Cornu cervi pantotrichum osteogenesis center polypeptide extract with effect of improving osteoporosis and application thereof

Abstract

The invention belongs to the technical field of biotechnology and tissue engineering, particularly relates to a pilose antler osteogenesis center polypeptide extract with an effect of improving osteoporosis and application of the pilose antler osteogenesis center polypeptide extract, and provides and prepares an active substance for precisely applying pilose antler for the first time, namely the pilose antler osteogenesis center polypeptide extract. The polypeptide extract is derived from a pilose antler osteogenesis center at a position 5-10 cm away from the top end of an in-vitro pilose antler; tissue powder is obtained by separating and crushing the cartialgenous osteogenesis center, and the tissue powder is extracted with water. The pilose antler osteogenesis center polypeptide extract provided by the invention has a remarkable anti-osteoporosis prevention and treatment effect, the action effect is remarkably better than that of a whole-branch pilose antler polypeptide extract, and the pilose antler osteogenesis center polypeptide extract has the characteristics of naturalness, safety and no side effect. The method disclosed by the invention is subsidized by the national natural science fund committee project, and the project approval number is 32300708.

Description

Technical Field

[0001] The present invention belongs to the technical fields of biotechnology and tissue engineering, and particularly relates to a polypeptide extract from the osteogenic center of pilose antler with the function of improving osteoporosis and its application. Background Art

[0002] Osteoporosis syndrome is a group of metabolic bone diseases caused by various reasons, characterized by impaired bone strength and increased fracture risk. Its main features are reduced bone tissue volume per unit volume, thinning of the bone cortex, reduction in the number and size of cancellous bone trabeculae, widening of the medullary cavity, and weakened bone load-bearing capacity, resulting in pain in the waist, back and limbs, spinal deformity and even fractures. Osteoporosis is the most common in metabolic bone diseases and is an important senile disease.

[0003] Pilose antler is a precious traditional Chinese medicine with the efficacy of "strengthening tendons and bones". Pilose antler is the only large bone organ in mammals that can be completely regenerated periodically. It starts to grow in spring every year, develops rapidly in summer and ossifies into antlers in autumn. The growth rate of pilose antler during the rapid growth period can be as high as 2.75 cm / day, and the mineral deposition rate is about 3.2 µm / day.

[0004] Previous studies on the osteogenic components of pilose antler and its anti-osteoporosis effects mainly focused on the whole pilose antler, but the anti-osteoporosis effect of the whole pilose antler is limited. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides a polypeptide extract from the osteogenic center of pilose antler with the function of improving osteoporosis and its application. The polypeptide extract from the osteogenic center of pilose antler provided by the present invention has a significant anti-osteoporosis prevention and treatment effect, and the effect is significantly stronger than that of the polypeptide extract of the whole pilose antler. It can be used to prepare drugs for preventing / treating osteoporosis or health products for increasing bone density.

[0006] In the first aspect of the present invention, there is provided a polypeptide extract from the osteogenic center of pilose antler with the function of improving osteoporosis, and the polypeptide extract from the osteogenic center of pilose antler is obtained through the following preparation steps: Cut the tissue at the top 5-10 cm of the pilose antler to obtain the osteogenic center tissue of the pilose antler; Crush the osteogenic center tissue of the pilose antler and add water for extraction to obtain the polypeptide extract from the osteogenic center of the pilose antler.

[0007] Furthermore, the material-liquid ratio of the osteogenic center tissue of the pilose antler to water is 1 g: 10-20 mL.

[0008] Preferably, the material-liquid ratio of the osteogenic center tissue of the pilose antler to water is 1 g: 10 mL.

[0009] Further, the extraction time is 12 to 48 hours.

[0010] Preferably, the extraction time is 24 hours.

[0011] Further, the extraction temperature is 4 to 10 °C.

[0012] Further, the extraction temperature is 4 °C.

[0013] Further, the extraction needs to be carried out 2 to 3 times.

[0014] Further, a protease inhibitor needs to be added during the extraction.

[0015] Further, the addition amount of the protease inhibitor is 0.01% to 0.05% of the mass of the antler osteogenic center tissue.

[0016] Preferably, the addition amount of the protease inhibitor is 0.02% of the mass of the antler osteogenic center tissue.

[0017] The second aspect of the present invention provides an application of the above-mentioned antler osteogenic center polypeptide extract in the preparation of drugs for preventing or treating osteoporosis.

[0018] Further, the osteoporosis is senile osteoporosis, postmenopausal osteoporosis and other pathological osteoporosis.

[0019] Further, the drug further includes pharmaceutically acceptable excipients.

[0020] The third aspect of the present invention provides an application of the above-mentioned antler osteogenic center polypeptide extract in the preparation of health products or medical devices or functional foods for increasing bone density.

[0021] Further, the health product further includes excipients acceptable for health products.

[0022] Further, the medical device for increasing bone density prepared by the antler osteogenic center polypeptide extract is that the antler osteogenic center polypeptide extract can be used to prepare bone tissue engineering scaffold materials or medical device coatings.

[0023] The present invention has the following beneficial effects: (1) The present invention first proposed and isolated a kind of active substance for precise application of pilose antler, namely osteogenic center polypeptide extract of pilose antler (O-VAP). By comparing the internal tissue structure and morphological characteristics of the longitudinal section of pilose antler, as well as the characteristic indexes and digital indexes of the components of tissues in different sections from the base to the tip, and analyzing the gene expression profiles of different sections of pilose antler, it was found that the deposition efficiency of Ca, P, and Mg at 5-10 cm at the tip of pilose antler (osteogenic center of pilose antler) was relatively high, and the enriched osteogenic development and bone metabolism signaling pathways were the strongest. An osteogenic center polypeptide extract of pilose antler was obtained by crushing the osteogenic center tissue of pilose antler and extracting it with water. The osteogenic center polypeptide extract of pilose antler provided by the present invention has a significant anti-osteoporosis prevention and treatment effect, and the effect is significantly stronger than that of the whole pilose antler polypeptide extract (T-VAP), and has the characteristics of naturalness, safety, and no side effects. The osteogenic center polypeptide extract of pilose antler provided by the present invention has important application value in the fields such as preparing drugs for preventing / treating osteoporosis or health products for increasing bone density.

[0024] (2) When preparing the osteogenic center polypeptide extract of pilose antler in the present invention, through water extraction, the soluble active protein polypeptides in the tissue are fully extracted, and the biological activity of the extracted polypeptides is retained to the greatest extent; and through experiments, it is verified that if the extraction time is too long, the product will ferment, become sticky and deteriorated, affecting the activity of the product, and if the extraction time is too short, the yield of the polypeptide extract obtained by extraction is small, resulting in waste of resources; by adding protease inhibitors, the degradation and denaturation of polypeptides during the extraction process can be effectively prevented, and the dissolution of active polypeptides in the osteogenic center polypeptide extract of pilose antler can be further improved. Brief Description of the Drawings

[0025] Figure 1 It is the longitudinal section of the three-branched pilose antler of sika deer and the deposition efficiency of Ca, P, and Mg in each section, and the part where the black circle is located is the osteogenic center of pilose antler.

[0026] Figure 2 It is the flow chart of the animal experiment on the anti-osteoporosis effect of the osteogenic center polypeptide extract of pilose antler (O-VAP).

[0027] Figure 3 It is the micro-CT image of the distal femur of a mouse; from top to bottom, each row is in turn: the overall longitudinal section, the cross-section of the primary osteogenic center, cancellous bone, and cortical bone; PBS represents the negative control group (treated with PBS after ovariectomy), BPs represents the positive control group (treated with bisphosphonate after ovariectomy), T-VAP represents the whole pilose antler polypeptide extract group (treated with the whole pilose antler polypeptide extract after ovariectomy), and O-VAP represents the osteogenic center polypeptide extract group of pilose antler (treated with the osteogenic center polypeptide extract of pilose antler after ovariectomy).

[0028] Figure 4Figure 1 shows the experimental results of the anti-osteoporosis effect of the peptide extract of antler osteogenic center (O-VAP). Figures A to F are the test results of bone density, bone volume fraction, trabecular number, trabecular thickness, trabecular separation and cortical bone density of the primary osteogenic center, respectively. CTRL represents the normal control group, PBS represents the negative control group (PBS treatment after ovarian removal), BPs represents the positive control group (bisphosphonate treatment after ovarian removal), T-VAP represents the whole antler peptide extract group (the whole antler peptide extract was treated after ovarian removal), and O-VAP represents the antler osteogenic center peptide extract group (the antler osteogenic center peptide extract was treated after ovarian removal). * indicates that there is a difference between the corresponding group and the O-VAP group, and * indicates p <0.05,** indicates p <0.01, *** indicates p <0.001, **** means p <0.0001.

[0029] Figure 5 Histological staining was used to detect the anti-osteoporosis effect of antler osteoblastic center polypeptide extract (O-VAP); from top to bottom, each row is: HE staining of distal femur of mice (whole, enlarged view of secondary osteoblastic center, enlarged view of primary osteoblastic center) and Safranin O fast green staining (whole, enlarged view of secondary osteoblastic center, enlarged view of primary osteoblastic center); CTRL represents normal control group, PBS represents negative control group (PBS treatment after ovary removal), BPs represents positive control group (bisphosphonate treatment after ovary removal), T-VAP represents whole antler polypeptide extract group (whole antler polypeptide extract treatment after ovary removal), O-VAP represents antler osteoblastic center polypeptide extract group (antler osteoblastic center polypeptide extract treatment after ovary removal).

[0030] Figure 6 To present the results of component analysis of O-VAP and T-VAP using HPLC technique; Figure 6 Figure A is the peak diagram of O-VAP, and the red arrow indicates the unique substance of O-VAP at a retention time of 3 min; Figure 6 Figure B is the peak diagram of T-VAP. DETAILED DESCRIPTION

[0031] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings.

[0032] In the description of the present invention, unless otherwise specified, all reagents used are commercially available and all methods used are conventional techniques in the art.

[0033] In some embodiments of the present invention, the pilose antler is sourced from Shuangyang Sika Deer Farm in Jilin Province, and all the sika deer are artificially farmed.

[0034] Osteoporosis syndrome is a group of skeletal metabolic diseases caused by various factors, characterized by impaired bone strength and increased fracture risk. Pilose antler is a precious traditional Chinese medicine with the efficacy of "strengthening tendons and bones", and it is also the only large bone organ in mammals that can be completely regenerated periodically. Pilose antler polypeptide, as a key regulatory factor for the rapid growth and osteogenic development of pilose antler, has a very strong effect on promoting cell osteogenic differentiation. Previous studies on the osteogenic components of pilose antler and its anti-osteoporosis effects mainly focused on the whole pilose antler, but the anti-osteoporosis effect of the whole pilose antler is limited.

[0035] The present invention provides a polypeptide extract from the osteogenic center of pilose antler with the effect of improving osteoporosis and its application. The present invention first systematically compared the internal tissue structure and morphological characteristics of the longitudinal section of pilose antler, as well as the characteristic indexes and digital indexes of the components of tissues in different sections from the base to the tip. According to the index characteristics, the osteogenic center of pilose antler was discovered, and a polypeptide extract from the osteogenic center of pilose antler was obtained by crushing the osteogenic center of pilose antler and extracting it with water. Then, animal experiments verified that the polypeptide extract from the osteogenic center of pilose antler provided by the present invention has a significant anti-osteoporosis prevention and treatment effect, and the effect is significantly stronger than that of the polypeptide extract of the whole pilose antler, and it can be used to prepare drugs for preventing / treating osteoporosis or health products for increasing bone density.

[0036] Example 1: Determination of the osteogenic center of pilose antler By systematically comparing the internal tissue structure and morphological characteristics of the longitudinal section of pilose antler, as well as the characteristic indexes and digital indexes of the components of tissues in different sections from the base to the tip, the results of the longitudinal section of the three-branched pilose antler of fresh sika deer and the deposition efficiency of Ca, P, and Mg in each section were obtained. Deposition efficiency = (content of the next section - content of the previous section) / content of the previous section × 100%; the deposition efficiency value of Ca at the base (the leftmost) was set as 1.

[0037] It was found that the deposition efficiency of Ca, P, and Mg in pilose antler from the base to the tip showed a trend of first increasing and then decreasing, and a peak appeared in the area 5 - 10 cm from the tip of the pilose antler, as Figure 1 shown; at the same time, by analyzing the gene (mRNA and protein) expression profiles of each section, it was also found that the osteogenic development and bone metabolism signaling pathways enriched in this area were the strongest; therefore, this area is the osteogenic center of pilose antler.

[0038] Example 2: Preparation of polypeptide extract from the osteogenic center of pilose antler (O-VAP) S1. Obtaining of osteogenic center tissue of pilose antler: Select artificially farmed adult male sika deer. After anesthesia, disinfect the bifurcated pilose antler with iodophor and alcohol in gradient, then harvest it. Under sterile environment, peel off the skin, find the osteogenic center part of the pilose antler, and cut the tissue at the top 5 - 10 cm of the pilose antler.

[0039] S2. Double-distilled water extraction: Cut the osteogenic center tissue obtained in S1 into pieces with side length of 4 mm. After washing 3 times with PBS, crush the tissue pieces to obtain tissue powder with particle size of 100 μm. Then mix 50 g of tissue powder with 500 mL of double-distilled water, place it at 4 °C, and stir and extract with a magnetic stirrer at a speed of 180 r / min for 24 h. Add 10 mg of protease inhibitor (purchased from thermofisher company) to prevent degradation of protein polypeptides. Extract 2 times, and finally mix the obtained aqueous solutions of osteogenic center polypeptides of pilose antler together.

[0040] S3. Filtration and drying: First, filter the polypeptide solution obtained in S2 with a 0.22-μm filter membrane to remove tissue residues, cells, bacteria and other impurities, and then vacuum freeze-dry at -70 °C for 48 h to obtain the osteogenic center polypeptide extract of pilose antler.

[0041] Example 3: Preparation of osteogenic center polypeptide extract (O-VAP) of pilose antler S1. Obtaining of osteogenic center tissue of pilose antler: Select artificially farmed adult male sika deer. After anesthesia, disinfect the trifurcated pilose antler with iodophor and alcohol in gradient, then harvest it. Under sterile environment, peel off the skin, find the osteogenic center part of the pilose antler, and cut the tissue at the top 5 - 10 cm of the pilose antler.

[0042] S2. Double-distilled water extraction: Cut the osteogenic center tissue obtained in S1 into pieces with side length of 4 mm. After washing 3 times with PBS, crush the tissue pieces to obtain tissue powder with particle size of 50 μm. Then mix 50 g of tissue powder with 750 mL of double-distilled water, place it at 6 °C, and stir and extract with a magnetic stirrer at a speed of 180 r / min for 12 h. Add 25 mg of protease inhibitor, extract 3 times, and finally mix the obtained aqueous solutions of osteogenic center polypeptides of pilose antler together.

[0043] S3. Filtration and drying: First, filter the polypeptide solution obtained in S2 with a 0.22-μm filter membrane to remove tissue residues, cells, bacteria and other impurities, and then vacuum freeze-dry at -50 °C for 96 h to obtain the osteogenic center polypeptide extract of pilose antler.

[0044] Example 4: Preparation of osteogenic center polypeptide extract (O-VAP) of pilose antler S1. Obtaining the osteogenic center tissue of antler: Select adult male sika deer raised artificially, anesthetize them, disinfect the two-bar antlers with iodine and alcohol, and then harvest them. Peel off the skin in a sterile environment, find the osteogenic center of the antler, and cut the tissue 5 to 10 cm from the top of the antler.

[0045] S2. Double-distilled water extraction: The osteogenic center tissue obtained in S1 was cut into pieces with a side length of 4 mm. After being washed with PBS for 3 times, the small pieces of tissue were crushed to obtain tissue powder with a particle size of 200 μm. Then, 50 g of tissue powder was mixed with 1000 mL of double-distilled water, placed at 10°C, and stirred and extracted with a magnetic stirrer at a speed of 180 rpm for 48 h. 5 mg of protease inhibitor was added, and the extraction was performed twice. Finally, the obtained antler osteogenic center polypeptide aqueous solutions were mixed together.

[0046] S3, filtration and drying: first, the polypeptide solution obtained in S2 is filtered with a 0.22 μm filter membrane to remove tissue residues and impurities such as cells and bacteria, and then vacuum freeze-dried at -70°C for 72 hours to obtain the antler osteogenic center polypeptide extract.

[0047] In order to verify the anti-osteoporosis effect of the velvet antler osteogenic center polypeptide extract prepared by the present invention, the inventors conducted the following experiments: Since Examples 2 to 4 have similar effects, for the convenience of subsequent discussion and reference, the test results of the antler osteogenic center polypeptide extract prepared in Example 2 shall prevail. The following are the test results of Example 2.

[0048] Experimental Example 1: Functional verification of anti-osteoporosis effect of antler osteogenic center polypeptide extract (O-VAP) like Figure 2 As shown, female BALB / c mice aged 6 to 8 weeks were selected for ovariectomy. Four weeks later, they were treated with PBS (PBS was given after ovariectomy as a negative control), BPs (1 mg / kg bisphosphonate was given after ovariectomy as a positive control), T-VAP (1 mg / kg whole antler polypeptide extract was given after ovariectomy), and O-VAP (1 mg / kg antler osteogenic center polypeptide extract prepared in Example 2 was given after ovariectomy), twice a week for 8 weeks. The mice were euthanized and the femoral tissues were removed for detection of osteoporosis indicators.

[0049] 1. Micro-CT detection The distal femur tissue was taken for micro-CT scanning, and the results were as follows: Figure 3 and Figure 4As shown in the figure, it can be found that O-VAP can significantly improve bone mineral density, bone volume fraction, trabecular bone number, trabecular bone thickness, trabecular bone separation degree and cortical bone density, and the effect is significantly stronger than that of T-VAP ( p <0.05), and the effects of some indicators are comparable to those of bisphosphonate treatment.

[0050] 2. Histological detection After the distal femur tissue was fixed with 4% paraformaldehyde, dehydrated with gradient ethanol, cleared with xylene, embedded in paraffin and sectioned, HE staining and safranin O-fast green staining were performed respectively.

[0051] The results are as Figure 5 shown. The results show that O-VAP significantly increased the trabecular bone area of the primary and secondary osteogenic centers of the mouse femur, and the effect was significantly stronger than that of T-VAP, even reaching the level of bisphosphonate treatment.

[0052] 3. Component analysis of O-VAP using high performance liquid chromatography technology Weighed the same mass of O-VAP and T-VAP and dissolved them in the same volume of deionized water respectively, and used HPLC to analyze their components. The specific method is as follows: Select a C18 reverse-phase chromatographic column, use acetonitrile-water (containing 0.1% trifluoroacetic acid) as the mobile phase for gradient elution, and detect at a wavelength of 214 nm. Qualitative and quantitative analysis of the polypeptides extracted from velvet antler was carried out based on the retention time and peak area.

[0053] The results are as Figure 6 shown. By comparing Figure 6 Figure A with Figure 6 Figure B, it was found that Figure 6 Figure A had a unique peak with a relatively large peak area at a retention time of 3 min, which indicated that O-VAP contained unique components.

[0054] In summary, the O-VAP provided by the present invention has a strong anti-osteoporosis effect. At the same dose, its anti-osteoporosis effect is better than that of T-VAP. This is because O-VAP contains unique components, which play an important role in the exertion of the anti-osteoporosis effect.

[0055] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that any value between the two endpoints of each numerical range and the two endpoints can be selected. To avoid repetition, the present invention describes preferred embodiments.

[0056] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they know the basic creative concepts. Therefore, the appended claims are intended to be construed to include the preferred embodiments and all changes and modifications falling within the scope of the present invention.

[0057] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and its equivalent technologies, the present invention also intends to include these modifications and variations.

Claims

1. A pilose antler osteogenic center polypeptide extract with the effect of improving osteoporosis, characterized in that, The polypeptide extract of the antler osteogenic center is obtained through the following preparation steps: Cut the tissue at the top 5-10 cm of the excised antler to obtain the antler osteogenic center tissue; Crush the antler osteogenic center tissue and add water for extraction to obtain the polypeptide extract of the antler osteogenic center.

2. The polypeptide extract of pilose antler osteogenic center according to claim 1, characterized in that, The ratio of the antler osteogenic center tissue to water is 1 g: 10-20 mL.

3. The polypeptide extract of pilose antler osteogenic center according to claim 1, characterized in that, The extraction time is 12-48 h.

4. The polypeptide extract of pilose antler osteogenic center according to claim 1, wherein The extraction temperature is 4-10 °C.

5. The polypeptide extract of pilose antler osteogenic center according to claim 1, characterized in that The extraction needs to be carried out 2-3 times.

6. The polypeptide extract of pilose antler osteogenic center according to claim 1, wherein A protease inhibitor needs to be added during the extraction, and the addition amount of the protease inhibitor is 0.01%-0.05% of the mass of the antler osteogenic center tissue.

7. Use of the polypeptide extract of the antler osteogenic center according to any one of claims 1-6 in the preparation of a drug for preventing or treating osteoporosis.

8. Use of the polypeptide extract of the antler osteogenic center according to any one of claims 1-6 in the preparation of a health product or medical device or functional food for increasing bone density.

Images