CN116082483B - Preparation method and application of polypeptide capable of improving osteoporosis and enhancing bone density - Google Patents
Preparation method and application of polypeptide capable of improving osteoporosis and enhancing bone density Download PDFInfo
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- CN116082483B CN116082483B CN202211104415.4A CN202211104415A CN116082483B CN 116082483 B CN116082483 B CN 116082483B CN 202211104415 A CN202211104415 A CN 202211104415A CN 116082483 B CN116082483 B CN 116082483B
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- polypeptide
- osteoporosis
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- medicament
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/461—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from fish
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
- A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Physical Education & Sports Medicine (AREA)
- Organic Chemistry (AREA)
- Rheumatology (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Pharmacology & Pharmacy (AREA)
- Toxicology (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Gastroenterology & Hepatology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Mycology (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a preparation method and application of a polypeptide capable of improving osteoporosis and enhancing bone mineral density, and belongs to the field of bioactive peptides. The polypeptide has an amino acid sequence shown in SEQ ID NO.1 (MNKKREAEFQ, P-GM-1), a molecular weight of 1280Da and is derived from a heavy chain of the cod myosin. The polypeptide is obtained by enzymolysis, separation (ion exchange fine separation is carried out according to different charges), molecular butt screening and solid phase synthesis. In an in vivo ovariectomized mouse model, the polypeptide can remarkably increase the number of trabeculae of an osteoporosis mouse and improve the bone density level by continuous 12-week intragastric administration. Meanwhile, the hardness of the bone tissue of the ovariectomized mice is also improved by 40%. In conclusion, the polypeptide has wide sources and simple preparation method, has the capability of obviously improving osteoporosis, and can be used as an active ingredient in the fields of biological medicine, food health care and the like related to bone mineral density regulation.
Description
Technical Field
The invention relates to a preparation method and application of polypeptide with the functions of improving osteoporosis and enhancing bone density, and belongs to the field of bioactive peptides.
Background
Osteoporosis (osteoporosis, OP) is a chronic disease of systemic bone metabolic disorder, mainly manifested by reduced bone mineral content, sparse trabecular structure, thin cortical bone, increased bone fragility, etc. The incidence of osteoporosis increases year by year with the prolongation of human life and the increase in the degree of population aging. At the same time, the fracture rate due to osteoporosis is also increasing year by year, causing serious economic burden, and becoming a serious public health problem. Osteoporosis often occurs in association with a variety of factors including age, eating habits, hormone secretion, exercise habits, and genetic factors. The existing medicines for treating osteoporosis clinically mainly comprise bisphosphate, estrogen activator/inhibitor, parathyroid hormone analogue, calcitonin and the like, and the medicines can relieve the pains of patients, but also can often cause side effects such as anaphylactic reaction, gastrointestinal side effects, liver and kidney function damage and the like. Numerous studies have shown that rational intake of certain foods containing active ingredients that regulate bone growth function can effectively prevent the occurrence of osteoporosis, and is also an effective adjunct to medical treatment.
Normal bone homeostasis in humans is maintained by both osteoblast-mediated bone formation and osteoclast-mediated bone resorption. Osteoblasts are responsible for new bone formation, while osteoclasts are responsible for bone breakdown and resorption, which work together to maintain the constant remodeling process of bone tissue. Mouse embryonic osteoblast precursor cells (MC 3T 3-E1) can specifically express alkaline phosphatase (ALP) and early osteogenic differentiation phenotype markers such as an osteogenic related transcription factor Runx-2 and the like, and are widely applied to the research of osteoblast differentiation at present. Integrins (integrins) consist of non-covalently linked alpha and beta heterodimeric subunits, transmembrane adhesion receptors, mediating cell adhesion dependence and adhering to extracellular matrix to promote cell proliferation. Studies have shown that β1 and αvβ containing integrins are important for osteoblast migration, adhesion, proliferation and differentiation and mediate cell interactions with some bone matrix proteins. Certain peptides and proteins can promote bone formation processes in vivo by modulating cellular signal integrins.
More and more researches show that the bioactive peptide has various biological activities, can regulate organism metabolism, plays various functions, has the characteristics of easy digestion and absorption and the like, and plays an increasing role in preventing or treating diseases. In recent years, research on searching polypeptides with bone mineral density regulating activity from different food-borne proteins is focused on, and substances such as tilapia collagen polypeptides, antarctic krill peptides, walleye pollack skin collagen polypeptides, cucumber seed polypeptides and the like are reported to have good bone mineral density regulating activity, but industrial production and utilization of the polypeptides are limited due to the technical limitations, high process cost and the like. The cod (Gadus morhua) is used as one of the fishes with the largest fishing amount in the world year and has higher economic value. It is rich in protein and amino acid components with rich variety, and has high nutritive value and medicinal value. Early researches show that the cod enzymolysis product has strong bone density regulating activity, and can effectively slow down the occurrence of the osteoporosis of the ovary mice. Therefore, the development and identification of active polypeptide with stronger bone density regulation by taking the cod as the raw material has important significance for the development of osteoporosis health-care food and medicines.
Disclosure of Invention
The invention provides a polypeptide preparation method with the functions of improving osteoporosis and enhancing bone density through separation preparation, molecular butt joint and a chemical method combined artificial synthesis method, and the polypeptide preparation method can be applied to the fields of biological pharmacy and the like to regulate bone density or prevent osteoporosis.
The invention provides a polypeptide P-GM-1 with the functions of improving osteoporosis and enhancing bone mineral density, the amino acid sequence of the polypeptide P-GM-1 is Met-Asn-Lys-Lys-Arg-Glu-Ala-Glu-Phe-Gln (MNKKREAEFQ is shown as SEQ ID NO. 1), and the molecular weight of the polypeptide P-GM-1 is 1280Da.
The invention also provides a medicine containing the polypeptide.
In one embodiment, the medicament further comprises a pharmaceutically acceptable carrier.
In one embodiment, the pharmaceutically acceptable carrier comprises one or more of fillers, binders, wetting agents, disintegrants, lubricants, flavoring agents commonly used in medicine.
In one embodiment, the purity of the polypeptide in the medicament is greater than or equal to 98%.
In one embodiment, the polypeptide is administered at a dose of 20 mg/kg/day.
The invention also provides application of the polypeptide in preparing a medicament for relieving and/or treating osteoporosis.
In one embodiment, the alleviating and/or treating osteoporosis comprises:
(a) Recovering the decrease in bone density caused by osteoporosis;
(b) Restoring the reduction in bone mass due to osteoporosis;
(c) Restoring the reduction in the number of trabeculae due to osteoporosis.
The invention also provides application of the polypeptide in preparing health care products which are helpful for improving bone mineral density.
The invention also provides a preparation method of the polypeptide, which comprises the following steps:
1) Preparing cod enzymolysis liquid;
2) And (3) separating and preparing the potential bone density regulating active peptide according to the charge of the polypeptide by using an AKTA protein purifier and a Hitrap Q-HP column.
In one embodiment, the polypeptide may also be prepared by chemical synthesis.
The invention also provides a medicine containing the polypeptide, wherein the polypeptide is added in the preparation process of the medicine, and the amino acid sequence of the polypeptide is shown as SEQ ID NO. 1.
The beneficial effects are that:
1. The invention synthesizes the peptide for the first time, is a novel active peptide for improving osteoporosis, has stronger bone density regulating activity through detection, can obviously increase the number of bone trabeculae of an osteoporosis mouse from 0.47 to 0.73/mm, improves the bone density level from 0.42mg/cm 3 to 0.46mg/cm 3, improves the hardness of femoral tissue by 40%, and can be applied to the development of medicines with bone density regulation;
2. the active polypeptide is derived from the heavy chain of the cod myosin, has the characteristics of wide source and high safety, avoids the problems of obvious side effects and the like of the existing osteoporosis treatment medicines, and simultaneously provides a new possibility for preventing osteoporosis.
Drawings
FIG. 1 is a graph showing the results of bone mineral density-regulating active peptides as determined by high performance liquid chromatography;
FIG. 2 is a diagram of the results of liquid phase-mass spectrometry identification of a polypeptide synthesized by a solid phase method;
FIG. 3 is a three-dimensional reconstruction of trabeculae of mice in each group (blank: sham; ovariectomized: OVX; positive drug teriparatide: OVX+ TPTD; polypeptide sample: OVX+P-GM-1);
FIG. 4 is a graph of the number of mouse bone trabeculae in each group (blank: sham; ovariectomized: OVX; positive drug teriparatide: OVX+ TPTD; polypeptide sample: OVX+P-GM-1);
FIG. 5 is a graph of bone density results for each group (blank: sham; ovariectomized: OVX; positive drug teriparatide: OVX+ TPTD; polypeptide sample: OVX+P-GM-1; representing significant differences, P < 0.05);
FIG. 6 is a graph of the results of bone tissue hardness measurements for mice of each group (blank: sham; ovariectomized: OVX; positive drug teriparatide: OVX+ TPTD; polypeptide sample: OVX+P-GM-1; representing significant differences, P < 0.05)
Detailed Description
The alkaline proteases referred to in the examples below were purchased from Norwestin (China) Biotechnology Co., ltd; mice were purchased from Liaoning long Biotechnology Co., ltd; teriparatide was purchased from Shanghai Meilin Biochemical technologies Co.
Example 1: acquisition of polypeptides with improved osteoporosis and increased bone density
The method for obtaining the active peptide for improving osteoporosis and enhancing bone density is realized by the following steps:
(1) Preparation of common cod (Gadus morhua) peptide powder
A. Grinding cod: cutting cod meat with fish scales, fish fins, fish heads and viscera removed into small pieces with the size of about 5cm, wherein the mass ratio is 1:1.5 adding water and grinding to obtain a cod meat mixed solution;
b. Enzymolysis: adding alkali into the cod meat mixed solution obtained in the step (1), adjusting the pH value to 8.0, heating to 55-60 ℃, adding alkaline protease (the enzyme activity is 20 ten thousand U/g) according to the mass ratio of 0.20%, stirring uniformly, carrying out enzymolysis for 2h, heating to 85-100 ℃ for enzyme deactivation for 15min, cooling to 60-65 ℃, carrying out filter pressing, centrifuging through a 120-mesh vibrating screen, carrying out membrane filtration separation, and finally concentrating to obtain the cod enzymolysis solution;
c. spray drying: and c, carrying out spray drying on the cod enzymolysis liquid in the step b to obtain common cod peptide powder.
(2) The potential bone density regulating active peptide is prepared by utilizing an Akta protein purifier and a Hitrap Q-HP column according to the charge size of the polypeptide, and the preparation method is specifically as follows:
Taking a Hitrap Q-HP column, and balancing the volume of the column by two times with water at a flow rate of 0.2-2 mL/min; preparing the peptide powder in the step (1) into a solution with the concentration of 5mg/mL by using water, filtering the solution by using a filter membrane with the concentration of 0.22-0.45 mu m, and loading the solution onto the Hitrap Q-HP column with the volume of 0.2-0.5 times of the column volume; then water as mobile phase A and 1mM NaCl as mobile phase B, and elution was performed as follows: 0-30 min:0% of B, 30-45 min:25% of B, 45-53 min:35% of B, 53-63 min:45% of B, 63-87: 100% of B, 87-100 min:0% B. Collecting each eluting peak (recorded as a-e) according to the sequence of the peak outlet time, and drying to obtain bone-promoting small molecular peptide powder; wherein the collecting temperature is 3-5 ℃, and the drying is spray drying or freeze drying;
(3) Identifying the polypeptide sequence in the collected elution peak c contributing bone micromolecular peptide powder by utilizing a liquid chromatography-mass spectrometry analysis method;
(4) And (3) carrying out molecular docking analysis on the sequences and integrins identified in the step (3) by using Discovery Studio software, docking the identified polypeptides with the integrins (PDB: 3VI4 and 1L 5G) by adopting a CDocker method in semi-flexible docking, evaluating the docking result by adopting a-CDOCKER _energy scoring function, and selecting the polypeptide with the strongest interaction with the selected integrins (highest scoring function) as a potential osteogenic active peptide to carry out subsequent activity verification samples.
(5) And (3) artificially synthesizing the polypeptide sequence obtained in the step (4), wherein the polypeptide with the amino acid sequence shown in SEQ ID NO.1 is named as P-GM-1.
Example 2: solid phase synthesis of polypeptides
The polypeptide is prepared by adopting a solid phase method synthesis mode, and the synthesis sequence is as follows: from the C end to the N end of the sequence, the specific steps are as follows:
a. N equivalents of resin were weighed into a reactor, swollen for half an hour with DCM (dichloromethane) and then the DCM was pumped off, 2n equivalents of the first amino acid in the sequence were added, 2n equivalents of DIEA, the appropriate amount of DMF, DCM, DIEA (diisopropylethylamine), DMF (dimethylformamide), DCM, nitrogen bubbling for 60min. Then adding about 5n equivalent of methanol, reacting for half an hour, pumping out the reaction solution, and cleaning with DMF and MEOH;
b. The second amino acid in the sequence (also 2N equivalents), 2N equivalents HBTU (1-hydroxy, benzo, trichloraz tetramethyl hexafluorophosphate) and DIEA, N2 were added to the reactor and the liquid was purged and the ninhydrin was detected and then capped with pyridine and acetic anhydride. Finally, cleaning, adding a proper amount of uncapping liquid to remove Fmoc (9-fluorenylmethoxycarbonyl) protecting group, cleaning, and detecting ninhydrin;
c. sequentially adding different amino acids in the sequence according to the mode of the step b and carrying out various modifications;
d. The resin was taken out of the reaction column after blow-drying with nitrogen, poured into a flask, and then a certain amount of cutting fluid (composition 95% tfa,2% ethanedithiol, 2% triisopropylsilane, 1% water) was added to the flask (cutting fluid and resin were in a ratio of about 10 ml/g), shaken, and the resin was filtered off;
e. Obtaining filtrate, adding a large amount of diethyl ether into the filtrate to separate out crude products, centrifuging, cleaning to obtain crude products of the sequence, purifying, desalting, and freeze-drying to obtain white powder, wherein the polypeptide content is more than or equal to 98%. The solid phase method is used for synthesizing the polypeptide by adopting the combination of liquid phase and liquid phase-mass spectrum identification, and the result is shown in fig. 1 and 2.
Example 3: evaluation of safety of Polypeptides
The polypeptide prepared in the embodiment 2 has no obvious adverse reaction to the experimental object and has certain medicinal safety.
Example 4: evaluation of the preventive/ameliorating Effect of polypeptide on osteoporosis in ovariectomized mice
The polypeptide prepared in example 2 was taken, the ovariectomized induced mouse osteoporosis model was subjected to active peptide gastric lavage for 12 weeks, and the anti-osteoporosis activity of the polypeptide in vivo was evaluated by measuring the changes in the microstructure of mouse bone and trabecular bone density.
1. Establishment of ovariectomized mouse model
Female C57BL/6 mice of 10 weeks of age were selected and randomly placed in a cage. The mice were allowed to receive water and mouse food freely throughout the study, with a light/dark cycle of 12 hours in the animal room, the temperature was maintained at 22±2 ℃, and the relative humidity was maintained at 55±5% for one week. One week later, the ovariectomy procedure was performed as follows: after the mice are anesthetized by the carbamic acid ester, the mice are fixed by the body, the legs are taken to be about 1cm away from the upper part of the fingers for skin preparation, then the sterilized surgical scissors are used for opening in the middle area, the outer skin is firstly cut off, the abdominal membrane is cut off, and the wound is about 2-3mm. Deep taking out fat oviduct from the opening with forceps, finding ovary at the far end of oviduct, wherein the ovary is bean-shaped composed of multiple particles, and one of left and right sides. After ligating the oviduct with suture, the ovaries can be removed by shearing off the oviduct connected with the ovaries by using surgical scissors. The blank group removed fat of the same size as the ovaries, as a sham group. Finally, the wound is sutured and disinfected. After one week of recovery, other mice, except for the sham group, were randomized into 3 groups, specifically designed as follows:
Blank group: sham, sustained lavage saline;
Model group: OVX, continuous lavage saline;
Positive drug group: ovx+ TPTD, neck injection of teriparatide (40 μg/kg) once every two days;
Polypeptide sample group: OVX+P-GM-1, continuous lavage of P-GM-1 peptide (20 mg/kg/day).
After a 12-week continuous gavage, the mice were sacrificed under anesthesia using urethane and their femur was taken for further analysis and evaluation.
2. Mouse femur Micro-CT tomographic analysis
After removal of the femoral tissue of the mice, they were scanned using a Micro-CT scanning system (SKYSCAN 1272, bruker, germany). The scanner operating parameters were as follows: x-ray voltage, 50kV; the filter sheet is 0.5 mm aluminum, the rotation angle is 360 degrees, the interval is 0.2 degrees, and the scanning is carried out twice at each interval. The scanned femur trabecular part of the instrument is subjected to three-dimensional reconstruction through NRecon software, all trabecular bones are converted into a 3D model by CTAn software, and the number and the bone density of each group of mouse bone trabecular are calculated.
The three-dimensional reconstruction result of the femoral tissue of the mice is shown in fig. 3, and compared with the normal group, the bone mass of the ovariectomized mice in the model group is obviously reduced, and the bone mass of the mice after the gastric lavage of the polypeptides is obviously improved. Further analysis and measurement of bone trabecular tissue shows that after 12 weeks of ovariectomy, the number of trabecular in the model group of mice is obviously reduced from 0.91/mm to 0.47/mm, and the bone density is obviously reduced from 0.49mg/cm 3 to 0.42mg/cm 3 (p < 0.05). However, the number of bone trabeculae was significantly increased to 0.73.1/mm and bone density was also significantly increased to 0.46mg/cm 3 in ovariectomized mice after 12 weeks of continuous administration of the polypeptide (P-GM-1, 20 mg/kg/day) compared to the model group (FIGS. 4 and 5).
3. Determination of the hardness of the bone tissue of mice
The isolated femur was placed on a texture analyzer and biomechanical testing was performed using a three-point bending method. Selecting a support with a supporting point span of 10mm, setting the middle femur section as a loading point, vertically and downwards extruding the middle femur section at a constant speed of 1mm/min until the femur breaks, and recording the maximum bending load (N).
The hardness of the femoral tissue of the mice was measured by a texture analyzer, and as shown in fig. 6, the hardness of the femoral tissue of the mice in the model group was significantly reduced from 19N to 12N (p < 0.05) and the brittleness was increased as compared with the blank group. And after 12 weeks of continuous gastric lavage of active polypeptide, the overall hardness of the femoral tissue of the mouse is increased to 17N. The activity effect is slightly weaker than that of a positive drug teriparatide, but the polypeptide has potential application value in the aspect of developing medicines for preventing osteoporosis, particularly bone density reduction caused by estrogen deficiency as food-borne active polypeptide.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (9)
1. A polypeptide with the functions of improving osteoporosis and enhancing bone density is characterized in that the amino acid sequence is shown as SEQ ID NO. 1.
2. A medicament comprising the polypeptide of claim 1.
3. The medicament according to claim 2, wherein the purity of the polypeptide in the medicament is not less than 98%.
4. A medicament according to claim 2 or 3, characterized in that it further comprises a pharmaceutically acceptable carrier.
5. The medicament according to claim 4, wherein the pharmaceutically acceptable carrier comprises one or more of fillers, binders, wetting agents, disintegrants, lubricants, flavoring agents commonly used in medicine.
6. Use of the polypeptide of claim 1 for the preparation of a medicament for alleviating and/or treating osteoporosis.
7. The use according to claim 6, wherein said alleviation and/or treatment of osteoporosis comprises (a): at least one of the following actions:
(a) Recovering the decrease in bone density caused by osteoporosis;
(b) Restoring the reduction in bone mass due to osteoporosis;
(c) Restoring the reduction in the number of trabeculae due to osteoporosis.
8. Use of the polypeptide of claim 1 for the preparation of a health product useful for improving bone mineral density.
9. A method for preparing the polypeptide according to claim 1, wherein the C-terminal carboxyl group of the target polypeptide is covalently linked to an insoluble polymer resin, and then reacts with the carboxyl group of another molecular amino acid to form a peptide bond using the amino group of the amino acid in which the C-terminal carboxyl group is located as a starting point; the process is repeated continuously, and the target polypeptide product can be obtained; after the synthesis reaction is completed, the protecting group is removed, and the peptide chain is separated from the resin.
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