Application of deer blood polypeptide in reverse phase of osteoporosis in preparation of osteoporosis treatment preparation

Abstract

The application belongs to the technical field of biotechnology and tissue engineering, and particularly relates to application of an osteoporosis-reversing period deer blood polypeptide in preparation of an osteoporosis treatment preparation. The application first proposes and isolates and prepares an active substance of precisely applied deer blood, i.e., an osteoporosis-reversing period deer blood polypeptide RDBP, and provides a preparation method of the RDBP. The deer blood polypeptide obtained through the preparation method has a significant anti-osteoporosis prevention and treatment effect, and the effect is significantly stronger than that of deer blood polypeptides in other periods. The deer blood polypeptide has the characteristics of being natural, safe and free of side effects. The osteoporosis-reversing period deer blood polypeptide has important application value in the fields of preparation of medicines for preventing / treating osteoporosis or health care products and medical products for increasing bone density.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and tissue engineering technology, specifically relating to the application of a deer blood polypeptide in the osteoporosis reversal phase in the preparation of osteoporosis treatment agents. Background Technology

[0002] Osteoporosis, a common skeletal disease, is characterized by decreased bone density and destruction of bone microstructure. It is a prevalent chronic disease, with a prevalence rate of 36% in people over 60 years of age, and over 60% in women. Osteoporosis can lead to bony pain, spinal deformities, multiple organ dysfunction, and osteoporotic fractures. Although various medications are available for treating osteoporosis, such as bisphosphonates, denosumab, calcitonin, osteocalcin, selective estrogen receptor modulators (SERMs), and parathyroid hormones (PTHs), their effectiveness is extremely limited. They can only slow the progression of osteoporosis to a limited extent, and cannot completely inhibit or even reverse it. Most importantly, they have side effects such as gastrointestinal irritation and jawbone necrosis, highlighting the urgent need to develop new treatment strategies. Summary of the Invention

[0003] To provide a novel strategy for treating osteoporosis with high safety, this invention provides the application of deer blood polypeptide in the osteoporosis reversal phase in the preparation of osteoporosis treatment agents.

[0004] The technical solution adopted in this invention is:

[0005] This invention provides the application of deer blood polypeptide in the osteoporosis reversal phase in the preparation of osteoporosis treatment agents, and the preparation method of the deer blood polypeptide in the osteoporosis reversal phase is as follows:

[0006] The freeze-dried blood powder from deer blood in the osteoporosis reversal period was pulverized, dissolved in water, and then extracted with protease inhibitors.

[0007] The extract was collected, filtered, and freeze-dried to obtain deer blood polypeptides in the osteoporosis reversal phase.

[0008] Preferably, the mass-to-volume ratio of the blood freeze-dried powder to water is 100g~120g:1L.

[0009] Preferably, the mass ratio of the blood lyophilized powder to the protease inhibitor is 5000~5500:1.

[0010] Preferably, the extraction conditions are 2℃~6℃, 160rpm~200rpm for 22h~26h.

[0011] Preferably, the extraction conditions are 4°C, 180 rpm for 24 hours.

[0012] Preferably, the extraction is performed 2 to 4 times.

[0013] Preferably, the osteoporosis treatment formulation further includes pharmaceutically acceptable excipients.

[0014] Preferably, the pharmaceutically acceptable excipient is one or more of the following: diluent, disintegrant, precipitation inhibitor, flow aid, binder, dispersant, suspending agent, isotonic agent, thickener, emulsifier, preservative, and stabilizer.

[0015] Preferably, the diluent is physiological saline.

[0016] Preferably, the disintegrant comprises any one of starch, microcrystalline cellulose, and low-substituted hydroxypropyl cellulose.

[0017] Preferably, the precipitation inhibitor comprises any one of sodium dodecyl sulfate, Tween-80, polyvinylpyrrolidone, and hydroxypropyl methylcellulose.

[0018] Preferably, the flow aid comprises any one of cationic polyacrylamide, polydiallyldimethylammonium chloride, and cationic starch.

[0019] Preferably, the adhesive comprises any one of starch paste, hydroxypropyl methylcellulose, and povidone.

[0020] Preferably, the dispersant comprises any one of sodium dodecyl sulfate, polyvinylpyrrolidone, and sodium carboxymethyl cellulose.

[0021] Preferably, the suspending agent includes any one of gum arabic, tragacanth gum, sodium carboxymethyl cellulose, and hydroxypropyl methylcellulose.

[0022] Preferably, the isotonic agent includes any one of sodium chloride, glucose, and mannitol.

[0023] Preferably, the thickener includes any one of gum arabic, xanthan gum, and sodium carboxymethyl cellulose.

[0024] Preferably, the emulsifier comprises any one of sodium dodecyl sulfate, benzalkonium chloride, and fatty acid sorbitan.

[0025] Preferably, the preservative includes any one of benzoic acid, sorbic acid, methylparaben, and benzalkonium bromide.

[0026] Preferably, the stabilizer comprises any one of sodium sulfite, sodium bisulfite, tocopherol, and disodium EDTA.

[0027] Preferably, the acceptable dosage form of the preparation for treating osteoporosis includes one of the following: tablets, capsules, granules, injections, pills, powders, ointments, and oral liquids.

[0028] Preferably, the formulation of the osteoporosis treatment preparation is as follows: dissolve the osteoporosis reversal phase deer blood polypeptide in physiological saline, mix well, and the osteoporosis treatment preparation is obtained.

[0029] Preferably, the concentration of the osteoporosis treatment preparation is 0.2 g / L to 1 g / L.

[0030] Compared with the prior art, the beneficial effects of the present invention are:

[0031] This invention provides an application of deer blood polypeptide in the osteoporosis reversal stage in the preparation of osteoporosis treatment agents. This invention is the first to propose and isolate an active substance from deer blood for precise application, namely, deer blood polypeptide RDBP in the osteoporosis reversal stage, and provides a method for preparing RDBP. The deer blood polypeptide obtained by the method provided by this invention has significant anti-osteoporosis prevention and treatment effects, and its effect is significantly stronger than that of deer blood polypeptides at other stages, such as deer ADBP; it is also natural, safe, and has no side effects. The deer blood polypeptide in the osteoporosis reversal stage provided by this invention has important application value in the fields of preparing drugs for the prevention / treatment of osteoporosis or health products and medical products that increase bone density.

[0032] This invention involves extracting freeze-dried deer blood powder from the osteoporosis reversal phase (i.e., freeze-dried deer blood powder from antler regeneration 100-130 days after regeneration) at a material-to-liquid ratio of 1:10 with double-distilled water at 4°C for 24 hours, repeating the extraction three times. This ensures the full extraction of soluble active protein peptides from the tissue while maintaining the bioactivity of the extracted peptides. Prolonged extraction can lead to fermentation, stickiness, and deterioration of the product, affecting its activity; conversely, insufficient extraction results in low peptide yield and resource waste. The addition of protease inhibitors effectively prevents peptide degradation and denaturation during extraction; water extraction minimizes variable factors, maximizing the retention of bioactivity in the extracted peptides. The preparation method provided by this invention is simple to operate and yields peptides that retain relatively complete activity. Attached Figure Description

[0033] Figure 1 This is a micro-CT image of the distal femur of a mouse. From top to bottom, each row represents: the overall longitudinal section, the central cross-section of primary osteogenic bone in cancellous and cortical bone, the central cross-section of primary osteogenic bone in cortical bone, and the central cross-section of primary osteogenic bone in cancellous bone.

[0034] Figure 2Statistical analysis of CT results. A: Trabecular separation (Tb.Sp); B: Trabecular thickness (Tb.Th); C: Bone volume fraction (BV / TV); D: Number of trabeculae (Tb.N). * indicates... p <0.05, ** indicates p <0.01, *** indicates p <0.001, **** indicates p <0.0001.

[0035] Figure 3 The images show the HE-alicin blue staining results of sections from the distal femur of mice. From top to bottom, each row represents: the overall longitudinal section, a magnified view of region a within the overall longitudinal section, a magnified view of region b within the overall longitudinal section, and a magnified view of region c within the overall longitudinal section. The blue portion represents cartilage. Detailed Implementation

[0036] The present invention will be further illustrated below with specific embodiments, but these embodiments do not limit the scope of the invention. Modifications or substitutions to the details and form of the technical solutions of the present invention may be made without departing from the spirit and scope of the invention, but all such modifications or substitutions fall within the protection scope of the present invention.

[0037] The inventive concept of this invention is as follows:

[0038] This invention provides the application of deer blood polypeptide in the osteoporosis reversal phase in the preparation of osteoporosis treatment agents.

[0039] Deer blood is widely used in traditional Chinese medicine for tonifying the kidneys and strengthening bones and muscles. Scientific research has also revealed its potential value in combating osteoporosis. These components exert their effects through multiple mechanisms, including promoting osteoblast proliferation and differentiation, inhibiting osteoclast activity, and improving overall immune function. It is important to note that deer bones suffer severe physiological osteoporosis every year, with loss exceeding 30%, yet their bones possess an astonishing regenerative capacity, with lost minerals being fully recovered within a month. Research has found that this remarkable osteoporosis reversal potential depends on the combined effects of the deer's endocrine environment and tissue microenvironment. However, previous research and applications of deer blood have not considered the differences in the deer's endocrine environment and tissue microenvironment at different stages, especially considering that deer experience osteoporosis and its reversal annually. Based on the understanding of the link between deer osteoporosis and its reversal, and the knowledge of the potential mechanisms of bone metabolism, this invention demonstrates that deer blood in the reversal period has a better effect in combating osteoporosis compared to deer blood at other stages. Deer blood can be said to be the most effective source of deer products for treating osteoporosis, because the active substances that stimulate antler ossification and reverse bone osteoporosis can be carried by circulating blood. The present invention provides a better alternative for the precise application and development of deer blood, as well as for the clinical treatment of osteoporosis.

[0040] Deer antlers are the bony organs of deer. During the summer, as deer grow antlers, calcium and minerals are transported from all parts of the body to the antlers, causing other bones in the deer's body to become very porous, leading to severe osteoporosis. Therefore, deer blood during this period is called growth period deer blood or osteoporosis period deer blood. In autumn, as antler growth stops, the other porous bones in the body become very healthy within a month, or even less. The previously porous bones become healthy and can replenish lost minerals; therefore, deer blood during this period is called osteoporosis reversal period deer blood, and the corresponding peptides are called osteoporosis reversal period deer blood peptides. Based on this biological phenomenon, it is inferred that the anti-osteoporosis effect of deer blood peptides in the osteoporosis reversal period is far stronger than that of deer blood peptides in the antler growth period. Therefore, based on the unique biological characteristics of deer, this invention proposes the precise application of deer blood peptides in the osteoporosis reversal period. This invention confirms that polypeptides extracted from deer blood during the osteoporosis reversal period possess very strong anti-osteoporosis effects and the potential to promote bone differentiation. This invention is the first to discover this function. In the prior art, deer blood from any stage is uniformly called deer blood. This invention is the first to propose the precise application of deer blood during the osteoporosis reversal period, treating it as deer blood from a special period, which has better effects.

[0041] Existing research indicates that while deer blood peptides possess osteogenic activity, their preclinical testing results have not met expectations. This phenomenon is speculated to be due to overlooking the unique "physiological osteoporosis-reversal" biorhythm in deer. During the rapid antler growth period, specifically the first 100 days after antler regeneration, the deer's skeleton mobilizes a large amount of calcium to support the rapid growth of the antler, resulting in severe osteoporosis, with a rate exceeding 30%. During the osteoporosis reversal period, which occurs 30 days after the end of rapid antler growth (100-130 days after antler regeneration), the osteoporosis in the deer's skeleton reverses rapidly. During this period, specific bioactive peptides may accumulate in the blood. These components, through synergistic regulation of the bone resorption / bone formation balance, hold promise for achieving precise regulation of bone metabolism.

[0042] Based on this, the present invention innovatively proposes to develop a novel osteoporosis treatment agent and a new strategy that can restore bone mass by extracting RDBP.

[0043] Deer blood peptides in the reversal phase of osteoporosis, abbreviated as RDBP.

[0044] To enable those skilled in the art to better understand and implement the technical solutions of this invention, the invention will be further described below with reference to specific embodiments. Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0045] Example 1

[0046] The application of deer blood polypeptides in the osteoporosis reversal phase in the preparation of osteoporosis treatment agents is as follows:

[0047] 1. Extraction and preparation of deer blood polypeptides at different stages.

[0048] Deer blood peptide RDBP for osteoporosis reversal: Extracted from customized freeze-dried deer blood powder purchased from Mountain River Venison, New Zealand. The freeze-dried deer blood powder was obtained from blood collected from adult male sika deer slaughtered at 100-130 days of antler regeneration.

[0049] Osteoporosis-prone deer blood polypeptide ADBP: lyophilized blood powder derived from deer blood during the antler growth period, specifically blood from 0 to 100 days after antler regeneration. Deer blood polypeptide from the osteoporosis-prone period was used as a control for subsequent experiments.

[0050] The two types of blood freeze-dried powders were processed as follows:

[0051] After vacuum freeze-drying at -70℃ for 48 h, the powder was pulverized to a particle size of 100 μm. Then, 50 g of powder was mixed with 500 mL of double-distilled water and placed at 4℃. The mixture was stirred and extracted at 180 rpm for 24 h using a magnetic stirrer. 10 mg of protease inhibitor was added simultaneously with the addition of double-distilled water to prevent protein and peptide degradation. The extraction was repeated three times, and the extract was collected. Finally, the extract was filtered and dried using a 0.22 μm filter membrane to remove tissue residues, cells, bacteria, and other impurities. The extract was then vacuum freeze-dried at -70℃ for 48 h to obtain osteoporosis reversal deer blood peptide RDBP and osteoporosis-stage deer blood peptide ADBP.

[0052] 2. The therapeutic effect of RDBP on osteoporosis in ovariectomized mice.

[0053] Eight-week-old female C57 / BL6 mice underwent ovariectomy to establish an osteoporosis model. Four weeks later, they were given different treatments by gavage, as detailed in Table 1.

[0054] The RDBP and ADBP obtained above were dissolved in physiological saline to prepare solutions of different concentrations, which were then administered to mice via gavage.

[0055] Table 1 Different processing methods

[0056] Note: In Table 1, "-" indicates that this item is not present. The Normal group consists of individuals who did not undergo oophorectomy.

[0057] The results are as follows:

[0058] Three-dimensional bone structure analysis of the distal femur in ovariectomized mice using a Micro-CT imaging system revealed significant differences in bone tissue morphology among different treatment groups. Figure 1 The normal control group (Normal) exhibited typical healthy bone structure characteristics: the overall longitudinal section showed uniform bone density distribution and a continuous and intact trabecular network. In stark contrast, the negative control group (NS) displayed typical osteoporotic features, with significantly lower overall bone density than the Normal group, reduced trabecular bone density in the cancellous bone region, and significantly thinner cortical bone. These changes are consistent with the characteristics of estrogen deficiency-induced bone loss due to ovariectomy. The positive control groups (BPs and ADBP) showed some anti-osteoporosis effects; while the RDBP group showed superior bone repair efficacy, exhibiting a dose-dependent characteristic. The 4 mg / kg dose group showed effects approaching those of the BPs group and significantly better than the ADBP group.

[0059] according to Figure 2 Micro-CT statistical analysis showed that the RDBP group exhibited a significant advantage in improving bone microstructural parameters. Compared with ADBP, 4 mg / kg RDBP treatment increased trabecular bone thickness (Tb.Th) by 23% (p<0.0001), trabecular bone number (Tb.N) by 25% (p<0.001), and trabecular bone separation (Tb.Sp) by 19% (p<0.01). In terms of bone volume fraction (BV / TV), the 4 mg / kg RDBP group reached 92% of the normal group level (p<0.0001) and was 18% higher than the same dose ADBP group (p<0.05).

[0060] Dose-response analysis showed that 4 mg / kg RDBP was significantly superior to its 2 mg / kg dose group in all measured parameters (p < 0.05); while ADBP only showed dose-dependent improvement in the BV / TV index. Notably, 4 mg / kg RDBP achieved 85%–90% improvement in Tb.N and BV / TV parameters compared to the bisphosphonate treatment group (p > 0.05), but showed superior trabecular bone thickening in the Tb.Th index, 15% higher than the bisphosphonate group (p < 0.05). Cortical bone mineral density testing further confirmed that 4 mg / kg RDBP restored bone mineral density to 95% of the normal level (p < 0.0001), an increase of 12% compared to the same dose ADBP group (p < 0.01).

[0061] Figure 3 HE-allicin blue staining results of distal femoral bone tissue from mice in different treatment groups are presented. Bone tissue sections prepared by fixation with 4% v / v paraformaldehyde, gradient dehydration with ethanol, xylene clearing, and paraffin embedding showed that, in the overall longitudinal section, the 4 mg / kg RDBP treatment group exhibited significant improvement in trabecular bone structure, with a significantly increased trabecular area in both primary and secondary osteogenic centers compared to the control group. Regions a and b show that the number of trabeculae in the 4 mg / kg RDBP group was approximately 1.5 times higher than that in the same dose ADBP group, and its trabecular connectivity was close to that of the bisphosphonate treatment group. Quantitative analysis in region c indicates that 4 mg / kg RDBP significantly promoted secondary trabecular bone formation, with a dose-dependent osteogenic effect, while ADBP only showed a moderate degree of bone formation promotion at a dose of 4 mg / kg. Notably, the bone formation indices of the 2 mg / kg RDBP group exceeded those of the 4 mg / kg ADBP group, suggesting that RDBP has stronger bone metabolism regulatory activity.

[0062] These results confirm that RDBP has a very strong anti-osteoporosis effect, and its effect is better than ADBP, and can even reach the level of treatment with commonly used clinical drugs such as bisphosphonates.

[0063] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0064] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. The application of deer blood polypeptide in the osteoporosis reversal phase in the preparation of osteoporosis treatment agents, characterized in that, The preparation method of the osteoporosis reversal phase deer blood polypeptide is as follows: The freeze-dried blood powder from deer blood in the osteoporosis reversal period was pulverized, dissolved in water, and then extracted with protease inhibitors. The extract was collected, filtered, and freeze-dried to obtain deer blood polypeptides in the osteoporosis reversal phase.

2. The application as described in claim 1, characterized in that, The mass-to-volume ratio of the blood freeze-dried powder to water is 100g~120g:1L.

3. The application according to claim 1, characterized in that, The mass ratio of the blood lyophilized powder to the protease inhibitor is 5000~5500:

1.

4. The application as described in claim 1, characterized in that, The extraction conditions are 2℃~6℃, 160rpm~200rpm for 22h~26h.

5. The application as described in claim 4, characterized in that, The extraction conditions were 4°C, 180 rpm for 24 hours.

6. The application as described in claim 1, characterized in that, The extraction is performed 2 to 4 times.

7. The application as described in claim 1, characterized in that, The osteoporosis treatment formulation also includes pharmaceutically acceptable excipients.

8. The application as described in claim 7, characterized in that, The pharmaceutically acceptable excipients are one or more of the following: diluents, disintegrants, precipitation inhibitors, flow aids, binders, dispersants, suspending agents, isotonic agents, thickeners, emulsifiers, preservatives, and stabilizers.

9. The application as described in claim 1, characterized in that, The acceptable dosage forms of the osteoporosis treatment preparation include one of the following: tablets, capsules, granules, injections, pills, powders, ointments, and oral liquids.

10. The application as described in claim 9, characterized in that, The formulation of the osteoporosis treatment preparation is as follows: The osteoporosis reversal phase deer blood polypeptide is dissolved in physiological saline and mixed well to obtain the osteoporosis treatment preparation.

Images