu.M, 30.0. Mu.M, 60.0. Mu.M, and the normal control group and the model control group were set at the same time, with a capacity of 3mL per well. Each of the remaining groups, except the normal control group, was water-soluble administered dexamethasone (Dex, 10 μm) for 4 days with a single daily replacement of the fluid, to establish a zebra fish osteogenic injury model. The 5dpf liquid change is performed once. During sample treatment, the number of zebra fish deaths per experimental group was counted daily and removed in time. After 4 days of treatment at 28 ℃, the effect of CPP on toxicity of young zebra fish was examined.

The experimental results are shown in table 1.

Table 1: toxicity results of oligopeptide CPP on young zebra fish (n=30)

The result shows that the oligopeptide CPP does not die when the highest concentration is 60 mu M, and the application concentration window of the CPP is large and the safety is high.

Example 3: experiment of influence of oligopeptide CPP on bone density of spine vertebra of zebra fish

The influence of oligopeptide CPP on the bone density of the spine of the zebra fish is detected in the embodiment, and the specific detection method is as follows:

(1) Feeding wild zebra fish (AB type) into 28 deg.C water (200 mg instant sea salt is added into 1L reverse osmosis water, conductivity is 450-550 mu S/cm, pH is 6.5-8.5, hardness is 50-100 mg/L CaCO) ₃ ). The AB type zebra fish is carried out in a natural pairing mating propagation mode. Zebra fish aged 4 days after fertilization (4 dpf) were selected for use in detecting the effect of oligopeptide CPP on the bone density of the zebra fish bone vertebrae.

(2) Several 4dpf zebra fish were selected, randomly divided into 6 groups, and placed in 24 well plates with 6 fish per well. CPP solutions (10 nM,100nM, 1. Mu.M, 10. Mu.M, 20. Mu.M) were prepared at various concentrations using embryo culture media. Embryo culture solutions in 24 well plates were changed to CPP solutions of different concentrations (where control group was not added), administered continuously for 5 days, changed daily, and zebra fish mortality was recorded. After staining at 9dpf with 0.1% calcein (ph=8.2) for 90 min, excess dye was removed by washing three times with embryo culture solution, the growth of the zebra fish backbone bone was photographed under a fluorescence microscope, and the fluorescence area of the vertebrae was counted with imageJ. Statistical treatment results are expressed in means.+ -. SD. Statistical analysis using SPSS25.0 software, P <0.05 (labeled "×") indicated that the differences were statistically significant.

The experimental results are shown in fig. 2, and it can be seen from the graph that CPP treatment can effectively increase the vertebral generation of zebra fish ridges and significantly increase the bone density of zebra fish.

Example 4: experiment of influence of oligopeptide CPP on dexamethasone-induced vertebral bone density of zebra fish

The influence of oligopeptide CPP on dexamethasone-induced spine bone density of zebra fish is detected in the embodiment, and the specific detection method is as follows:

(1) Feeding wild zebra fish (AB type) into 28 deg.C water (200 mg instant sea salt is added into 1L reverse osmosis water, conductivity is 450-550 mu S/cm, pH is 6.5-8.5, hardness is 50-100 mg/L CaCO) ₃ ). The AB type zebra fish is carried out in a natural paired mating propagation mode, and the zebra fish with the age of 5 days after fertilization (5 dpf) is selected for detecting the influence of oligopeptide CPP on the vertebra bone density of the zebra fish.

(2) 5dpf AB zebra fish were randomly selected in 12-well plates, 5 zebra fish per well. Oligopeptides CPP (0.01. Mu.M, 0.1. Mu.M, and 1. Mu.M) were given in water while setting the normal control group and the model control group at a capacity of 2mL per well. The other groups, except the normal control group, were each water-soluble given dexamethasone (Dex, 10. Mu.M) to establish a zebra fish osteogenic injury model. The liquid is changed once a day. After treatment at 28 ℃ for 4 days, zebra fish were stained with 1% calcein solution and observed under a fluorescence microscope to detect the fluorescence area of the zebra fish spine vertebrae. Statistical treatment results are expressed in means.+ -. SD. Statistical analysis was performed with SPSS25.0 software, P <0.05 indicated that the differences were statistically significant.

The experimental results are shown in fig. 3 and 4, dex significantly inhibits the formation of the zebra fish spine vertebrae, and the CPP treatment can effectively reverse the inhibition of the zebra fish spine vertebrae formation, thereby effectively increasing the bone density of the zebra fish.

Example 5: experiment of Effect of oligopeptide CPP on promotion of osteogenesis of transgenic zebra fish (osx:cy 25)

The experiment shows that the oligopeptide CPP has the effect of promoting the bone formation of transgenic zebra fish (osx:cy 25), and the specific detection method is as follows:

(1) Feeding zebra fish into water (200 mg of instant sea salt is added into 1L of reverse osmosis water, the water quality is that the water is 200mg of instant sea salt is added into each 1L of reverse osmosis water), and the conductivity is 450-550Mu S/cm; the pH is 6.5-8.5; caCO with the hardness of 50-100 mg/L ₃ ). Transgenic hard bone green fluorescent zebra fish (osx:cy25) is bred in a natural pairing mating breeding mode. Zebra fish aged 3 days after fertilization (3 dpf) were selected for use in detecting the effect of oligopeptide CPP on toxicity of young zebra fish.

(2) 3dpf transgenic hard bone green fluorescent zebra fish (osx: cy 25) were randomly selected in 6-well plates, and 30 zebra fish were treated per well. CPP treatment was given in water, and the CPP solution concentrations were set to 7.5. Mu.M, 15. Mu.M and 30. Mu.M, respectively, and the positive control alendronate sodium was 5.00. Mu.g/mL, while the normal control group and the model control group were set to 3mL per well. Except for the normal control group, the other groups were all water-soluble to Dex (10. Mu.M) to establish a zebra fish osteogenic injury model. The 5dpf liquid change is performed once. After treatment at 28 ℃ for 4 days, 10 zebra fish were randomly selected from each experimental group, photographed under a fluorescence microscope, analyzed and data were collected using NIS-Elements D3.20 advanced image processing software, analyzed for the fluorescence intensity of the hard bones of the zebra fish, and the statistical analysis results of the index were used to evaluate the bone-promoting efficacy of the sample. Statistical treatment results are expressed in means.+ -. SD. Statistical analysis was performed with SPSS25.0 software, P <0.05 indicated that the differences were statistically significant.

The experimental results are shown in fig. 5 and 6, and it can be seen from the figures that CPP can effectively promote zebra fish bone formation.

In conclusion, the oligopeptide CPP provided by the invention has excellent effects in the aspects of promoting osteogenesis, increasing bone density and the like, can promote the proliferation of osteoblast precursor cells MC3T3-E1 at a low dose, and does not show obvious toxicity at a higher concentration; the oligopeptide CPP provided by the invention has no obvious toxic effect on juvenile zebra fish; the oligopeptide CPP provided by the invention can effectively increase the bone density of the zebra fish spine vertebrae; the oligopeptide CPP provided by the invention can effectively reverse dexamethasone-induced reduction of the spine bone density of the zebra fish; the oligopeptide CPP provided by the invention significantly promotes the bone formation of transgenic zebra fish (osx:cy 25).

While the embodiments of the present invention have been described in detail, the present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.

Claims (10)

1. The application of the small molecular peptide and the derivative thereof in preparing bone health products is characterized in that the small molecular peptide is dipeptide Cyclo (L-Phe-L-Phe).

2. Use of a small molecule peptide or derivative thereof for the preparation of a product for promoting proliferation of osteoblast precursor cells, wherein the small molecule peptide is the dipeptide Cyclo (L-Phe-L-Phe).

3. Use of a small molecule peptide or derivative thereof for the preparation of a product for promoting osteogenesis in an animal, wherein the small molecule peptide is the dipeptide Cyclo (L-Phe-L-Phe).

4. Use of a small molecule peptide or a derivative thereof for the preparation of a product for increasing bone mineral density in an animal, wherein the small molecule peptide is the dipeptide Cyclo (L-Phe-L-Phe).

5. The use according to any one of claims 1 to 4, wherein the products include pharmaceuticals and functional foods.

6. The use according to claim 5, wherein the pharmaceutical product is in the form of a solid, semi-solid or liquid; preferably an aqueous solution, non-aqueous solution, suspension or paste; more preferably, the composition is in the form of tablet, capsule, granule, pill, oral liquid, emulsion, dry suspension, dry extract or injection.

7. The use according to claim 6, wherein the raw materials for the preparation of the medicament further comprise pharmaceutical excipients; preferably, the pharmaceutical excipients are selected from at least one of disintegrants, diluents, lubricants, binders, wetting agents, flavoring agents, suspending agents, surfactants, preservatives.

8. The use according to claim 7, wherein the disintegrant is at least one selected from the group consisting of corn starch, potato starch, cross-linked polyvinylpyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, cross-linked sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, alginic acid; more preferably, the diluent is selected from at least one of lactose, sucrose, mannitol, corn starch, potato starch, calcium phosphate, calcium citrate, crystalline cellulose; more preferably, the lubricant is at least one selected from the group consisting of silica gel micropowder, magnesium stearate, calcium stearate, stearic acid, talc, and anhydrous silica gel; more preferably, the binder is at least one selected from acacia, gelatin, dextrin, hydroxypropyl cellulose, methylcellulose, polyvinylpyrrolidone; more preferably, the wetting agent is selected from sodium dodecyl sulfate; more preferably, the flavoring agent is at least one selected from aspartame, stevioside, sucrose, maltitol, citric acid; more preferably, the suspending agent is at least one selected from acacia, gelatin, methylcellulose, sodium carboxymethylcellulose, hydroxymethyl cellulose, and aluminum stearate gel; more preferably, the surfactant is at least one selected from lecithin, sorbitan monooleate, and glyceryl monostearate; more preferably, the preservative is at least one selected from the group consisting of methyl paraben and propyl paraben.

9. The use according to claim 5, wherein the functional food is in the form of at least one of a decoction, a beverage, a candy, an oral liquid, a capsule, a tablet, a powder.

10. The use according to any one of claims 1 to 4, wherein the dipeptide Cyclo (L-Phe-L-Phe) is prepared by the following method: the chicken is taken as a raw material and extracted by adopting an organic solvent.

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