Preparation method, composition and application of yak bone collagen hydrolysate capable of preventing and treating osteoporosis

Abstract

The invention belongs to the field of biology, particularly relates to a preparation method, a composition and application of yak bone collagen hydrolysate capable of preventing and treating osteoporosis, and further provides an oral liquid composition which comprises the following raw materials in percentage by mass: 6% of yak bone collagen hydrolysate, 2% of glucosamine sulfate, 0.4% of chondroitin sulfate and the balance of water. The yak bone collagen hydrolysate and oral liquid composition prepared by the method can improve pathological changes of bone tissues of mice with osteoporosis, promote regeneration of collagen in osteoporosis tissues, improve BMD, BV / TV, Tb.N and Tb.Th of cortical bones, improve osteoporosis, improve osteoporosis, improve osteoporosis, improve osteoporosis, improve osteoporosis, improve osteoporosis, improve osteoporosis, improve osteoporosis, improve osteoporosis, improve osteoporosis, improve osteoporosis, improve osteoporosis, improve osteoporosis, improve osteoporosis, improve osteoporosis, improve osteoporosis, improve osteoporosis, improve osteoporosis, improve osteoporosis, improve osteoporosis, improve osteoporosis, improve osteoporosis, improve osteoporosis, and improve osteoporosis. The Tb.Sp and Po of cortical bones are reduced, and the excellent function of preventing and treating osteoporosis is achieved.

Description

Technical Field

[0001] The invention belongs to the biological field, and specifically relates to a preparation method, a composition and an application of a yak bone collagen hydrolyzate capable of preventing and treating osteoporosis. Background Art

[0002] Osteoporosis (OP) is a systemic bone metabolic disease, the main pathological features of which include decreased bone mass, destruction of bone tissue microstructure, increased bone brittleness and high incidence of fractures. Osteoporosis is considered a direct manifestation of bone aging, which seriously affects the structure and function of bones, and thus affects the overall health of the body. Bad living habits, long-term use of glucocorticoid drugs, and lack of calcium and vitamin D in the diet can accelerate the process of bone aging and have an adverse effect on bone health.

[0003] Collagen, as the main component of bone matrix, accounts for about one-third of the dry weight of bones, of which type I collagen is the main one, accounting for 80% to 90% of the organic components of bones. Bone collagen hydrolysate is collagen that has been further hydrolyzed, and it has excellent biocompatibility and antioxidant properties. The sources of bone collagen hydrolysate are diverse and varied, but there are significant differences in their quality and efficacy. How to prepare collagen hydrolysate that can effectively prevent and treat osteoporosis is still a great challenge. Among them, small molecular weight bone collagen hydrolysate is considered to have high application potential because it is easy to absorb.

[0004] Yaks are precious biological resources unique to the Qinghai-Tibet Plateau in China. Because they grow in an alpine, oxygen-deficient and pollution-free environment, the collagen produced by them has unique properties. Invention patent CN113151391B discloses a method for preparing yak bone collagen peptides. The yak bone collagen peptides prepared by this method can significantly improve the abundance of intestinal microorganisms, regulate the structure of intestinal flora, and optimize the intestinal environment. The yak collagen peptides prepared by this method have a wide range of molecular weights. Given the complex composition of yak bone tissue, it is still a huge technical challenge to accurately control the molecular weight of the prepared yak bone collagen hydrolysate and ensure its high activity.

[0005] In view of the above technical problems, the present invention provides a novel method for preparing yak collagen hydrolysate. The method can accurately control the molecular weight distribution of yak collagen hydrolysate, and the yak collagen hydrolysate with different molecular weight distribution has significant differences in the therapeutic effect on osteoporosis, among which the yak collagen hydrolysate with medium molecular weight shows the best effect in treating osteoporosis. The oral product prepared by the method can promote the regeneration of collagen in the bone tissue of osteoporotic mice, and significantly improve the bone density (BMD), bone volume fraction (BV / TV), trabecular number (Tb.N), and trabecular thickness (Tb.Th) of cortical bone, while effectively reducing the trabecular interval (Tb.Sp) and bone tissue porosity (Po), with significant effects on preventing and treating osteoporosis, and has broad market prospects in the field of health food. Summary of the invention

[0006] In order to solve the above technical problems existing in the prior art, the object of the present invention is to provide a method for preparing yak collagen hydrolyzate capable of preventing and treating osteoporosis, comprising the following steps:

[0007] (1) Yak bone pretreatment

[0008] The yak bones are crushed by a bone crusher, defatted, decalcified, and impurity removed, and washed with water until the pH value is 7 to obtain pre-treated yak bones;

[0009] (2) Preparation of yak collagen filtrate

[0010] The yak bone pretreated in step (1) is added to a 0.5 M citric acid solution with a solid-liquid ratio of 1:10, pepsin is added to react under low temperature conditions, filtered, and the pH value is adjusted to neutral to obtain a yak bone collagen filtrate;

[0011] (3) Preparation of yak collagen hydrolysate

[0012] Add 8 to 120 U / mL of papain to the yak bone collagen filtrate obtained in step (2), and hydrolyze at 50° C. for 2 to 6 hours to obtain a yak bone collagen hydrolyzate;

[0013] (4) Enzyme inactivation

[0014] The yak bone collagen hydrolysate obtained in step (3) is placed in boiling water;

[0015] (5) Purification of yak collagen hydrolysate

[0016] Take 100 mL of the yak bone collagen hydrolyzate obtained in step (4) and elute it in the chromatography column. The absorbance of the eluate is measured at 220 nm, and an elution curve is drawn. The molecular weight distribution of the eluate is determined by size exclusion chromatography. According to the measurement results, the target product is collected, freeze-dried, and stored at -20°C for future use.

[0017] Preferably, the concentration of papain in step (3) is 16-120 U / mL.

[0018] Preferably, the concentration of papain in step (3) is 80-120 U / mL.

[0019] Preferably, the hydrolysis time in step (3) is 3 to 6 hours.

[0020] Preferably, the hydrolysis time in step (3) is 5 to 6 hours.

[0021] The second object of the present invention is to provide yak collagen hydrolysate prepared by the preparation method.

[0022] The third object of the present invention is to provide the use of the yak collagen hydrolysate in the preparation of a drug for treating osteoporosis.

[0023] The fourth object of the present invention is to provide an oral liquid composition capable of preventing and treating osteoporosis, wherein the oral liquid composition comprises the following raw materials in percentage by mass: 6% yak bone collagen hydrolysate, 2% glucosamine sulfate, 0.4% chondroitin sulfate, 0.0066% hyaluronic acid, 0.02% black wolfberry powder, 0.02% snow chrysanthemum extract, and the balance is water.

[0024] Preferably, the yak bone collagen hydrolysate is a medium molecular weight yak bone collagen hydrolysate.

[0025] The fifth object of the present invention is to provide the use of the oral liquid composition in the preparation of medicines or health products for preventing and treating osteoporosis.

[0026] The beneficial effects of the present invention are as follows: (1) The present invention provides a method for preparing yak bone collagen hydrolysate with the efficacy of preventing and treating osteoporosis, wherein the method is a molecular weight-oriented enzymatic hydrolysis-chromatography combination, which has a simple process, is easy to operate, and has a low cost. The prepared yak bone collagen hydrolysate improves the pathological changes in the bone tissue of osteoporotic mice, promotes the proliferation of osteoblasts, regulates the content of alkaline phosphatase, reduces the bone resorption of osteoclasts, promotes the regeneration of collagen in osteoporotic tissues, affects bone morphological and mechanical indicators, and improves osteoporosis.

[0027] (2) The present invention also provides an oral liquid composition of yak bone collagen hydrolyzate for improving osteoporosis, comprising the following raw materials in percentage by mass: 6% yak bone collagen hydrolyzate, 2% glucosamine sulfate, 0.4% chondroitin sulfate, 0.0066% hyaluronic acid, 0.02% black wolfberry powder, 0.02% snow chrysanthemum extract, and the balance is water; the oral liquid composition prepared by the method can improve the pathological changes of bone tissue in osteoporotic mice, accelerate the expression of collagen fibers in osteoporotic tissue, promote the increase in the number of trabeculae in mouse tibial tissue, reduce the gap, restore the trabecular network structure, increase the BMD, BV / TV, Tb.N and Tb.Th of cortical bone, and reduce the Tb.Sp and Po of cortical bone, and has the effect of repairing osteoporosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Staining results of paraffin sections of tibia tissue of osteoporotic mice at different repair times.

[0029] Note: a. H&E staining results; b. Masson staining results.

[0030] Figure 2 Collagen fiber content in tibia tissue of osteoporotic mice at different repair times.

[0031] Figure 3 Relative content of Hyp in tibia tissue of osteoporotic mice at different repair times.

[0032] Figure 4 Micro-CT evaluation of the effect of yak bone collagen hydrolysate in repairing osteoporosis.

[0033] Note: ag. 3D image; h. bone density; i. bone volume fraction; j. number of trabeculae; k. trabecular thickness; l. trabecular gap; m. total porosity.

[0034] Figure 5 Staining results of paraffin sections of tibia tissue of osteoporotic mice at different repair times.

[0035] Note: a. H&E staining results; b. Masson staining results.

[0036] Figure 6 Collagen fiber content in tibia tissue of osteoporotic mice at different repair times.

[0037] Figure 7 Relative content of Hyp in tibia tissue of osteoporotic mice at different repair times. DETAILED DESCRIPTION

[0038] The technical solution of the present invention is further described below in conjunction with embodiments, but the protection scope of the present invention is not limited to the following.

[0039] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those of ordinary skill in the art to which the methods belong. Although any method similar or equivalent to that described herein can also be used for the precise preparation of yak collagen hydrolysate, it is not limited to the representative exemplary methods described in the following examples. It should also be understood that the terms used in the present invention are only for the purpose of describing specific embodiments and do not limit the scope of protection of the present invention and the appended claims.

[0040] In the following examples, the methods used are conventional methods unless otherwise specified, and the reagents used are conventional reagents that can be obtained from commercial sources.

[0041] Example 1. Preparation of yak collagen hydrolysate

[0042] The yak collagen hydrolysates are all prepared by a green method of molecular weight-directed enzymatic hydrolysis-chromatography, and the molecular weight-directed enzymatic hydrolysis-chromatography method is specifically as follows:

[0043] 1. Yak Bone Pretreatment

[0044] The meat and bone marrow of yak bones are removed and crushed with a bone crusher to obtain yak bone particles. The yak bone particles are pretreated with 10% n-butanol defatting, 0.5M hydrochloric acid decalcification, and 10% sodium hydroxide impurity removal, and then washed with water to pH=7 to obtain pretreated yak bone particles.

[0045] 2. Preparation of Yak Bone Collagen Filtrate

[0046] The pretreated yak bone was added to a 0.5M citric acid solution with a solid-liquid ratio of 1:10. Pepsin was added to react for 48 hours under low temperature conditions to extract collagen from the yak bone tissue. After the reaction was completed, the filtrate was filtered and the pH value of the filtrate was adjusted to neutral for use.

[0047] 3. Preparation of Yak Bone Collagen Hydrolysate

[0048] The conditions for preparing ultra-high molecular weight yak bone collagen hydrolysate (UYBCH) were explored by adding 8U / mL papain to the filtrate and hydrolyzing it at 50℃ for different times. The results of SDS-PAGE gel electrophoresis showed that the product was yak bone collagen at 0h, most of the yak bone collagen in the hydrolysate was not hydrolyzed at 1h, and most of the yak bone collagen was hydrolyzed at 2h and 3h. In summary, considering factors such as production cost, the hydrolysis time was determined to be 2h.

[0049] Table 1 Preparation of ultra-high molecular weight yak bone collagen hydrolysate (UYBCH)

[0050]

[0051] The optimal conditions for preparing high molecular weight yak bone collagen hydrolysate (HYBCH) were explored by adding different concentrations of papain to the filtrate and hydrolyzing it at 50°C for a certain period of time. According to the liquid phase results, the optimal conditions for preparing high molecular weight yak bone collagen hydrolysate (HYBCH) were 100% filtrate concentration, 16U / mL papain was added and hydrolyzed at 50°C for 3 hours.

[0052] Table 2 Preparation of high molecular weight yak collagen hydrolysate (HYBCH)

[0053]

[0054]

[0055] The optimal conditions for preparing medium molecular weight yak bone collagen hydrolysate (MYBCH) were explored by adding different concentrations of papain to the filtrate and hydrolyzing it at 50°C for a certain time. According to the liquid phase results, the filtrate concentration was 100%, and 80U / mL of papain was added and hydrolyzed at 50°C for 5 hours, which was the optimal condition for preparing medium molecular weight yak bone collagen hydrolysate (MYBCH).

[0056] Table 3 Preparation of medium molecular weight yak bone collagen hydrolysate (MYBCH)

[0057]

[0058] The optimal conditions for preparing low molecular weight yak bone collagen hydrolysate (LYBCH) were explored by adding different concentrations of papain to the filtrate and hydrolyzing it at 50°C for a certain period of time. According to the liquid phase results, the optimal conditions for preparing low molecular weight yak bone collagen hydrolysate (LYBCH) were 75% filtrate concentration, adding 120U / mL papain and hydrolyzing it at 50°C for 6 hours.

[0059] Table 4 Preparation of low molecular weight yak collagen hydrolysate (LYBCH)

[0060]

[0061] 4. Inactivate enzymes

[0062] After the hydrolysis, the reaction system was placed in boiling water for 10 minutes to stop the enzymatic reaction.

[0063] 5. Purification of Yak Bone Collagen Hydrolysate

[0064] After the Sephadex G25 gel was pretreated, it was packed into a prepared polytetrafluoro sand-core glass chromatography column, and the column was ensured to be balanced and stable. A certain volume of the enzymolysis solution was loaded onto the column, and distilled water was used as the eluent to control the elution flow rate. During the elution process, every 5 mL of the eluent was taken as one tube. The absorbance of each tube of the eluent was measured at 220 nm with a UV spectrophotometer, and an elution curve of absorbance versus the number of tubes was plotted. Size exclusion chromatography was used to determine the molecular weight distribution of the eluent in each tube, and the target product was collected according to the measurement results for freeze-drying and stored at -20 °C for standby.

[0065] Example 2: Characterization of the Properties of Yak Bone Collagen Hydrolysate

[0066] According to the method provided by the national standard (GB 31648-2018), high performance liquid chromatography (HPLC) equipped with a TSK gel G2000 SWXL chromatographic column was used to determine the molecular weight distribution of yak bone collagen hydrolysate (YBCH) (Table 5). The molecular weight of low molecular weight yak bone collagen hydrolysate (LYBCH) was mainly concentrated below 1000 Da, and the proportion of M < 1000 Da could reach 64.28%. The molecular weight of medium molecular weight yak bone collagen hydrolysate (MYBCH) was mainly concentrated between 1000 Da and 3000 Da, and the proportion of 1000 Da < M < 3000 Da could reach 59.58%. The molecular weight of high molecular weight yak bone collagen hydrolysate (HYBCH) was mainly concentrated between 3000 Da and 10000 Da, and the proportion of 3000 Da < M < 10000 Da could reach 51.46%. The molecular weight distribution of ultra-high molecular weight yak bone collagen hydrolysate (UYBCH) was characterized by SDS-PAGE gel electrophoresis, and the results showed that it was mainly concentrated between 35 kDa and 180 kDa.

[0067] Table 5 Molecular Weight Distribution Table of Yak Bone Collagen Hydrolysate

[0068]

[0069] Example 3: Ability of Yak Bone Collagen Hydrolysate to Prevent and Treat Osteoporosis

[0070] 1. Modeling and Repair of Osteoporotic Mice

[0071] Two-month-old female Kunming mice were randomly divided into 7 groups, including normal group (Normal), model group (Model), positive control group, low molecular weight yak bone collagen hydrolysate group (LYBCH), medium molecular weight yak bone collagen hydrolysate group (MYBCH), high molecular weight yak bone collagen hydrolysate group (HYBCH) and ultra-high molecular weight yak bone collagen hydrolysate group (UYBCH), with 16 mice in each group. The construction of the mouse osteoporosis model was carried out by diluting dexamethasone sodium phosphate injection 50 times and injecting it into the gluteal muscle twice a week at a dose of 1 mg / kg BW. Dexamethasone sodium phosphate can inhibit bone formation, promote bone resorption, interfere with bone cell function and affect Ca in vivo. 2+ Balance and other ways to affect bone tissue, destroy the normal balance of bone metabolism, and then lead to the occurrence of osteoporosis. The mice in the normal group and the model group were orally administered with 0.4 mL of normal saline daily. The LYBCH group, MYBCH group, HYBCH group and UYBCH group were orally administered with the same volume of LYBCH, MYBCH, HYBCH and UYBCH solutions by gavage, respectively, at a dose of 300 mg / kg BW. The positive control group was orally administered with 0.4 mL of estradiol solution daily.

[0072] 2. H&E staining of mouse tibia tissue

[0073] The repair effect of yak bone collagen hydrolysate (YBCH) on osteoporosis was studied at the tissue level by H&E staining. Figure 1 As shown in a, during the whole experiment, the tibia tissue of the normal group mice has been in a healthy state, with high bone matrix density, orderly and tight trabecular reticular structure, full trabeculae, moderate number and thickness. The tibia tissue of the model group mice has low bone matrix density, incomplete trabecular reticular structure, significantly reduced number of trabeculae and widened trabecular spacing, indicating that the osteoporosis mouse model was successfully constructed. At week 4, the structure of the tibia tissue of the ES, LYBCH, MYBCH and HYBCH groups of mice was improved to a certain extent, and the density of bone matrix and the number of trabeculae increased. At week 8, except for the UYBCH group, the structure of the tibia tissue of the mice in the other experimental groups was further repaired, the trabeculae tended to be full, the trabecular gap was reduced, and the MYBCH group restored the arrangement of the trabecular reticular structure. The above experimental results show that the YBCH prepared in this application can improve the pathological changes of bone tissue in osteoporotic mice, MYBCH has the best effect, followed by LYBCH, and finally HYBCH, while UYBCH has almost no effect.

[0074] 3. Masson staining of mouse tibia tissue

[0075] Masson staining was used to further study the repair effect of yak bone collagen hydrolysate (YBCH) on osteoporosis from the tissue level. Figure 1 As shown in b, in Masson staining, blue indicates newly formed bone and red indicates mature bone tissue. Throughout the experiment, the tibia tissue of the normal group mice was always healthy, with complete trabecular reticular structure and full trabeculae, indicating that the collagen fibers of the mice were dense and neatly arranged. However, the trabecular reticular structure of the tibia tissue of the model group mice was missing, the number of trabeculae was significantly reduced, and the spacing between trabeculae became wider, indicating that the number of collagen fibers in the tibia tissue of the mice was reduced and broken and irregularly arranged. At week 4, a certain amount of new bone appeared in the tibia tissue of the ES, LYBCH, MYBCH and HYBCH groups of mice. This is because YBCH can promote the proliferation of osteoblasts, regulate the content of alkaline phosphatase, reduce the bone resorption of osteoclasts, and increase the expression of type I collagen in bone tissue. At week 8, except for the UYBCH group, the number of trabeculae in the tibia tissue of the mice in the other experimental groups increased further, the trabecular spacing decreased, and the number of trabeculae in the MYBCH group was significantly better than that in the other experimental groups. The above experimental results show that the YBCH prepared in this application can improve the pathological changes of bone tissue in osteoporosis mice, and MYBCH has the best effect.

[0076] 4. Quantitative evaluation of the ability of yak bone collagen hydrolysate to promote collagen regeneration

[0077] The collagen fiber content in the tibia tissue of mice at week 4 and week 8 was quantified by Image J software to evaluate the ability of yak bone collagen hydrolysate to repair osteoporotic bone. Figure 2As shown: At week 4, the collagen fiber contents in the tibia tissue of mice in the model group, ES, LYBCH, MYBCH, HYBCH and UYBCH groups were significantly lower than those in the normal group (66.82±1.16%), and were 38.45±1.16%, 51.58±1.56%, 43.29±1.91%, 47.69±1.68%, 40.94±1.60% and 38.53±1.19%, respectively. At week 8, the collagen fiber contents in the tibia tissue of mice in the ES, LYBCH, MYBCH and HYBCH groups increased to 58.00±1.17%, 46.57±2.21%, 54.55±1.34% and 43.09±3.86%. The collagen fiber contents in the tibia tissue of mice in the LYBCH and HYBCH groups were significantly lower than those in the MYBCH group. The collagen fiber content in the tibia tissue of mice in the UYBCH group (36.95±1.43%) was not significantly different from that in the model group (34.78±1.63%). The above results show that the yak bone collagen hydrolysate prepared in this application can accelerate the expression of collagen fibers in osteoporotic tissues, and the effect is MYBCH>LYBCH>HYBCH, while UYBCH has almost no effect.

[0078] The chloramine T method was used to further quantitatively analyze the relative content of hydroxyproline (Hyp) in the tibia tissue of each group of mice at different time periods to evaluate the ability of yak bone collagen hydrolysate to repair osteoporotic bones. Figure 3 As shown, the Hyp content of the normal group at week 4 was used as the benchmark. At week 4, the Hyp content in the tibia tissue of mice in the model group, ES, LYBCH, MYBCH, HYBCH and UYBCH groups was lower than that in the normal group, decreasing to 72.10±3.45%, 88.71±4.37%, 84.89±4.00%, 86.63±2.61%, 80.41±2.61% and 75.43±3.60%, respectively. At week 8, the Hyp content in the tibia tissue of mice in the ES, LYBCH, MYBCH and HYBCH groups was 95.65±3.49%, 85.59±2.26%, 93.94±2.76% and 81.00±2.56%, respectively, which was significantly better than that in the model group (53.51±3.01%). The Hyp content in the tibia tissue of mice in the UYBCH group (55.16±2.20%) was not significantly different from that in the model group. The above results show that the yak bone collagen hydrolysate prepared in this application can promote the regeneration of collagen in osteoporotic tissues, and the effect is MYBCH>LYBCH>HYBCH, while UYBCH has almost no effect.

[0079] 5. Effects of yak collagen hydrolysate on bone morphomechanical parameters

[0080] Micro-CT scanner was used to measure the 3D images of the bone microstructure of the tibia of mice in the normal group, model group, ES, LYBCH, MYBCH, HYBCH and UYBCH groups at week 8 to evaluate the effect of yak bone collagen hydrolysate (YBCH) with different specific molecular weights on repairing osteoporosis. Figure 4 As shown in Figures ac, the 3D imaging results of Micro-CT showed that the tibial tissue of the normal group of mice had dense trabeculae and a continuous and complete trabecular reticular structure ( Figure 4 a). Compared with the normal group, the number and density of trabeculae in the tibia tissue of the model group mice were significantly decreased, the gaps were larger, the continuity was poor, and the trabecular network structure was incomplete, indicating that the osteoporosis mouse model induced by dexamethasone sodium phosphate was successfully established ( Figure 4 b). Compared with the model group, except for the UYBCH group, the number of trabeculae in the tibia tissue of mice in the other experimental groups increased, the gaps decreased, and the trabecular network structure was restored ( Figure 4 cg). The above experimental results show that YBCH with different specific molecular weights prepared in this application has the effect of improving osteoporosis.

[0081] Micro-CT was used to measure the structural parameters of the tibia tissue of mice in the normal group, model group, ES, LYBCH, MYBCH, HYBCH and UYBCH groups at week 8, and further quantitatively evaluate the effect of yak bone collagen hydrolysate (YBCH) with different specific molecular weights on repairing osteoporosis. Figure 4 As shown in hm, bone tissue can be divided into two categories: cortical bone and cancellous bone. Cortical bone is mainly composed of dense bone tissue, with a relatively hard outer layer and a porous structure inside. Cancellous bone is mainly composed of loose reticular bone tissue, with many small holes and pores inside. The structural parameters of bone tissue mainly include bone density (BMD), bone volume fraction (BV / TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular gap (Tb.Sp) and total porosity (Po).

[0082] Cortical bone constitutes the main scaffold of the skeleton and is responsible for providing mechanical support, protecting internal organs and storing minerals. The BMD, BV / TV, Tb.N and Tb.Th values ​​of the cortical bone of mice in the normal group were higher, which were 1.31±0.05g / cc, 96.42±1.52%, 3.57±0.071 / mm and 300.98±10.64μm, respectively. Compared with the normal group, the BMD, BV / TV, Tb.N and Tb.Th values ​​of the cortical bone of mice in the model group were lower, which were 1.13±0.04g / cc, 54.81±3.00%, 3.21±0.091 / mm and 196.21±16.57μm, respectively. Compared with the model group, the BMD, BV / TV, Tb.N and Tb.Th values ​​of the cortical bone of mice in the LYBCH, MYBCH and HYBCH groups increased, while the BMD, BV / TV, Tb.N and Tb.Th values ​​of the cortical bone of mice in the UYBCH group showed no significant difference. Among them, the MYBCH group had the most significant effect, with the BMD, BV / TV, Tb.N and Tb.Th values ​​of its cortical bone being 1.26±0.04g / cc, 89.79±2.08%, 3.51±0.051 / mm and 271.01±12.67μm, respectively (Figure hk). In addition, the Tb.Sp and Po values ​​of the tibia cortical bone of mice in the normal group were both low, at 10.24±1.26μm and 1.82±0.05%, respectively. Compared with the normal group, the Tb.Sp and Po values ​​of the tibia cortical bone of mice in the model group were higher, which were 17.21±1.69μm and 2.32±0.11%, respectively. Compared with the model group, the Tb.Sp and Po values ​​of the cortical bone of mice in the LYBCH, MYBCH and HYBCH groups decreased, while there was no significant difference in the Tb.Sp and Po values ​​of the cortical bone of mice in the UYBCH group. Among them, the Tb.Sp and Po values ​​of the MYBCH group decreased most significantly, which were 12.55±1.61μm and 1.94±0.07%, respectively (Figure l, m). The above results show that the YBCH prepared in this application has the ability to repair osteoporosis, which is specifically manifested in the ability to increase the BMD, BV / TV, Tb.N and Tb.Th of cortical bone, and reduce the Tb.Sp and Po of cortical bone. The effects are MYBCH>LYBCH>HYBCH, and UYBCH has almost no effect.

[0083] Cancellous bone is located inside the cortical bone, and its main functions include participating in the production of blood cells, storing fat, and providing a bone marrow environment. The BMD, BV / TV, Tb.N, and Tb.Th values ​​of the cancellous bone of mice in the normal group were higher, which were 0.38±0.02g / cc, 11.89±0.27%, 1.39±0.031 / mm, and 89.97±1.32μm, respectively. Compared with the normal group, the BMD, BV / TV, Tb.N, and Tb.Th values ​​of the cancellous bone of mice in the model group were lower, which were 0.21±0.03g / cc, 4.25±0.46%, 0.57±0.081 / mm, and 71.73±2.01μm, respectively. Compared with the model group, the BMD, BV / TV, Tb.N and Tb.Th values ​​of the cancellous bone of mice in the LYBCH, MYBCH and HYBCH groups increased, while the BMD, BV / TV, Tb.N and Tb.Th values ​​of the cancellous bone of mice in the UYBCH group showed no significant difference. Among them, the BMD, BV / TV, Tb.N and Tb.Th values ​​of the cancellous bone of mice in the MYBCH group increased most significantly, which were 0.32±0.03g / cc, 9.69±0.41%, 1.17±0.061 / mm and 86.72±1.81μm, respectively (Figure hk). In addition, the Tb.Sp and Po values ​​of the cancellous bone of the normal group were lower, which were 195.57±20.60μm and 71.35±2.48%, respectively. Compared with the normal group, the Tb.Sp and Po values ​​of the cancellous bone of the mice in the model group were higher, which were 498.44±28.74μm and 96.29±2.98%, respectively. Compared with the model group, the Tb.Sp and Po values ​​of the cancellous bone of the mice in the LYBCH, MYBCH and HYBCH groups decreased, while there was no significant difference in the Tb.Sp and Po values ​​of the cancellous bone of the mice in the UYBCH group. Among them, the Tb.Sp and Po values ​​of the MYBCH group decreased most significantly, which were 268.16±26.24μm and 79.11±2.77%, respectively (Figure 1m). The above results show that the YBCH prepared in this application has the effect of repairing osteoporosis, which is specifically manifested in that it can increase the BMD, BV / TV, Tb.N and Tb.Th of cancellous bone, and reduce the Tb.Sp and Po of cancellous bone, among which MYBCH has the best effect.

[0084] Example 4: Preparation of yak collagen hydrolysate oral liquid composition

[0085] An oral liquid composition of yak bone collagen hydrolysate for repairing osteoporosis comprises the following raw materials in percentage by mass: 6% yak bone collagen hydrolysate, 2% glucosamine sulfate, 0.4% chondroitin sulfate, 0.0066% hyaluronic acid, 0.02% black wolfberry powder, 0.02% snow chrysanthemum extract, and the balance is water.

[0086] Preparation process: (1) accurately weigh each component and mix well; (2) add a certain volume of drinking water to homogenize and dissolve; (3) sterilize; (4) clean and disinfect the glass bottle; (5) fill.

[0087] Example 5. Ability of yak collagen hydrolysate oral liquid composition to prevent and treat osteoporosis

[0088] 1. Modeling and repair of osteoporosis in mice

[0089] Two-month-old female Kunming mice were randomly divided into three groups, including a normal group (Normal), a model group (Model) and a homemade oral solution group (Oral solution), with 16 mice in each group. The mouse osteoporosis model was constructed by diluting dexamethasone sodium phosphate injection 50 times and injecting it into the gluteal muscle twice a week at a dose of 1 mg / kg BW. Dexamethasone sodium phosphate can inhibit bone formation, promote bone resorption, interfere with bone cell function, and affect Ca in the body. 2+ Balance and other ways to affect bone tissue, destroy the normal balance of bone metabolism, and then lead to the occurrence of osteoporosis. The mice in the normal group and the model group were orally administered with 0.4 mL of normal saline every day. The homemade oral solution group was given the same volume of homemade oral solution by gavage.

[0090] 2. H&E staining of mouse tibia tissue

[0091] The repair effect of yak collagen hydrolysate oral product on osteoporosis was studied at the tissue level through H&E staining. Figure 5 As shown in a, at the 8th week, the tibia tissue of the normal group mice was always in a healthy state, with high bone matrix density, orderly and tight trabecular reticular structure, full trabeculae, moderate number and thickness. The tibia tissue of the model group mice had low bone matrix density, incomplete trabecular reticular structure, significantly reduced number of trabeculae and widened trabecular spacing, indicating that the osteoporosis mouse model was successfully constructed. The structure of the tibia tissue of the homemade oral liquid group mice was repaired, the trabeculae tended to be full, the trabecular spacing was reduced, and the arrangement of the trabecular reticular structure was restored. The above experimental results show that the oral product of yak bone collagen hydrolysate prepared by this application can significantly improve the pathological changes in the bone tissue of osteoporotic mice.

[0092] 3. Masson staining of mouse tibia tissue

[0093] Masson staining was used to further study the repair effect of yak collagen hydrolysate oral products on osteoporosis from the tissue level. Figure 5As shown in Figure 2, in the 8th week, the tibial tissue of the normal group mice was always in a healthy state, with a complete trabecular network structure and full trabeculae, indicating that the mouse collagen fibers were dense and neatly arranged. However, the trabecular network structure of the tibial tissue of the model group mice was missing, the number of trabeculae was significantly reduced, and the trabecular spacing was widened, indicating that the number of collagen fibers in the tibial tissue of the mice was reduced and fractures and irregular arrangements occurred. Compared with the model group, the number of trabeculae in the tibial tissue of the mice in the homemade oral liquid group increased significantly, and the trabecular spacing decreased. The above experimental results show that the oral product of yak bone collagen hydrolyzate prepared by this application can significantly improve the pathological changes in the bone tissue of osteoporotic mice.

[0094] 4. Quantitative evaluation of the ability of yak bone collagen hydrolysate oral product to promote collagen regeneration

[0095] The collagen fiber content in the tibia tissue of mice at week 8 was quantified by Image J software to evaluate the ability of yak bone collagen hydrolysate oral product to repair osteoporotic bone. Figure 6 As shown: At the 8th week, the collagen fiber content in the tibia tissue of mice in the model group (36.57±1.54%) was significantly reduced compared with the normal group (63.50±1.61%). Compared with the model group, the collagen fiber content in the tibia tissue of mice in the homemade oral liquid group (55.98±1.75%) was significantly increased. The above results show that the oral product of yak bone collagen hydrolyzate prepared in this application can significantly accelerate the expression of collagen fibers in osteoporotic tissue.

[0096] The chloramine T method was used to further quantitatively analyze the relative content of hydroxyproline (Hyp) in the tibia tissue of each group of mice at week 8 to evaluate the ability of yak collagen hydrolysate oral product to repair osteoporotic bones. Taking the Hyp content of the normal group at week 8 as the benchmark, the experimental results are as follows Figure 7 As shown. At the 8th week, compared with the normal group (100.62±2.69%), the Hyp content in the tibia tissue of mice in the model group (53.90±3.26%) decreased significantly. Compared with the model group, the Hyp content in the tibia tissue of mice in the homemade oral liquid group (94.85±2.44%) increased significantly. The above results show that the oral product of yak bone collagen hydrolyzate prepared in this application can significantly promote the regeneration of collagen in osteoporosis tissue.

[0097] In summary, the invention provides a method for preparing yak bone collagen hydrolyzate with the efficacy of treating osteoporosis. The method is a molecular weight-oriented enzymatic hydrolysis-chromatography combination, which has a simple process, is easy to operate, and has a low cost. The prepared yak bone collagen hydrolyzate improves the pathological changes in the bone tissue of osteoporotic mice, promotes the proliferation of osteoblasts, regulates the content of alkaline phosphatase, reduces the bone resorption of osteoclasts, and thereby increases the expression of type I collagen in bone tissue, promotes the regeneration of collagen in osteoporotic tissue, affects bone morphological and mechanical indicators, and improves osteoporosis. The present invention also provides an oral liquid composition of yak bone collagen hydrolysate for improving osteoporosis, comprising the following raw materials in percentage by mass: 6% yak bone collagen hydrolysate, 2% glucosamine sulfate, 0.4% chondroitin sulfate, 0.0066% hyaluronic acid, 0.02% black wolfberry powder, 0.02% snow chrysanthemum extract, and the balance is water; the oral liquid composition prepared by the method can improve the pathological changes of bone tissue of osteoporotic mice, promote the regeneration of collagen in osteoporotic tissue, promote the increase of the number of trabeculae in mouse tibia tissue, reduce the gap, restore the trabecular reticular structure, improve the BMD, BV / TV, Tb.N and Tb.Th of cortical bone, and reduce the Tb.Sp and Po of cortical bone, and has the effect of repairing osteoporosis.

[0098] The above description is only the details of individual exemplary implementation cases of the present invention. For those skilled in the art, the present invention may have various modifications and changes according to specific preparation conditions during actual application, and is not intended to limit the present invention. Anything within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing yak collagen hydrolyzate for preventing and treating osteoporosis, characterized in that: The steps include: (1) Yak bone pretreatment The yak bones are crushed by a bone crusher, defatted, decalcified, and impurity removed, and washed with water until the pH value is 7 to obtain pre-treated yak bones; (2) Preparation of yak collagen filtrate The yak bone pretreated in step (1) is added to a 0.5 M citric acid solution with a solid-liquid ratio of 1:10, pepsin is added to react under low temperature conditions, filtered, and the pH value is adjusted to neutral to obtain a yak bone collagen filtrate; (3) Preparation of yak collagen hydrolysate Add 8-120 U / mL of papain to the 75%-100% yak bone collagen filtrate obtained in step (2), and hydrolyze at 50° C. for 2-6 hours to obtain yak bone collagen hydrolyzate; (4) Enzyme inactivation The yak bone collagen hydrolysate obtained in step (3) is placed in boiling water; (5) Purification of yak collagen hydrolysate Take 100 mL of the yak bone collagen hydrolyzate obtained in step (4) and elute it in the chromatography column. The absorbance of the eluate is measured at 220 nm, and an elution curve is drawn. The molecular weight distribution of the eluate is determined by size exclusion chromatography. According to the measurement results, the target product is collected, freeze-dried, and stored at -20°C for future use.

2. The preparation method according to claim 1, characterized in that The concentration of papain in step (3) is 16-120 U / mL.

3. The preparation method according to claim 2, characterized in that: The concentration of papain in step (3) is 80-120 U / mL.

4. The preparation method according to claim 1, characterized in that: The hydrolysis time in step (3) is 3 to 6 hours.

5. The preparation method according to claim 4, characterized in that: The hydrolysis time in step (3) is 5 to 6 hours.

6. Yak collagen hydrolysate prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the yak collagen hydrolyzate as claimed in claim 6 in the preparation of a drug for treating osteoporosis.

8. An oral liquid composition capable of preventing and treating osteoporosis, characterized in that: The oral liquid composition comprises the following raw materials in percentage by mass: 6% yak bone collagen hydrolysate, 2% glucosamine sulfate, 0.4% chondroitin sulfate, 0.0066% hyaluronic acid, 0.02% black wolfberry powder, 0.02% snow chrysanthemum extract, and the balance is water.

9. The oral liquid composition according to claim 8, characterized in that The yak bone collagen hydrolysate is a yak bone collagen hydrolysate with a medium molecular weight.

10. Use of the oral liquid composition according to claim 8 or 9 in the preparation of medicines or health products for preventing and treating osteoporosis.

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