阅读说明：本技术 一种促进成骨细胞增殖的活性肽及其应用 (Active peptide for promoting osteoblast proliferation and application thereof ) 是由 郭燕川 王佳宁 刘俊丽 王富荣 刘芳 于 2019-10-12 设计创作，主要内容包括：本发明公开一种促进成骨细胞增殖的活性肽及其应用。本发明所述活性肽的氨基酸序列如SEQ ID NO:1-SEQ ID NO:5所示。本发明提供的活性肽可以促进成骨细胞增殖,从而预防或治疗骨质疏松。这些活性肽还可以开发成药物、食品或保健品。(The invention discloses an active peptide for promoting osteoblast proliferation and application thereof. The amino acid sequence of the active peptide is shown as SEQ ID NO. 1-SEQ ID NO. 5. The active peptide provided by the invention can promote osteoblast proliferation, thereby preventing or treating osteoporosis. The active peptides can also be developed into medicines, foods or health products.)

1. An active peptide, wherein the amino acid sequence of said active peptide comprises one or more of the following sequences:

GPAGPAGPR；

GPAGPSGPAGK；

GPMGPSGPR, wherein M is oxidized;

GPAGPQGPR；

GPPGSPGPR。

2. a polynucleotide encoding any one of the active peptides of claim 1.

3. Use of the active peptide of claim 1 for the preparation of a medicament, food or health product for promoting osteoblast proliferation.

4. Use of the active peptide of claim 1 for the preparation of a medicament, food or health product for the prevention and/or treatment of osteoporosis.

5. A medicament comprising the active peptide of claim 1.

6. A food product comprising the active peptide of claim 1.

7. A nutraceutical comprising the active peptide of claim 1.

Technical Field

The invention relates to the technical field of active peptides. More particularly, it relates to an active peptide for promoting osteoblast proliferation and its application.

Background

Senile Osteoporosis (OP) is a chronic, progressive disease characterized by a decrease in bone mass and bone microarchitecture and is prone to fracture. With age, the probability of suffering from osteoporosis will increase greatly. Based on WHO statistical data, there are approximately 7500 million OP patients in the united states, europe, and japan. The domestic situation is not optimistic, and the large-scale epidemiological survey in China shows that the prevalence rate of the population over 60 years old is obviously improved. However, most of the drugs for treating osteoporosis on the market have great side effects and common effects.

Bioactive peptides refer to peptide compounds that are beneficial to or have a physiological effect on the vital activities of the living organism. The structure of active peptides can range from smaller dipeptides to larger polypeptides. Moreover, the bioactive peptide has wide sources, including animals, plants, microorganisms and the like. The preparation method of the bioactive peptide comprises an acid method, an alkaline method and an enzyme method, wherein the preparation of the bioactive peptide by the enzyme method is common, at present, most bioactive peptides derived from ossein on the market are enzymolysis products of the ossein, but a single purified bioactive peptide product is not available, the product components are undefined, and various problems can be encountered in the development process of later-stage medicines and health-care products. Therefore, it is necessary to obtain a polypeptide with high-efficiency and safe anti-osteoporosis activity.

Disclosure of Invention

The first purpose of the invention is to provide active peptides derived from bovine bone collagen.

The second purpose of the present invention is to provide the application of the active peptide in promoting osteoblast proliferation and preventing and/or treating osteoporosis.

In order to achieve the purpose, the invention adopts the following technical scheme:

according to a first object of the present invention, the present invention provides an active peptide, the amino acid sequence of which comprises one or more of the following sequences:

GPAGPAGPR, as shown in SEQ ID NO: 1;

GPAGPSGPAGK, as shown in SEQ ID NO: 2;

GPMGPSGPR, wherein M is oxidized as shown in SEQ ID NO 3;

GPAGPQGPR, as shown in SEQ ID NO. 4;

GPPGSPGPR, as shown in SEQ ID NO: 5.

Alternatively, the active peptide may be an active peptide having 80% or more homology with the amino acid sequence of any of the above active peptides, and the function of the active peptide is the same as or similar to that of the above active peptide. For example, the amino acid sequence of the active peptide may be a sequence having 85%, 90%, 95% or 97% homology to the amino acid sequence of any of the above active peptides.

The present invention also provides polynucleotides encoding the above active peptides.

In the case of amino acid sequences of active peptides being known, one skilled in the art can design genes encoding active peptides with different nucleotide sequences based on the principle of degeneracy of codons and the usage preference of codons by different species, as required for the expression of active peptides.

In the invention, the nucleotide sequence of the active peptide is shown as SEQ ID NO 6-SEQ ID NO 10 of the sequence table.

The active peptide of the present invention can be obtained by separation and purification from bovine bone collagen enzymolysis products, and can also be prepared by other methods known in the art, such as a solid phase synthesis method, a biological engineering method, and the like.

According to a second object of the present invention, the present invention provides the use of the active peptide in the preparation of a medicament, food or health product for promoting osteoblast proliferation. Further provides the application of the active peptide in preparing medicaments, foods or health-care products for preventing and/or treating osteoporosis.

The invention further relates to a medicament, a food product or a health product comprising one or more active peptides of the invention, such as GPAGPAGPR, GPAGPSGPAGK, GPMGPSGPR (wherein M is oxidised), GPAGPQGPR, GPPGSPGPR.

The pharmaceutical and nutraceutical products of the present invention may be prepared in various forms as is common in the art, including, but not limited to, powders, tablets (including various coated tablets, sustained-release or controlled-release tablets), lozenges, capsules (including soft and hard capsules), granules, pills, dispersible powders, aqueous or oily suspensions, aqueous or oily solutions, emulsions, elixirs, syrups, and the like, suitable for oral use, creams, ointments, gels, aqueous or oily solutions, aqueous or oily suspensions, and the like; a powder or liquid aerosol suitable for inhalation use; suitable for parenteral administration, intravenous, subcutaneous or intramuscular injection, sterile aqueous or oily injection or lyophilized powder injection, suppository and the like.

The medicine and health care product of the invention can further contain various conventional auxiliary materials and/or other active ingredients. Suitable excipients include, but are not limited to, excipients, lubricants, binders, disintegrants, water soluble polymers, inorganic salts, solvents, dissolution aids, suspending agents, isotonic agents, buffers, preservatives, antioxidants, coloring agents, sweetening agents, acidulants, foaming agents, flavoring agents, and the like.

The invention has the following beneficial effects:

the invention adopts an MTT method, takes promotion of bone cell proliferation as an activity screening index, sequentially carries out reversed-phase silica gel column chromatography, ion exchange column chromatography, gel column chromatography and high performance liquid chromatography on bovine bone collagen enzymolysis products, finally purifies to obtain 5 active peptides with stronger activity, and carries out amino acid sequence identification and activity verification.

Drawings

The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.

FIG. 1 shows a rapid reverse phase silica gel column chromatography chromatogram of the ultrafiltration fraction CP 1.

FIG. 2 shows the results of MTT cell proliferation activity assay of 6 fractions obtained by subjecting the ultrafiltrate fraction CP1 to ODS-AQ rapid reverse phase silica gel column chromatography.

FIG. 3 shows a chromatogram of a rapid reverse phase silica gel column chromatography component CP1-5 ion exchange column chromatography.

FIG. 4 shows the results of MTT cell proliferation activity test of 2 fractions obtained by subjecting fraction CP1-5 to ion exchange column chromatography on a rapid reverse phase silica gel column.

FIG. 5 shows a gel column chromatography chromatogram of ion exchange column chromatography component CP 1-5-1.

FIG. 6 shows the results of MTT cell proliferation activity assay of 2 fractions obtained by purifying the ion-exchange column chromatography fraction CP1-5-1 through gel column chromatography.

FIG. 7 shows a first high performance liquid column chromatography chromatogram of gel column chromatography component CP 1-5-1-A.

FIG. 8 shows the results of MTT cell proliferation activity assay of 4 fractions obtained from the first high performance liquid column chromatography purification of the gel column chromatography fraction CP 1-5-1-A.

FIG. 9 shows a first high performance liquid chromatography component CP1-5-1-A-2 second high performance liquid chromatogram.

FIG. 10 shows the results of MTT cell proliferation activity assay of 2 fractions obtained by the first high performance liquid column chromatography purification of fraction CP 1-5-1-A-2.

FIG. 11 shows the results of cell proliferation activity assay for verifying the 5 active peptides of the present invention using MTT method.

Detailed Description

In order to more clearly illustrate the invention, the invention is further described below with reference to preferred embodiments and the accompanying drawings. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.