Pharmaceutical composition for preventing osteoporosis as well as preparation method and application thereof

Abstract

The invention discloses a pharmaceutical composition for preventing osteoporosis as well as a preparation method and application of the pharmaceutical composition, and relates to the technical field of pharmaceutical compositions. The pharmaceutical composition for preventing osteoporosis comprises the following components in parts by weight: 9-14 parts of bovine embryonic muscle stem cell mitochondrial polypeptide extract, 5-10 parts of bovine embryonic spinal stem cell mitochondrial polypeptide extract, 5-10 parts of bovine thyroid and testis polypeptide extract, 5-10 parts of polysaccharide chondroitin sulfate, 3-7 parts of vitamin D and the like. According to the pharmaceutical composition for preventing osteoporosis, all the components cooperate with one another, the treatment effect on an osteoporosis rat model is remarkably improved, and a new treatment means is provided for preventing osteoporosis diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical compositions, and in particular to a pharmaceutical composition for preventing osteoporosis, and a preparation method and application thereof. Background Art

[0002] Osteoporosis is a bone disease that is prevalent worldwide. Its main characteristics are decreased bone density and destruction of bone microstructure, which significantly increase the risk of fractures. This disease not only seriously affects the quality of life of patients, but also brings a heavy economic burden to families and society. At present, both traditional Chinese medicine and Western medicine have their own unique theoretical systems and treatment methods for the prevention and treatment of osteoporosis.

[0003] Traditional Chinese medicine theory attributes osteoporosis to multiple factors such as kidney essence deficiency, spleen and stomach weakness, and qi and blood stasis. Chinese medicine believes that the kidney is responsible for bone and marrow production, and the fullness of kidney essence is directly related to the health of bones. Kidney essence deficiency will lead to insufficient bone marrow, which cannot fully nourish the bones, and then cause osteoporosis. At the same time, the spleen and stomach, as the foundation of acquired constitution, are responsible for transporting and transforming the essence of water and grain, and are an important source of nourishment for muscles and bone marrow. If the spleen and stomach are damaged, the essence of water and grain will not be produced enough, the muscles and bone marrow will not be nourished, the limbs will not be used, and the health of bones will naturally be damaged. In addition, the liver is responsible for regulating and storing blood. If the liver qi is strong and the liver blood is full, the tendons will be strong and the bones will be healthy; otherwise, the tendons will be weak and the bones will be atrophied. Liver depression and qi stagnation, qi depression and fire, are easy to burn liver yin, which is closely related to the occurrence and development of osteoporosis. Blood stasis, as a pathological product of osteoporosis, blocks the meridians and further aggravates the condition.

[0004] Based on the above theory, the principle of TCM in treating osteoporosis is mainly "differentiation and treatment, combination of disease and syndrome, overall regulation, combination of prevention and treatment", and improving symptoms and quality of life through methods such as tonifying the kidney, strengthening the spleen, and promoting blood circulation. However, although traditional Chinese medicine treatment is effective, its mechanism of action is often not clear, and it takes a long time for the efficacy to take effect.

[0005] In Western medicine, osteoporosis is mainly prevented and treated by supplementing calcium, vitamin D and using drugs such as bisphosphonates to increase bone density and reduce the risk of fractures. At the same time, regulating parathyroid function and testosterone substances is also an important means to promote bone formation and maintain bone density. Although these methods can relieve the symptoms of osteoporosis to a certain extent, long-term use may have side effects, and for some patients, the effect is not ideal.

[0006] In recent years, with the in-depth study of Chinese medicine monomers and compound prescriptions, a variety of Chinese medicine ingredients such as paeoniflorin, total flavonoids of bone drynaria, catalpol, icariin, etc., as well as Chinese medicine compound prescriptions such as Zuogui Pills, Yishen Gugu Recipe, and Eucommia Bone Recipe, etc., have been proven to promote osteogenic differentiation of bone marrow mesenchymal stem cells, osteoblast proliferation and differentiation by activating the Wnt signaling pathway, thereby repairing bone damage and treating osteoporosis. Activation of the Wnt signaling pathway leads to increased expression of a series of target genes, including β-catenin, Cyclin D1, c-Myc, Axin2, etc., which play an important role in bone formation and bone metabolism.

[0007] Therefore, developing a pharmaceutical composition that combines traditional Chinese and Western medicine theories, can quickly relieve symptoms, fundamentally regulate bone metabolism, and has few side effects is of great significance for the prevention and treatment of osteoporosis. Summary of the invention

[0008] In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing osteoporosis and a preparation method and application thereof.

[0009] In a first aspect, the present invention provides a pharmaceutical composition for preventing osteoporosis, wherein the pharmaceutical composition for preventing osteoporosis comprises the following components in parts by weight:

[0010] 9-14 parts of bovine embryonic muscle stem cell mitochondrial polypeptide extract, 5-10 parts of bovine embryonic spinal cord stem cell mitochondrial polypeptide extract, 5-10 parts of bovine thyroid and testicular polypeptide extract, 5-10 parts of polysaccharide chondroitin sulfate, 3-7 parts of vitamin D, 2-5 parts of vitamin K, 1-3 parts of curcumin, 3-7 parts of epimedium, 3-7 parts of melon seeds, 1-3 parts of rhodiola rosea, 5-10 parts of astragalus and 1-5 parts of seabuckthorn.

[0011] Furthermore, the pharmaceutical composition for preventing osteoporosis comprises the following components in parts by weight:

[0012] 10 parts of bovine embryonic muscle stem cell mitochondrial polypeptide extract, 8 parts of bovine embryonic spinal cord stem cell mitochondrial polypeptide extract, 8 parts of bovine thyroid and testicular polypeptide extract, 8 parts of polysaccharide chondroitin sulfate, 5 parts of vitamin D, 3 parts of vitamin K, 2 parts of curcumin, 5 parts of epimedium, 3 parts of melon seeds, 2 parts of rhodiola rosea, 8 parts of astragalus and 3 parts of seabuckthorn.

[0013] Furthermore, the method for preparing the bovine embryonic muscle stem cell mitochondrial polypeptide extract comprises the following process:

[0014] 1) Isolation of bovine embryonic muscle stem cells:

[0015] Healthy bovine embryos at 60-90 days of gestation were selected, and skeletal muscle tissues of the limbs were obtained under sterile conditions;

[0016] Digestion was performed with a mixture of 0.1% collagenase IV and 0.05% trypsin at 37°C for 30 minutes under shaking, and then neutralized with DMEM medium containing 10% fetal bovine serum. The fibroblasts were removed by the differential adhesion method at 37°C for 30 minutes, and the suspended cells were collected as muscle stem cells.

[0017] 2) Stem cell expansion and mitochondrial activation:

[0018] The basal medium was DMEM / F12 supplemented with 1% fetal bovine serum substitute and maintained at 37°C and 5% CO 2 Three-dimensional suspension culture was cultured under hypoxia for 1 week with 5% O 2 , circulates once every 24 hours, enhancing mitochondrial biogenesis;

[0019] 3) Cell disruption and mitochondrial separation:

[0020] High-pressure homogenization was used, with 800 bar and 4 °C for three cycles, using a mitochondrial protection buffer containing 0.25 M sucrose and 10 mM HEPES, pH 7.4;

[0021] Gradient centrifugation: First centrifugation: 1000 × g, 4 ° C for 10 minutes to remove cell debris; second centrifugation: 12000 × g, 4 ° C for 15 minutes to collect mitochondrial precipitate;

[0022] 4) Mitochondrial freeze-thaw lysis:

[0023] Lysis buffer: Tris-HCl buffer containing 1% Triton X-100, 5 mM DTT, pH 8.0;

[0024] Procedure: -80℃ quick freezing for 30 minutes → 37℃ water bath rapid thawing, repeat 3 times to destroy the mitochondrial membrane structure;

[0025] 5) Enzymatic hydrolysis:

[0026] The mixed protease of trypsin: pepsin = 3:1 was treated in stages: the first stage: pH 7.5, 37°C enzymolysis for 2 hours; the second stage: pH 2.0, 42°C enzymolysis for 1 hour; inactivation: 80°C water bath for 10 minutes to terminate the reaction;

[0027] 6) Purification:

[0028] Ultrafiltration classification: 10kDa ultrafiltration membrane retains large molecular proteins, and fractions <10kDa are collected for concentration and drying.

[0029] Furthermore, the method for preparing the bovine embryonic spinal cord stem cell mitochondrial polypeptide extract comprises the following steps:

[0030] 1) Isolation of spinal cord stem cells:

[0031] Healthy bovine embryos at 60-75 days of gestation were selected, and the spinal cord central canal region tissue was isolated under sterile conditions;

[0032] The cells were digested with a mixture of 0.15% collagenase IV and 0.02% hyaluronidase at 37°C for 20 minutes under shaking. The stop solution was DMEM / F12 medium containing 10% low-fat acid bovine serum albumin (BSA).

[0033] Spinal cord stem cells were obtained by CD133+ / Nestin+ labeled immunomagnetic bead sorting and pre-attachment at 37°C for 1 hour to remove foreign cells.

[0034] 2) Three-dimensional dynamic amplification:

[0035] The basal medium DMEM / F12 was supplemented with 15% fetal bovine serum replacement, 10 ng / mL FGF2, and 5 μM Wnt signal inhibitor XAV939;

[0036] 37°C, 5% CO 2 Under the environment, three-dimensional dynamic suspension culture was performed in a rotating bioreactor at a speed of 12 rpm for 1 week, and 0.1 μM Y-27632ROCK inhibitor was supplemented every 48 h;

[0037] 3) Mitochondrial-directed activation:

[0038] Introduce intermittent hypoxia-reoxygenation cycles during the 2 days following the expansion: 5% O 2 6 hours → 21% O 2 Conduct for 2 hours;

[0039] 4) Crushing and separation:

[0040] Cell disruption: Nitrogen cavitation method, pressure 450 psi, 4 ° C for 5 minutes, buffer containing 0.3 M mannitol, 10 mM EDTA and 1 mM PMSF, pH 7.2;

[0041] Gradient centrifugation: initial centrifugation: 800 × g, 4°C for 10 min) to remove nuclear debris;

[0042] Separation: centrifugation at 15000×g, 4°C for 20 min) to collect mitochondrial pellet;

[0043] 5) Cracking:

[0044] Lysis buffer: Tris-HCl buffer, pH 7.8, containing 0.5% sodium deoxycholate and 2 mM DTT;

[0045] Lysis procedure: Continuous shear lysis was achieved through a microfluidic chip with a channel diameter of 50 μm, and lysis was performed at a flow rate of 0.5 mL / min for 1 hour;

[0046] 6) Enzymatic hydrolysis:

[0047] Step 1: neutral protease, pH 7.0, hydrolysis at 45°C for 1.5 hours;

[0048] Step 2: hydrolysis with pancreatic elastase, pH 8.5, 37°C for 2 h;

[0049] Inactivation: ultrafiltration using a 30 kDa molecular weight cutoff membrane, followed by instantaneous high temperature, maintained at 70°C for 30 seconds;

[0050] 7) Purification:

[0051] Ultrafiltration fractionation: pass through 100kDa, 30kDa, and 10kDa ultrafiltration membranes in sequence, collect fractions <10kDa for concentration and drying.

[0052] Furthermore, the preparation method of the bovine thyroid and testicular polypeptide extract comprises the following process:

[0053] 1) Raw material processing:

[0054] Thyroid gland processing: Take the thyroid gland of healthy adult cattle, remove the fascia and cut into 3mm pieces 3 Tissue blocks were immersed in 0.1% β-mercaptoethanol + 0.05% EDTA solution for 30 min;

[0055] Testis processing: Take fresh bovine testis tissue, peel off the white membrane and cut into 5mm pieces 3 The pellet was washed three times with PBS containing 0.2% sodium citrate;

[0056] 2) Low temperature crushing:

[0057] Thyroid disruption: cryogenic grinding with liquid nitrogen, -196°C, 3000 rpm for 2 minutes, followed by high pressure homogenization, 600 bar cycle 3 times, disruption buffer containing 0.15 M NaCl + 10 mM Tris-HCl with a pH of 7.4;

[0058] Testis disruption: 40kHz ultrasound for 30 minutes, with simultaneous addition of 0.1% neutral protease;

[0059] 3) Thyroid-specific enzymatic hydrolysis:

[0060] The first stage: hydrolysis with trypsin at pH 7.0, 1:50 enzyme-substrate ratio, 50°C for 2 hours;

[0061] Stage 2: Adjust pH to 4.5, add pepsin, 1:100 enzyme-substrate ratio, hydrolyze at 37°C for 3 hours;

[0062] 4) Testis-directed enzymolysis:

[0063] Enzyme combination: bromelain (0.5%) + elastase (0.3%) segmented treatment;

[0064] Pretreatment: pH 6.8, 45°C pre-activation for 15 minutes;

[0065] Main enzymatic hydrolysis: pH 7.5, 55°C shaking hydrolysis for 4 hours;

[0066] 5) Inactivation and purification:

[0067] The product was inactivated at 85°C for 15 seconds, and then passed through 100 kDa, 30 kDa, and 10 kDa ultrafiltration membranes in sequence to collect fractions <10 kDa for concentration and drying.

[0068] In a second aspect, based on the same inventive concept, the present invention provides a method for preparing the pharmaceutical composition for preventing osteoporosis as described in any one of the first aspects, such as Figure 1 As shown, the preparation method comprises the following steps:

[0069] Epimedium, melon seeds, rhodiola rosea, astragalus and seabuckthorn are mixed and fermented to obtain a traditional Chinese medicine fermentation extract;

[0070] The traditional Chinese medicine fermentation extract and the remaining components are mixed, crushed and sieved to obtain the pharmaceutical composition for preventing osteoporosis.

[0071] Furthermore, the step of mixing and fermenting epimedium, melon seeds, rhodiola rosea, astragalus and seabuckthorn to obtain the traditional Chinese medicine fermentation extract comprises the following process:

[0072] Mixing and grinding epimedium, melon seeds, rhodiola rosea, astragalus and seabuckthorn according to a certain proportion, and passing through a 50-mesh sieve to obtain a mixed powder; mixing the mixed powder with deionized water in a weight ratio of 1:(8-10), soaking the mixture for 12-16 hours, and then centrifuging and drying the obtained solid to obtain a traditional Chinese medicine powder;

[0073] Wash the rice and grind it into fine powder, then mix it with rice flour and glucose in a mass ratio of 3:2:1 as a basal culture medium;

[0074] According to the mass ratio of basal culture medium to Chinese medicine powder of 2:1, the two were fully mixed, and an appropriate amount of deionized water was added to make the water content of the culture medium reach 60%-65%, and the pH value of the culture medium was adjusted to 7.4;

[0075] Lactobacillus rhamnosus, Lactobacillus casei and Bifidobacterium longum were prepared and mixed in an inoculation ratio of 3:1:1;

[0076] Add 4% yeast powder and 4% glucose by mass to the culture medium, inoculate the mixed bacteria into the culture medium at an inoculum volume of 5%, and stir well;

[0077] The inoculated culture medium was placed in a fermentation tank and the fermentation temperature was controlled at 37°C. During the fermentation process, the culture medium was dispersed with a stirring paddle every 8 hours, and the fermentation time was set to 3 weeks;

[0078] After fermentation is complete, filter the fermentation product with a 200-mesh filter to remove larger residues. Then centrifuge the filtrate at 4000r / min for 15-20 minutes to further remove fine residues and impurities.

[0079] The supernatant after centrifugation was transferred to a freeze dryer, pre-frozen at -50°C to -40°C for 2-3 hours, and then freeze-dried at a vacuum degree of 10-20 Pa until the moisture content was less than 5%;

[0080] The freeze-dried fermentation product is crushed and passed through an 80-mesh sieve to prepare a uniform powder product, namely the traditional Chinese medicine fermentation extract.

[0081] In the third aspect, based on the same inventive concept, the present invention provides a pharmaceutical composition for preventing osteoporosis as described in any one of the first aspect or a pharmaceutical composition for preventing osteoporosis prepared by the preparation method of the pharmaceutical composition for preventing osteoporosis as described in any one of the second aspect, and its use in the preparation of drugs for preventing osteoporosis.

[0082] Furthermore, the dosage form of the drug for preventing osteoporosis includes capsules.

[0083] The above technical solution provided by the embodiment of the present invention has at least the following advantages compared with the prior art:

[0084] The embodiments of the present invention provide a pharmaceutical composition for preventing osteoporosis, and a preparation method and application thereof. The present invention provides a pharmaceutical composition for preventing osteoporosis, wherein the components work synergistically with each other, significantly improving the therapeutic effect on an osteoporosis rat model, and providing a new treatment method for preventing osteoporosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0086] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0087] Figure 1 A schematic flow chart of a method for preparing a pharmaceutical composition for preventing osteoporosis provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0088] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0089] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0090] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually measured according to national standards. If there is no corresponding national standard, then the conditions recommended by the manufacturer are followed.

[0091] Example 1

[0092] This example provides a pharmaceutical composition for preventing osteoporosis, which comprises the following components in parts by weight:

[0093] 10 parts of bovine embryonic muscle stem cell mitochondrial polypeptide extract, 8 parts of bovine embryonic spinal cord stem cell mitochondrial polypeptide extract, 8 parts of bovine thyroid and testicular polypeptide extract, 8 parts of polysaccharide chondroitin sulfate, 5 parts of vitamin D, 3 parts of vitamin K, 2 parts of curcumin, 5 parts of epimedium, 3 parts of melon seeds, 2 parts of rhodiola rosea, 8 parts of astragalus and 3 parts of seabuckthorn;

[0094] The method for preparing the bovine embryonic muscle stem cell mitochondrial polypeptide extract comprises the following steps:

[0095] 1) Isolation of bovine embryonic muscle stem cells:

[0096] Healthy bovine embryos at 60-90 days of gestation were selected, and skeletal muscle tissues of the limbs were obtained under sterile conditions;

[0097] Digestion was performed with a mixture of 0.1% collagenase IV and 0.05% trypsin at 37°C for 30 minutes under shaking, and then neutralized with DMEM medium containing 10% fetal bovine serum. The fibroblasts were removed by the differential adhesion method at 37°C for 30 minutes, and the suspended cells were collected as muscle stem cells.

[0098] 2) Stem cell expansion and mitochondrial activation:

[0099] The basal medium was DMEM / F12 supplemented with 1% fetal bovine serum substitute and maintained at 37°C and 5% CO 2 Three-dimensional suspension culture was cultured under hypoxia for 1 week with 5% O 2 , circulates once every 24 hours, enhancing mitochondrial biogenesis;

[0100] 3) Cell disruption and mitochondrial separation:

[0101] High-pressure homogenization was used, with 800 bar and 4 °C for three cycles, using a mitochondrial protection buffer containing 0.25 M sucrose and 10 mM HEPES, pH 7.4;

[0102] Gradient centrifugation: First centrifugation: 1000 × g, 4 ° C for 10 minutes to remove cell debris; second centrifugation: 12000 × g, 4 ° C for 15 minutes to collect mitochondrial precipitate;

[0103] 4) Mitochondrial freeze-thaw lysis:

[0104] Lysis buffer: Tris-HCl buffer containing 1% Triton X-100, 5 mM DTT, pH 8.0;

[0105] Procedure: -80℃ quick freezing for 30 minutes → 37℃ water bath rapid thawing, repeat 3 times to destroy the mitochondrial membrane structure;

[0106] 5) Enzymatic hydrolysis:

[0107] The mixed protease of trypsin: pepsin = 3:1 was treated in stages: the first stage: pH 7.5, 37°C enzymolysis for 2 hours; the second stage: pH 2.0, 42°C enzymolysis for 1 hour; inactivation: 80°C water bath for 10 minutes to terminate the reaction;

[0108] 6) Purification:

[0109] Ultrafiltration classification: 10kDa ultrafiltration membrane retains large molecular proteins, and fractions <10kDa are collected for concentration and drying;

[0110] The method for preparing the bovine embryonic spinal cord stem cell mitochondrial polypeptide extract comprises the following steps:

[0111] 1) Isolation of spinal cord stem cells:

[0112] Healthy bovine embryos at 60-75 days of gestation were selected, and the spinal cord central canal region tissue was isolated under sterile conditions;

[0113] The cells were digested with a mixture of 0.15% collagenase IV and 0.02% hyaluronidase at 37°C for 20 minutes under shaking. The stop solution was DMEM / F12 medium containing 10% low-fat acid bovine serum albumin (BSA).

[0114] Spinal cord stem cells were obtained by CD133+ / Nestin+ labeled immunomagnetic bead sorting and pre-attachment at 37°C for 1 hour to remove foreign cells.

[0115] 2) Three-dimensional dynamic amplification:

[0116] The basal medium DMEM / F12 was supplemented with 15% fetal bovine serum replacement, 10 ng / mL FGF2, and 5 μM Wnt signal inhibitor XAV939;

[0117] 37°C, 5% CO 2 Under the environment, three-dimensional dynamic suspension culture was performed in a rotating bioreactor at a speed of 12 rpm for 1 week, and 0.1 μM Y-27632ROCK inhibitor was supplemented every 48 h;

[0118] 3) Mitochondrial-directed activation:

[0119] Introduce intermittent hypoxia-reoxygenation cycles during the 2 days following the expansion: 5% O 2 6 hours → 21% O 2 Conduct for 2 hours;

[0120] 4) Crushing and separation:

[0121] Cell disruption: Nitrogen cavitation method, pressure 450 psi, 4 ° C for 5 minutes, buffer containing 0.3 M mannitol, 10 mM EDTA and 1 mM PMSF, pH 7.2;

[0122] Gradient centrifugation: initial centrifugation: 800 × g, 4°C for 10 min) to remove nuclear debris;

[0123] Separation: centrifugation at 15000×g, 4°C for 20 min) to collect mitochondrial pellet;

[0124] 5) Cracking:

[0125] Lysis buffer: Tris-HCl buffer, pH 7.8, containing 0.5% sodium deoxycholate and 2 mM DTT;

[0126] Lysis procedure: Continuous shear lysis was achieved through a microfluidic chip with a channel diameter of 50 μm, and lysis was performed at a flow rate of 0.5 mL / min for 1 hour;

[0127] 6) Enzymatic hydrolysis:

[0128] Step 1: neutral protease, pH 7.0, hydrolysis at 45°C for 1.5 hours;

[0129] Step 2: hydrolysis with pancreatic elastase, pH 8.5, 37°C for 2 h;

[0130] Inactivation: ultrafiltration using a 30 kDa molecular weight cutoff membrane, followed by instantaneous high temperature, maintained at 70°C for 30 seconds;

[0131] 7) Purification:

[0132] Ultrafiltration classification: pass through 100kDa, 30kDa, and 10kDa ultrafiltration membranes in sequence, collect fractions <10kDa for concentration and drying;

[0133] The preparation method of the bovine thyroid and testis polypeptide extract comprises the following process:

[0134] 1) Raw material processing:

[0135] Thyroid gland processing: Take the thyroid gland of healthy adult cattle, remove the fascia and cut into 3mm pieces 3 Tissue blocks were immersed in 0.1% β-mercaptoethanol + 0.05% EDTA solution for 30 min;

[0136] Testis processing: Take fresh bovine testis tissue, peel off the white membrane and cut into 5mm pieces 3 The pellet was washed three times with PBS containing 0.2% sodium citrate;

[0137] 2) Low temperature crushing:

[0138] Thyroid disruption: cryogenic grinding with liquid nitrogen, -196°C, 3000 rpm for 2 minutes, followed by high pressure homogenization, 600 bar cycle 3 times, disruption buffer containing 0.15 M NaCl + 10 mM Tris-HCl with a pH of 7.4;

[0139] Testis disruption: 40kHz ultrasound for 30 minutes, with simultaneous addition of 0.1% neutral protease;

[0140] 3) Thyroid-specific enzymatic hydrolysis:

[0141] The first stage: hydrolysis with trypsin at pH 7.0, 1:50 enzyme-substrate ratio, 50°C for 2 hours;

[0142] Stage 2: Adjust pH to 4.5, add pepsin, 1:100 enzyme-substrate ratio, hydrolyze at 37°C for 3 hours;

[0143] 4) Testis-directed enzymolysis:

[0144] Enzyme combination: bromelain (0.5%) + elastase (0.3%) segmented treatment;

[0145] Pretreatment: pH 6.8, 45°C pre-activation for 15 minutes;

[0146] Main enzymatic hydrolysis: pH 7.5, 55°C shaking hydrolysis for 4 hours;

[0147] 5) Inactivation and purification:

[0148] The product was inactivated at 85°C for 15 seconds, and then passed through 100 kDa, 30 kDa, and 10 kDa ultrafiltration membranes in sequence to collect fractions <10 kDa for concentration and drying.

[0149] The preparation method of the above-mentioned pharmaceutical composition for preventing osteoporosis comprises the following steps:

[0150] Epimedium, melon seeds, rhodiola rosea, astragalus and seabuckthorn are mixed and crushed in proportion, and passed through a 50-mesh sieve to obtain a mixed powder; the mixed powder and deionized water are mixed in a weight ratio of 1:9, and then soaked for 14 hours, and then centrifuged and the obtained solid is dried to obtain a traditional Chinese medicine powder;

[0151] Wash the rice and grind it into fine powder, then mix it with rice flour and glucose in a mass ratio of 3:2:1 as a basal culture medium;

[0152] According to the mass ratio of basal culture medium to Chinese medicine powder of 2:1, the two were fully mixed, and an appropriate amount of deionized water was added to make the water content of the culture medium reach 62%, and the pH value of the culture medium was adjusted to 7.4;

[0153] Lactobacillus rhamnosus, Lactobacillus casei and Bifidobacterium longum were prepared and mixed in an inoculation ratio of 3:1:1;

[0154] Add 4% yeast powder and 4% glucose by mass to the culture medium, inoculate the mixed bacteria into the culture medium at an inoculum volume of 5%, and stir well;

[0155] The inoculated culture medium was placed in a fermentation tank and the fermentation temperature was controlled at 37°C. During the fermentation process, the culture medium was dispersed with a stirring paddle every 8 hours, and the fermentation time was set to 3 weeks;

[0156] After fermentation is complete, the fermentation product is filtered using a 200-mesh filter to remove larger residues. The filtrate is then centrifuged at 4000 r / min for 18 minutes to further remove fine residues and impurities.

[0157] The supernatant after centrifugation was transferred to a freeze dryer, pre-frozen at -45°C for 2.5 hours, and then freeze-dried at a vacuum degree of 15 Pa until the moisture content was less than 5%;

[0158] The freeze-dried fermentation product is crushed and passed through an 80-mesh sieve to prepare a uniform powder product, namely the traditional Chinese medicine fermentation extract;

[0159] The traditional Chinese medicine fermentation extract and the remaining components are mixed, crushed and passed through a 200-mesh sieve to obtain the pharmaceutical composition for preventing osteoporosis.

[0160] Example 2

[0161] On the basis of the above Example 1, this example provides a pharmaceutical composition for preventing osteoporosis and a preparation method thereof, the only difference being that: in parts by weight, the pharmaceutical composition for preventing osteoporosis comprises the following components: 9 parts of bovine embryonic muscle stem cell mitochondrial polypeptide extract, 5 parts of bovine embryonic spinal cord stem cell mitochondrial polypeptide extract, 5 parts of bovine thyroid and testicular polypeptide extract, 5 parts of polysaccharide chondroitin sulfate, 3 parts of vitamin D, 2 parts of vitamin K, 1 part of curcumin, 3 parts of epimedium, 3 parts of melon seeds, 1 part of rhodiola rosea, 5 parts of astragalus and 1 part of seabuckthorn.

[0162] Example 3

[0163] On the basis of the above Example 1, this example provides a pharmaceutical composition for preventing osteoporosis and a preparation method thereof, the only difference being that: in parts by weight, the pharmaceutical composition for preventing osteoporosis comprises the following components: 14 parts of bovine embryonic muscle stem cell mitochondrial polypeptide extract, 10 parts of bovine embryonic spinal cord stem cell mitochondrial polypeptide extract, 10 parts of bovine thyroid and testicular polypeptide extract, 10 parts of polysaccharide chondroitin sulfate, 7 parts of vitamin D, 5 parts of vitamin K, 3 parts of curcumin, 7 parts of epimedium, 7 parts of melon seeds, 3 parts of rhodiola rosea, 10 parts of astragalus and 5 parts of seabuckthorn.

[0164] Test Case

[0165] This example investigates the therapeutic effect of the pharmaceutical composition obtained in Examples 1 to 3 above on osteoporosis.

[0166] The test method is as follows:

[0167] Animals: Male Wistar outbred rats. Fifty Wistar rats (2 months old, average weight 151.73±10.11g) were randomly divided into 6 groups, 10 rats in each group. The groups were normal control group, model control group, experimental group, small, medium and large dose groups of the prescription, respectively. The 10 rats in the normal control group were fed freely without modeling, and the remaining 40 rats were gavaged with 120mg / kg of sodium pentobarbital. After anesthesia, the scrotal skin was disinfected, and both testicles were completely removed. After surgery, 20,000 u / kg of potassium penicillin G was injected intramuscularly to make the OP model of castrated rats. After one week of observation after surgery, all castrated rats survived healthily.

[0168] The experimental preparations were 0.25 g of the pharmaceutical composition obtained in Examples 1 to 3, and each group of drugs was diluted with water at a ratio of 1:3 to a corresponding concentration. The drugs were administered for 90 days, once a day, with a volume of about 1.0 ml each time. The normal control group and the blank control group were gavaged with the same volume of tap water. 30 minutes after the last administration, the rats in each group were decapitated and blood was collected to determine the changes in serum calcitonin (CT) and parathyroid hormone (PTH) in each group of rats. The lumbar vertebrae (L1-4) of each group of rats were removed, and the bone density was measured using an X-ray bone density instrument.

[0169] The test results are shown in Table 1 and Table 2.

[0170] Table 1 Changes in bone density of rats in group (X+S)

[0171] Group Number of cases Lumbar spine bone density (X±S) Normal control group 10 0.164±0.013 Model control group 10 0.141±0.039 Example 2 Group 10 0.154±0.013 Example 3 Group 10 0.159±0.005 Example 1 Group 10 0.165±0.008

[0172] Table 2 Effects on calcitonin and parathyroid hormone in rats (X+S)

[0173] Group Number of cases Calcitonin (ng / L) Parathyroid hormone (ng / L) Normal control group 10 449.2±142.1 324.9±80.9 Model control group 10 383.5±134.2 450.6±146.4 Example 2 Group 10 510.0±162.2 427.7±109.3 Example 3 Group 10 553.5±186.2 380.7±47.4 Example 1 Group 10 670.7±198.1 335.8±76.2

[0174] As can be seen from Table 1 and Table 2, the present invention provides a pharmaceutical composition for preventing osteoporosis, and the components work synergistically with each other to significantly improve the therapeutic effect on the osteoporosis rat model, thereby providing a new therapeutic means for preventing osteoporosis.

[0175] Various embodiments of the present invention may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity, and should not be understood as a rigid limitation on the scope of the present invention; therefore, the range description should be considered to have specifically disclosed all possible sub-ranges and single numerical values ​​within the range. For example, the range description from 1 to 6 should be considered to have specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which apply regardless of the range. In addition, whenever a numerical range is indicated herein, it is meant to include any cited number (fractional or integer) within the indicated range.

[0176] The foregoing is merely a specific embodiment of the present invention, which enables those skilled in the art to understand or implement the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A pharmaceutical composition for preventing osteoporosis, characterized in that: The pharmaceutical composition for preventing osteoporosis comprises the following components in parts by weight: 9-14 parts of bovine embryonic muscle stem cell mitochondrial polypeptide extract, 5-10 parts of bovine embryonic spinal cord stem cell mitochondrial polypeptide extract, 5-10 parts of bovine thyroid and testicular polypeptide extract, 5-10 parts of polysaccharide chondroitin sulfate, 3-7 parts of vitamin D, 2-5 parts of vitamin K, 1-3 parts of curcumin, 3-7 parts of epimedium, 3-7 parts of melon seeds, 1-3 parts of rhodiola rosea, 5-10 parts of astragalus and 1-5 parts of seabuckthorn.

2. The pharmaceutical composition for preventing osteoporosis according to claim 1, characterized in that: The pharmaceutical composition for preventing osteoporosis comprises the following components in parts by weight: 10 parts of bovine embryonic muscle stem cell mitochondrial polypeptide extract, 8 parts of bovine embryonic spinal cord stem cell mitochondrial polypeptide extract, 8 parts of bovine thyroid and testicular polypeptide extract, 8 parts of polysaccharide chondroitin sulfate, 5 parts of vitamin D, 3 parts of vitamin K, 2 parts of curcumin, 5 parts of epimedium, 3 parts of melon seeds, 2 parts of rhodiola rosea, 8 parts of astragalus and 3 parts of seabuckthorn.

3. The pharmaceutical composition for preventing osteoporosis according to claim 1, characterized in that: The preparation method of the bovine embryonic muscle stem cell mitochondrial polypeptide extract comprises the following steps: 1) Isolation of bovine embryonic muscle stem cells: Healthy bovine embryos at 60-90 days of gestation were selected, and skeletal muscle tissues of the limbs were obtained under sterile conditions; Digestion was performed with a mixture of 0.1% collagenase IV and 0.05% trypsin at 37°C for 30 minutes under shaking, and then neutralized with DMEM medium containing 10% fetal bovine serum. The fibroblasts were removed by the differential adhesion method at 37°C for 30 minutes, and the suspended cells were collected as muscle stem cells. 2) Stem cell expansion and mitochondrial activation: The basal medium DMEM / F12 was supplemented with 1% fetal bovine serum substitute and cultured in three-dimensional suspension at 37°C and 5% CO2. Mitochondrial biogenesis was enhanced by periodic hypoxia treatment for 1 week, with 5% O2 and a cycle every 24 hours. 3) Cell disruption and mitochondrial separation: High-pressure homogenization was used, with 800 bar and 4 °C for three cycles, using a mitochondrial protection buffer containing 0.25 M sucrose and 10 mM HEPES, pH 7.4; Gradient centrifugation: First centrifugation: 1000 × g, 4 ° C for 10 minutes to remove cell debris; second centrifugation: 12000 × g, 4 ° C for 15 minutes to collect mitochondrial precipitate; 4) Mitochondrial freeze-thaw lysis: Lysis buffer: Tris-HCl buffer containing 1% Triton X-100, 5 mM DTT, pH 8.0; Procedure: -80℃ quick freezing for 30 minutes → 37℃ water bath rapid thawing, repeat 3 times to destroy the mitochondrial membrane structure; 5) Enzymatic hydrolysis: The mixed protease of trypsin: pepsin = 3:1 was treated in stages: the first stage: pH 7.5, 37°C enzymatic hydrolysis for 2 hours; the second stage: pH 2.0, 42°C enzymatic hydrolysis for 1 hour; Inactivation: Terminate the reaction in 80℃ water bath for 10 minutes; 6) Purification: Ultrafiltration classification: 10kDa ultrafiltration membrane retains large molecular proteins, and fractions <10kDa are collected for concentration and drying.

4. The pharmaceutical composition for preventing osteoporosis according to claim 1, characterized in that: The method for preparing the bovine embryonic spinal cord stem cell mitochondrial polypeptide extract comprises the following steps: 1) Isolation of spinal cord stem cells: Healthy bovine embryos at 60-75 days of gestation were selected, and the spinal cord central canal region tissue was isolated under sterile conditions; The mixture of 0.15% collagenase IV and 0.02% hyaluronidase was used for digestion at 37°C for 20 minutes with shaking. The stop solution was DMEM / F12 medium containing 10% low-fat acid bovine serum albumin BSA; Spinal cord stem cells were obtained by CD133+ / Nestin+ labeled immunomagnetic bead sorting and pre-attachment at 37°C for 1 hour to remove foreign cells. 2) Three-dimensional dynamic amplification: The basal medium DMEM / F12 was supplemented with 15% fetal bovine serum replacement, 10 ng / mL FGF2, and 5 μM Wnt signal inhibitor XAV939; Three-dimensional dynamic suspension culture was performed in a rotating bioreactor at 12 rpm at 37°C and 5% CO2 for 1 week, supplemented with 0.1 μM Y-27632ROCK inhibitor every 48 h; 3) Mitochondrial-directed activation: Introduce intermittent hypoxia-reoxygenation cycles during the 2 days of the late expansion period: 5% O2 for 6 hours → 21% O2 for 2 hours; 4) Crushing and separation: Cell disruption: Nitrogen cavitation method, pressure 450 psi, 4 ° C for 5 minutes, buffer containing 0.3 M mannitol, 10 mM EDTA and 1 mM PMSF, pH 7.2; Gradient centrifugation: initial centrifugation: 800 × g, 4°C for 10 min) to remove nuclear debris; Separation: centrifugation at 15000×g, 4°C for 20 min) to collect mitochondrial pellet; 5) Cracking: Lysis buffer: Tris-HCl buffer, pH 7.8, containing 0.5% sodium deoxycholate and 2 mM DTT; Lysis procedure: Continuous shear lysis was achieved through a microfluidic chip with a channel diameter of 50 μm, and lysis was performed at a flow rate of 0.5 mL / min for 1 hour; 6) Enzymatic hydrolysis: Step 1: neutral protease, pH 7.0, hydrolysis at 45°C for 1.5 hours; Step 2: hydrolysis with pancreatic elastase, pH 8.5, 37°C for 2 h; Inactivation: ultrafiltration using a 30 kDa molecular weight cutoff membrane, followed by instantaneous high temperature, maintained at 70°C for 30 seconds; 7) Purification: Ultrafiltration fractionation: pass through 100kDa, 30kDa, and 10kDa ultrafiltration membranes in sequence, collect fractions <10kDa for concentration and drying.

5. The pharmaceutical composition for preventing osteoporosis according to claim 1, characterized in that: The preparation method of the bovine thyroid and testis polypeptide extract comprises the following process: 1) Raw material processing: Thyroid gland processing: Take the thyroid gland of healthy adult cattle, remove the fascia and cut into 3mm pieces 3 Tissue blocks were immersed in 0.1% β-mercaptoethanol + 0.05% EDTA solution for 30 min; Testis processing: Take fresh bovine testis tissue, peel off the white membrane and cut into 5mm pieces 3 The pellet was washed three times with PBS containing 0.2% sodium citrate; 2) Low temperature crushing: Thyroid disruption: cryogenic grinding with liquid nitrogen, -196°C, 3000 rpm for 2 minutes, followed by high pressure homogenization, 600 bar cycle 3 times, disruption buffer containing 0.15 M NaCl + 10 mM Tris-HCl with a pH of 7.4; Testis disruption: 40kHz ultrasound for 30 minutes, with simultaneous addition of 0.1% neutral protease; 3) Thyroid-specific enzymatic hydrolysis: The first stage: hydrolysis with trypsin at pH 7.0, 1:50 enzyme-substrate ratio, 50°C for 2 hours; Stage 2: Adjust pH to 4.5, add pepsin, 1:100 enzyme-substrate ratio, hydrolyze at 37°C for 3 hours; 4) Testis-directed enzymatic hydrolysis: Enzyme combination: bromelain (0.5%) + elastase (0.3%) segmented treatment; Pretreatment: pH 6.8, 45°C pre-activation for 15 minutes; Main enzymatic hydrolysis: pH 7.5, 55°C shaking hydrolysis for 4 hours; 5) Inactivation and purification: The product was inactivated at 85°C for 15 seconds, and then passed through 100 kDa, 30 kDa, and 10 kDa ultrafiltration membranes in sequence to collect fractions <10 kDa for concentration and drying.

6. A method for preparing the pharmaceutical composition for preventing osteoporosis according to any one of claims 1 to 5, characterized in that: The preparation method comprises the following steps: Epimedium, melon seeds, rhodiola rosea, astragalus and seabuckthorn are mixed and fermented to obtain a traditional Chinese medicine fermentation extract; The traditional Chinese medicine fermentation extract and the remaining components are mixed, crushed and sieved to obtain the pharmaceutical composition for preventing osteoporosis.

7. The method for preparing the pharmaceutical composition for preventing osteoporosis according to claim 6, characterized in that: The step of mixing and fermenting epimedium, melon seeds, rhodiola rosea, astragalus and seabuckthorn to obtain a traditional Chinese medicine fermentation extract comprises the following process: Mixing and grinding epimedium, melon seeds, rhodiola rosea, astragalus and seabuckthorn according to a certain proportion, and passing through a 50-mesh sieve to obtain a mixed powder; mixing the mixed powder with deionized water in a weight ratio of 1:(8-10), soaking the mixture for 12-16 hours, and then centrifuging and drying the obtained solid to obtain a traditional Chinese medicine powder; Wash the rice and grind it into fine powder, then mix it with rice flour and glucose in a mass ratio of 3:2:1 as a basal culture medium; According to the mass ratio of basal culture medium to Chinese medicine powder of 2:1, the two were fully mixed, and an appropriate amount of deionized water was added to make the water content of the culture medium reach 60%-65%, and the pH value of the culture medium was adjusted to 7.4; Lactobacillus rhamnosus, Lactobacillus casei, and Bifidobacterium longum were prepared and mixed in an inoculation ratio of 3:1:1; Add 4% yeast powder and 4% glucose by mass to the culture medium, inoculate the mixed bacteria into the culture medium at an inoculum volume of 5%, and stir well; The inoculated culture medium was placed in a fermentation tank and the fermentation temperature was controlled at 37°C. During the fermentation process, the culture medium was dispersed with a stirring paddle every 8 hours, and the fermentation time was set to 3 weeks; After fermentation is complete, filter the fermentation product with a 200-mesh filter to remove larger residues. Then centrifuge the filtrate at 4000r / min for 15-20 minutes to further remove fine residues and impurities. The supernatant after centrifugation was transferred to a freeze dryer, pre-frozen at -50°C to -40°C for 2-3 hours, and then freeze-dried at a vacuum degree of 10-20 Pa until the moisture content was less than 5%; The freeze-dried fermentation product is crushed and passed through an 80-mesh sieve to prepare a uniform powder product, namely the traditional Chinese medicine fermentation extract.

8. Use of the pharmaceutical composition for preventing osteoporosis according to any one of claims 1 to 5 or the pharmaceutical composition for preventing osteoporosis prepared by the method for preparing the pharmaceutical composition for preventing osteoporosis according to any one of claims 6 to 7 in the preparation of drugs for preventing osteoporosis.

9. The use according to claim 8, characterized in that: The dosage form of the drug for preventing osteoporosis includes capsules.

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