WO2019061862A1

WO2019061862A1 - Shell nacre matrix protein, preparation method therefor, and use thereof

Info

Publication number: WO2019061862A1
Application number: PCT/CN2017/116615
Authority: WO
Inventors: 张键; 向亮; 李梦霞; 任培根
Original assignee: 深圳先进技术研究院
Priority date: 2017-09-26
Filing date: 2017-12-15
Publication date: 2019-04-04
Also published as: CN109553674A

Abstract

The present invention provides a shell nacre matrix protein and a preparation method therefor. The preparation method comprises: pulverizing shellfish nacre into a powder having a diameter of less than 250 μm, dissolving the shellfish nacre powder in a calcium ion chelating agent solution, removing insoluble matter in the calcium ion chelating agent solution, concentrating and dialyzing the shellfish nacre powder solution with water as a dialysate to remove small molecular impurities, and concentrating and drying the dialysis retention liquid to obtain a nacre matrix protein. The invention further provides a use of the nacre matrix protein for the preparation of a medicament for the treatment or prevention of a metabolic bone disease and a pharmaceutical composition comprising the nacre matrix protein.

Description

Shell nacre matrix protein, preparation method and use thereof

Technical field

The invention relates to the field of medicine, in particular to a shell nacre protein and a preparation method and use thereof.

Background technique

Osteoporosis is a chronic pathological process caused by a variety of factors, and its therapeutic effect is relatively slow, bringing great inconvenience to the patient, as well as both physical and psychological pain. There are about 84 million osteoporosis patients in China, and the number of patients is expected to reach 200 million by 2050. In the treatment of osteoporosis, it is necessary to inhibit bone resorption factors and factors that promote bone formation in order to achieve a better therapeutic effect. Although there are many kinds of related drugs for treating osteoporosis, they generally have certain side effects, and they cannot be taken for a long time as a preventive measure. Therefore, it has become important to find a biologically active substance capable of preventing osteoporosis which has a small side effect and can be taken for a long period of time. Research direction.

Pearl has always been regarded as a precious medicinal material by Chinese medicine, and has a long history as a medicinal and beauty. The pearl has the functions of calming the nerves, clearing away heat and relieving yin, clearing the eyes and detoxifying, eliminating inflammation, and relieving cough. It is suitable for stomach and duodenal ulcer, insomnia, neurasthenia, hepatitis, sore throat and other symptoms. Epilepsy, rheumatic heart disease, etc. have a certain effect, has been made into more than 20 kinds of proprietary Chinese medicines such as Huichundan, Liushen Pill, and Compound Asthma, but its efficacy in the treatment of osteoporosis has not been developed. The shell nacre powder has the same composition as the nacre, and the price is low, and the application prospect is broad. Shell nacre powder contains rich anti-osteoporosis activity protein and is an excellent natural calcium source. Shells may contain bioactive factors that inhibit bone resorption and promote bone formation. Therefore, the preparation of osteoinductive factors from shells not only increases the comprehensive utilization value of shells, but also provides a scientific basis for the development of new anti-osteoporosis drugs. It has very important practical significance.

In 1989, the group of French scholar Lopes was enlightened by implanting teeth made of pearls into the mouth, and began to use the pearls in the study of bone repair. Subsequently, scholars from France, Sweden, South Korea and China used the sheep lumbar vertebrae, the femur, the rat femur, and the upper and lower jaws of humans and dogs as bone defect models, and also carried out experiments on the transplantation and repair of bones for bone. Research and fruitful research results have been made, but the research on osteoporosis in nacre is progressing slowly.

The bone tissue is composed of highly crystalline calcium phosphate and an organic matrix, and the synergistic effect of osteoclasts and osteoblasts is required during bone repair. Bone resorption includes mineral dissolution and degradation of organic matrix, while bone formation includes Formation of new matrix and mineral deposition. Insufficient bone formation or excessive bone resorption can induce bone disease, especially osteoporosis. It is worth noting that drugs that prevent osteoporosis need to contain both factors that inhibit bone resorption and induce bone formation. A water-soluble matrix in the shell nacre may be a better choice.

Shell nacre has good biocompatibility, biodegradability and potential bone conduction properties, which can stimulate and induce the regeneration of bone tissue and the formation of new bone, and promote the mineralization of host bone. Matrix proteins (WSM) in water-soluble organic matrix components extracted from nacre are different in different types of bone cells. In vitro studies have shown that WSM can promote the mineralization of osteoblasts, and can also inhibit the activity of osteoclasts by inhibiting the activity of cathepsins, thereby inhibiting bone resorption. By contrast, calcium carbonate solution alone has almost no therapeutic effect on osteoporosis, and WSM can accelerate the calcification of osteoblasts and promote the differentiation of osteoblasts. WSM inhibits the proliferation of bone marrow cells and promotes the activity of alkaline phosphatase, which is similar to the effects of bone growth factors such as BMP and TGF-β, indicating that the water-soluble matrix protein may contain one or more signals. Molecules can promote the differentiation of bone marrow cells into osteoblasts. Through the analysis of signaling pathways, osteogenesis of WSM differs from well-known growth factors such as BMP-2 and osteocalcin. In the process of bone repair, the absorption of osteoclasts and the osteogenic interaction of osteoblasts are required. Experiments have shown that the signaling molecules of WSM may have both the dual effects of inhibiting osteoclast activity and promoting osteoblast mineralization: on the one hand, the water-soluble matrix in the nacre prevents bone resorption by inhibiting the activity of osteoclast cathepsin K; In one aspect, the water-soluble matrix stimulates the biomineralization of osteoblasts to form new bone by activating the signaling pathways of c-Jun NH2-terminal kinase (JNK) and Fos-related antigen-1 (Fra-1). So far, there have been few studies on shell nacre WSM. WSM contains at least 300 molecules of small molecular weight. The osteogenic factors of nacre are likely to be related to small molecular weight molecules, which may spread more easily from nacre to bone tissue. Around, it is good for osteogenesis. Although matrix proteins in shells have shown potential applications as therapeutic drugs for osteoporosis, their content is extremely low and the water extraction method allows a large number of effective active factors to be lost, which limits its application.

Since the shell matrix protein is enclosed in the shell and usually combined with the calcium carbonate crystal, there is a strong interaction between the matrix protein and the crystal inside the crystal, so it is difficult to extract the entire organic matrix. At present, there are mainly two methods according to the method of dissolving shell calcium carbonate, which are weak acid decalcification method and ultrapure water dissolution method. The extraction conditions of weak acid decalcification method are relatively severe, the protein may be degraded, the active ingredient is seriously lost, and the ultra-pure water dissolution method extracts the protein with low efficiency, long duration and easy loss of protein activity.

Summary of the invention

The EDTA decalcification method used in the invention can effectively dissolve the shell calcium carbonate crystal, and release the matrix protein inside the crystal in a large amount, and has the advantages of mild extraction condition, short duration, high matrix protein concentration, and the like, and the shell matrix protein is ensured. Biological activity. The shell matrix protein extracted according to the invention has high biological activity, can promote the mineralization activity of osteoblasts efficiently, and can effectively treat and alleviate the symptoms of osteoporosis in mice.

One aspect of the present invention provides a method for preparing a nacre matrix protein, comprising the steps of:

1) taking a shellfish nacre to be pulverized into a powder, the powder having a diameter of less than 250 μm;

2) dissolving the shellfish nacre powder in a calcium ion chelating agent solution;

3) removing the insoluble matter of the calcium ion chelating agent solution;

4) concentrating the shellfish nacre powder solution and dialysis to remove small molecular impurities, the dialysate being water;

5) The dialysis retention solution is concentrated and dried to obtain a nacre matrix protein.

In a solution of the invention, the powder in step 1) has a diameter of less than 200 μm, preferably less than 100 μm, more preferably less than 80 μm.

In the technical solution of the present invention, the calcium ion chelating agent solution has a concentration of 0.1-1 M and a pH of 7.5-8.5 (8.0).

In the technical solution of the present invention, the calcium ion chelating agent is selected from one or more of ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), and cyclohexanediaminetetraacetic acid (CDTA), or Its sodium or magnesium salt.

In the embodiment of the invention, the time of dissolution in step 2) is from 10 to 50 hours, preferably from 20 to 40 hours.

In the technical solution of the present invention, the shellfish nacre is obtained by mechanically removing the prism layer and the stratum corneum outside the shellfish, and does not use any chemical agent treatment when removing the prism layer and the stratum corneum outside the shellfish.

In the solution of the present invention, the shellfish is selected from any shellfish having a nacre layer.

In the technical solution of the present invention, the dialysate contains no salt and the pH is neutral, preferably about 6.5-7.5; more preferably, the dialysate is selected from pure water.

Another aspect of the present invention provides a nacre matrix protein obtained by the production method of the present invention.

In the present invention, the nacre matrix protein has a molecular weight of 14-97 kd of mixed protein.

In the present invention, the nacre matrix protein is mainly a mixed protein having a molecular weight of about 60 kd.

In an embodiment of the invention, the nacre matrix protein is obtained from a nacre of a shell, and in one embodiment of the invention, the shell is Hepu.

Still another aspect of the present invention provides the use of a nacre matrix protein for the preparation of a medicament for treating or preventing a bone metabolic disease.

Still another aspect of the present invention provides the use of a nacre matrix protein for the treatment or prevention of a drug for bone metabolism.

In the technical solution of the present invention, the bone metabolic disease is a disease associated with abnormal growth of bone growth or bone.

Among them, including osteoporosis, Paget, osteogenesis imperfecta, bone fiber dysplasia, phosphatase hypothyroidism, and humoral hypercalcemia of malignant tumors and multiple myeloma-induced osteolysis .

In a further aspect of the invention there is provided a method of increasing bone density and/or bone volume fraction of trabecular bone and/or number of trabecular bone, the method of administering a nacre matrix protein to a subject.

A further aspect of the invention provides the use of a nacre matrix protein for the manufacture of a medicament for increasing bone density and/or bone volume fraction of trabecular bone and/or the number of trabecular bone.

In still another aspect, the present invention provides a method of increasing mineralization activity of osteoblasts by administering to a subject a nacre matrix protein.

Still another aspect of the present invention provides the use of a nacre matrix protein for the preparation of a medicament for enhancing mineralization activity of osteoblasts.

According to still another aspect of the present invention, a pharmaceutical composition for a bone metabolic disease, wherein the active ingredient is a nacre matrix protein.

In the technical solution of the present invention, the pharmaceutical composition further comprises one or more pharmaceutical excipients.

In the technical solution of the present invention, the pharmaceutical composition further comprises one or more other active ingredients.

In the technical solution of the present invention, the pharmaceutical composition may be formulated in the form of a tablet, a capsule, a pill, a granule, a powder, an injection or a liquid, but the invention is not limited thereto.

Beneficial effect

1. The present invention provides a method for efficiently preparing a shell nacre matrix protein with consistent conditions, high yield and high purity.

2. The present invention proves that the nacre matrix protein extract can effectively treat osteoporosis symptoms and can effectively treat osteoporosis and related diseases.

DRAWINGS

Figure 1 is a separation of shell nacre and a preparation process of pearl powder.

Fig. 2 is a result of FTIR analysis of the crystal form of nacre powder by Fourier transform infrared spectrometer. The nacre powder is a typical aragonite characteristic peak with no impurities. The aragonite characteristic peak is 700cm ^-1 , 712cm ^-1 , 858cm ^-1 and 1082 cm ^-1 .

Figure 3 is a graph of SDS-PAGE results of EDTA soluble matrix proteins extracted from nacre powder. The matrix proteins are distributed in the 97-14 kDa region, and there is a highly abundant protein band around 60 kDa.

Figure 4 is a graph showing the results of matrix protein stimulation of osteoblast MC3T3-E1, in which A is the effect of different concentrations of matrix proteins on MC3T3-E1 activity, showing no toxicity to cells; B is matrix protein and shell protein stimulating MC3T3-E1 Bone differentiation, alizarin red staining showed that matrix proteins promoted the formation of MC3T3-E1 calcium nodules, while shell protein did not promote.

Figure 5 is a graph showing the results of micro CT scanning analysis of matrix proteins in the treatment of femurs in osteoporotic mice. Comparative analysis of bone parameters of the femur of the control group (Sham group), the shell protein treatment group (OVX+Shell group), the osteoporosis group (OVX group), and the matrix protein treatment group (OVX+Protein group): bone density ( BMD), bone volume fraction (BV/TV) and trabecular bone number (Tb.N), *, P < 0.05, **, P < 0.01, **, P < 0.001.

Detailed ways

The invention will now be illustrated by reference to specific examples and pharmacodynamic tests. The embodiments are only used to further illustrate the present invention, and do not represent the scope of the present invention. The non-essential modifications and adjustments made by others according to the present invention are still within the scope of the present invention.

Example 1 EDTA dissolved nacre powder experiment

The Hepu pearl motherfish was sacrificed for one week in the laboratory and the shells were sacrificed. Subsequently, the edge portion of the shell is cut with scissors, and the remaining shells are mechanically scraped off using a pair of tweezers to scrape off the outer prism layer and the stratum corneum, that is, a shell nacre sample. The specific steps of EDTA to dissolve the nacre powder are as follows:

1 The prepared nacre samples were pulverized by a high-speed pulverizer, and different sizes of powders (60 mesh = 250 μm, 180 mesh = 80 μm) were screened with different sieves to obtain particle diameters ≤ 80 μm, 80 μm < particle diameters < 250 μm, respectively. Powder with particle size ≥250μm, nacre powder is identified by FTIR spectroscopy;

2 Put the nacre powder into 0.5M EDTA (0.5M EDTA per 10g powder dissolved in 100ml) Medium, pH 8.0) agitation was dissolved to observe the dissolution time.

Example 2 Extraction of shell nacre matrix protein

The shells were from Hepu Pearl, in the waters of Daya Bay, Shenzhen, Guangdong Province. They were sacrificed for one week after they were cultured in the laboratory. Subsequently, the edge portion of the shell is cut with scissors, and the remaining shells are mechanically scraped off using a pair of tweezers to scrape off the outer prism layer and the stratum corneum, that is, a shell nacre sample. The specific steps for extracting EDTA soluble shell matrix protein are as follows:

1 The prepared nacre sample is broken into a powder having a diameter of less than 80 μm by a high-speed pulverizer, and the nacre powder is subjected to FTIR spectral crystal identification;

2 the nacre powder was placed in 0.5 M EDTA (10 g / 100 ml) and dissolved for 36 h;

Centrifuge at 13,000 rpm for 30 min at 34 ° C. This step was repeated twice to completely remove impurities that were not dissolved in EDTA;

4 The obtained supernatant was concentrated using a Milipore 15 ml 3KD ultrafiltration tube, and the solution volume was concentrated to one tenth.

5 The obtained solution was placed in a 3.5KD dialysis bag, dialyzed for 12h with ultrapure water, and changed to water every 2 hours. Then, the dialysis solution was again concentrated by ultrafiltration using Milipore 15ml 3KD ultrafiltration tube. The mixture was concentrated to one tenth, and then freeze-dried with a lyophilizer to obtain a protein powder, which was stored in a refrigerator at -80 ° C to obtain a nacre matrix protein.

Example 3 Identification of SDS-PAGE Protein Size Distribution

1 The protein powder is formulated into a 3 mg/ml protein solution with ultrapure water, centrifuged at 25,000 rpm for 15 min at 4 ° C, the supernatant is taken, and the supernatant is drained;

2 The supernatant and the precipitate after the extraction were combined, 200 μl of SDS L3, PMSF with a final concentration of 1 mM, 2 mM EDTA, and mixed, placed on ice for 5 min, and then added to a final concentration of 10 mM DTT (PMSF, EDTA, DTT) The volume is 1% of the volume of the protein solution);

3 Ultrasonic on ice for 5 min (working 5 s, interval 5 s, power 100 W), centrifugation at 2 ° C, 25000 rpm for 20 min;

Transfer the supernatant into a new 1.5 ml centrifuge tube, the final concentration of 10 mM DTT (1% of the volume of protein solution), water bath at 56 ° C for 1 h;

4 cooled to room temperature, adding a final concentration of 55 mM IAM (1% of the volume of protein solution), and placed in a dark room for 45 min;

Centrifuge at 25000 rpm for 15 min at 54 ° C, the supernatant is the sample protein solution;

6 Run the gel analysis according to the SDS-PAGE standard procedure (separation gel concentration is 12%).

Example 4 Comparison of Shell Protein Extraction

The shells were from Hepu Pearl, in the waters of Daya Bay, Shenzhen, Guangdong Province. They were sacrificed for one week after they were cultured in the laboratory. Subsequently, the edge portion of the shell was cut with scissors, and the remaining shells were mechanically scraped using a forceps, and the outer prism layer and the stratum corneum were scraped off, and the shells were treated with NaOH for three days. As a control, a shell nacre sample was obtained. The specific steps for extracting the hull protein are as follows:

1 The prepared nacre sample is crushed into a powder having a diameter of more than 200 μm by a grinder;

2 each 10g of nacre powder was dissolved in 100ml of 0.5M EDTA (pH 8.0) and dissolved for 36h;

5 The obtained solution was placed in a 3.5KD dialysis bag, dialyzed against Tris-Cl pH 8.0 for 12 hours, and the dialysate was changed once every 2 hours. Then, the dialyzed solution was again subjected to ultrafiltration concentration using Milipore 15 ml 3KD ultrafiltration tube. The solution volume was concentrated to one tenth, and then freeze-dried with a lyophilizer to obtain a protein powder, which was stored in a refrigerator at -80 ° C to obtain a nacre shell protein.

Example 5 Analysis of mineralization function of matrix protein cells

1 Culture of osteoblast MC3T3-E1:

The medium of osteoblast MC3T3-E1 was α-MEM (10% fetal bovine serum, 100 μg/ml Penicillin, 100 μg/ml Streptomycin), and the osteoblast MC3T3-E1 was cultured in an incubator at 37 ° C (5% CO _{2 ).} ), the medium was changed every 3 days and passaged once every 7 days. MC3T3-E1 cell suspension (100 μl/well) was seeded in 96-well plates, and different concentrations of matrix proteins (5, 10, 15, 30 μg/ml) were added to the medium, and incubated for 72 hours. Osteoblasts were measured every 24 hours. Activity.

2 Determination of the activity of osteoblasts:

Cell viability was determined according to the Dojindo Cell Counting Kit (CCK-8) cell proliferation kit step. 10 μl of CCK-8 solution was added to each well, and the plate was incubated in an incubator for 2 h, and the absorbance at 450 nm was measured with a microplate reader. .

Analysis of calcium nodules by 3 alizarin red staining:

Osteoblast MC3T3-E1 was cultured for 21 days in osteogenic induction medium (50 μM vitamin C, 10 mM β-glycerophosphate, 100 nM dexamethasone, 30 μg/ml shell protein or 30 μg/ml matrix protein), and the medium was discarded. Wash 3 times with PBS; fix it with 4% paraformaldehyde for 30 min at room temperature; discard the solid The solution was washed 3 times with ultrapure water; the alizarin red staining (Shanghai source leaf organism, item number R20794) was added, and the mixture was incubated at room temperature for 30 min; the staining solution was discarded, and the ultrapure water was washed 3 times, and photographed.

(4) Anti-osteoporosis function of matrix proteins

1 Control group (Sham group): 9-week-old C57BL/6J wild-type female mice were bilaterally cut from the back, and a part of the fat pad around the ovary was taken to ensure that the bilateral ovaries were intact and intact. After 5 days of recovery of the wound in mice, 100 μl of physiological saline was intraperitoneally injected, and injected every 3 days for 30 days;

2 Osteoporosis group (OVX group): 9-week-old C57BL/6J wild-type female mice underwent bilateral ovariectomy from the back to ensure that the bilateral ovaries were removed. After 5 days of recovery of the wound in mice, 100 μl of physiological saline was intraperitoneally injected, and injected every 3 days for 30 days;

3 Shell protein treatment group (OVX+Shell group): 9-week-old C57BL/6J wild-type female mice underwent bilateral ovariectomy from the back to ensure that both ovaries were removed. After 5 days of mouse wound recovery, 100 μl of a concentration of 4 mg/ml of seashell protein was intraperitoneally injected, and injected every 3 days for 30 days.

4 Matrix protein treatment group (OVX+Protein group): 9-week-old C57BL/6J wild-type female mice underwent bilateral ovariectomy from the back to ensure that both ovaries were removed. Five days after the wound healing in mice, 100 μl of a matrix protein of 4 mg/ml was intraperitoneally injected, and injected every 3 days for 30 days.

At the end of the experiment, the mice were sacrificed by cervical dislocation, and the bones of the mice were fixed with 70% ethanol for CT analysis. The bones of the mice were scanned using BRUKER's SkyScan 1176 MicroCT, and the position of the bones was fixed using a foam plate for scanning. CT scanning was performed using a voltage of 60 kV, a current of 425 μA, and a resolution of 9 μm. After the scan reconstruction was completed, the structural analysis of the trabecular bone of the femoral metaphysis was performed. After the disappearance of the metaphyseal growth plate at the distal end of the femur, 50 layers were discarded, and 200 layers were selected as the trabecular reconstruction area for reconstruction. The reconstruction was performed using MicroCT's own software with a threshold of 80. The main indicators included bone mineral density (BMD), bone volume fraction (BV/TV), and trabecular bone number (Tb.N).

result

1 separation of shell nacre, preparation of nacre powder and identification of crystal form

The invention uses artificial mechanical method to separate the nacre layer from the shell, the nacre layer is colorful (Fig. 1), similar to the color and luster of the pearl, and the mechanical separation does not add any chemical treatment steps to ensure the biological activity of the nacre.

The use of high-speed pulverizer to break into different diameters has a large difference in dissolution rate, and the processing time in the protein extraction process directly affects the protein activity. It can be seen that the pearl layer powder has a particle size of ≤80 μm, and when dissolved in EDTA, the dissolution time is 36 hours, and the nacre powder particle size is 80 μm<particle size <250 μm. When EDTA is dissolved, the dissolution time is 50 hours, and the powder dissolution time of the particle size ≥ 250 μm is higher than 90 hours. It can be seen from the results of Example 1 that the powder having a diameter of less than 80 μm (Fig. 1) increases the surface area of the nacre powder particles, which is advantageous for the high-efficiency chelation dissolution of EDTA, and the nacre powder crystal form prepared by the method is fully identified. It is aragonite with a purity of 100% (Figure 2). In the comparative test, the particle size was too large to cause rapid dissolution, and the chelating efficiency of EDTA was also low.

2 molecular size distribution of shell EDTA soluble matrix protein

SDS-PAGE separates proteins according to the molecular weight of the protein. The soluble matrix proteins extracted by the inventors by EDTA dissolution method were analyzed by SDS-PAGE. The results of Fig. 3 showed that matrix proteins with different molecular weights were distributed in the 14-97kD segment. Among them, there is a highly abundant protein band around 60 kDa, and the method of the invention can effectively separate and extract the matrix protein.

3 Identification of the biological activity of shell matrix protein and its evaluation in the treatment of osteoporosis mice

MC3T3-E1 is a mouse embryonic osteoblast precursor cell that can be used to screen for bioactive substances that stimulate osteogenic differentiation. The results in Figure 4 show that matrix proteins are not toxic to MC3T3-E1 cells and promote mineralization of MC3T3-E1, demonstrating that matrix proteins are an effective pro- osteogenic factor. We injected the matrix protein intraperitoneally into osteoporotic mice. Compared with osteoporotic mice, the matrix protein treatment group can effectively alleviate the symptoms of osteoporosis in mice. Figure 5 shows the bone mineral density of BMD and trabecular bone in mice. Significant increases in bone volume fraction (BV/TV) and trabecular bone number (Tb.N) were observed. The shell protein treatment group did not effectively alleviate the symptoms of osteoporosis in mice. The results showed that there was no change in bone mineral density BMD, bone trabecular bone volume fraction (BV/TV) and trabecular bone number (Tb.N).

Claims

A method for preparing a nacre matrix protein, comprising the steps of:

1) taking a shellfish nacre to be pulverized into a powder, the powder having a diameter of less than 250 μm;

2) dissolving the shellfish nacre powder in a calcium ion chelating agent solution;

3) removing the insoluble matter of the calcium ion chelating agent solution;

4) concentrating the shellfish nacre powder solution and dialysis to remove small molecular impurities, the dialysate being water;

5) concentrating and drying the dialysis retention solution to obtain a nacre matrix protein;

Preferably, the calcium ion chelating agent is selected from the group consisting of ethylenediaminetetraacetic acid, ethylene glycol tetraacetic acid, cyclohexanediaminetetraacetic acid or a combination of one or more of the sodium or magnesium salts thereof.
The preparation method according to claim 1, wherein the powder in the step 1) has a diameter of less than 200 μm, preferably less than 100 μm, more preferably less than 80 μm.
The preparation method according to claim 1, wherein the shellfish nacre is obtained by mechanically removing the prism layer and the stratum corneum outside the shellfish.
A nacre matrix protein obtained by the production method according to any one of claims 1 to 3.
The nacre matrix protein according to claim 4 is a mixed protein having a molecular weight of 14 to 97 kd.
The use of the nacre matrix protein according to claim 4 or 5 for the preparation of a medicament for treating or preventing a bone metabolic disease.
The use according to claim 6, wherein the bone metabolic disease is a disease associated with abnormal growth of bone growth or bone; preferably, the bone metabolic disease is osteoporosis, Paget, osteogenesis Incomplete, bone fiber dysplasia, phosphatase hypothyroidism, and humoral hypercalcemia of malignant tumors and multiple myeloma-induced osteolysis.
Use of a nacre matrix protein according to claim 4 or 5 in the manufacture of a medicament for increasing bone density and/or bone volume fraction of trabecular bone and/or the number of trabecular bone.
The use of the nacre matrix protein according to claim 4 or 5 for the preparation of a medicament for promoting mineralization activity of osteoblasts.
A pharmaceutical composition for a bone metabolic disease, wherein the active ingredient is the nacre matrix protein of claims 1-3.