Calf bone marrow hydrolyzate liposome freeze-dried powder and its application in hematopoiesis-promoting and anti-aging products

Abstract

The invention relates to a calf bone marrow hydrolysate liposome freeze-dried powder and its application in hematopoiesis-promoting and anti-aging products, belonging to the field of biotechnology. The calf bone marrow hydrolysate liposome freeze-dried powder is a product obtained by using calf bone marrow as a raw material, and enzymolyzing the product step by step with pancreatic lipase, alkaline phosphatase, trypsin, elastase, and ficin to obtain an enzymolyzate below 3 kDa, and then coating the product with liposomes. The product is rich in multiple small and medium molecule nutritional active substances, growth factors, and small molecule peptide components. The combination of each component can enhance the efficacy, can more effectively promote hematopoiesis, and also has excellent antioxidant ability, removes free radicals in the body, reduces the damage of oxidation reactions to cells and tissues, and effectively delays the aging process.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a calf bone marrow hydrolyzate liposome freeze-dried powder and application thereof in hematopoiesis-promoting and anti-aging products. Background Art

[0002] In the field of life sciences, research on promoting hematopoiesis and anti-aging has always been a focus of attention. As a substance with potential health benefits, bovine bone marrow shows its unique value in promoting hematopoiesis and anti-aging.

[0003] Hematopoiesis is an important physiological process for the human body to maintain life activities, ensuring the normal generation and renewal of various cells in the blood. Bone marrow is the main site of hematopoiesis, which contains hematopoietic stem cells and a variety of hematopoietic regulatory factors. Bovine bone marrow has certain similarities with human bone marrow in composition and function, so it has become an important object for studying the mechanism of promoting hematopoiesis.

[0004] In terms of ingredients, bovine bone marrow is rich in proteins, minerals, lipids, amino acids, and a variety of growth factors, such as stem cell factor, granulocyte colony stimulating factor, etc. These ingredients play a key regulatory role in the proliferation, differentiation, and maturation of hematopoietic cells. Certain proteins in bovine bone marrow can stimulate the self-renewal of hematopoietic stem cells and increase their number, thereby providing sufficient "seed cells" for hematopoiesis. Bovine bone marrow is also rich in mineral elements such as iron, copper, and zinc, which can provide the necessary material basis for hematopoiesis and promote the synthesis of hemoglobin and the maturation of red blood cells. At the same time, the lipid components in bovine bone marrow also provide support for the energy metabolism of hematopoietic cells.

[0005] Aging is a complex biological process involving cell damage, oxidative stress, telomere shortening, inflammatory response and other aspects. Among them, chronic inflammation is considered to be one of the important factors that accelerate aging. With the increase of age, the body's hematopoietic function gradually declines, resulting in a decrease in the number and function of immune cells in the blood, which in turn affects the overall health and aging process. The nutrients and bioactive substances in bovine bone marrow have antioxidant and anti-inflammatory properties. Antioxidants can neutralize free radicals in the body, reduce cellular oxidative damage, and slow down the rate of cell aging; anti-inflammatory ingredients help to reduce chronic inflammatory responses, thereby delaying cell aging.

[0006] Although in traditional medicine, cow bone marrow has long been considered to have a tonic effect, the effect is not obvious enough and the treatment process is slow. However, in order to transform the potential advantages of cow bone marrow in promoting hematopoiesis and anti-aging into more effective treatment and health products, it is necessary to transform and improve the components of cow bone marrow. Summary of the invention

[0007] Although bovine bone marrow has the nourishing and strengthening effects of promoting hematopoiesis and anti-aging, the effect is not prominent enough and the treatment course is slow. The present invention provides a calf bone marrow hydrolyzate liposome freeze-dried powder and its application in products promoting hematopoiesis and anti-aging. The bone marrow of young yaks under 1 year old is used as a raw material. The product obtained through multi-stage enzymatic hydrolysis is rich in multiple small and medium molecule nutritional active substances, growth factors, and small molecule peptide components. The combination of various components can enhance the efficacy and can more effectively promote hematopoiesis. It also has excellent antioxidant capacity, removes free radicals in the body, reduces the damage of oxidation reactions to cells and tissues, and effectively delays the aging process. The specific technical scheme is as follows:

[0008] A calf bone marrow hydrolysate liposome freeze-dried powder, wherein the calf bone marrow hydrolysate liposome freeze-dried powder is prepared by using calf bone marrow as a raw material, performing step-by-step enzymolysis to obtain an enzymolysate below 3 kDa, and then coating the enzymolysate with liposomes to obtain a product; the specific preparation method comprises the following steps:

[0009] S1: Take calf bone marrow, add 6 to 8 times the weight of water, stir to make a slurry, adjust the pH to 7.5 to 8.5, add 0.5% to 1.5% of the weight of pancreatic lipase from calf bone marrow, enzymolyze at 35℃ to 40℃ for 2h to 3h, then adjust the pH to 8.5 to 9.0, add 0.3% to 0.8% of the weight of alkaline phosphatase from calf bone marrow, enzymolyze at 40℃ to 45℃ for 1.5h to 2h, then adjust the pH to 8.0 to 8.5, add 0.8% to 2% of the weight of trypsin from calf bone marrow, and enzymolyze at 35℃ to 40℃. Enzyme hydrolyze for 1 h to 1.5 h, inactivate the enzyme, cool to room temperature, add 1% to 2.5% by weight of elastase from calf bone marrow, enzyme hydrolyze at 25°C to 28°C for 1 h to 1.5 h, inactivate the enzyme, cool to room temperature, adjust the pH to 6.8 to 7.2, add 1.5% to 2.0% by weight of ficin from calf bone marrow, enzyme hydrolyze at 60°C to 65°C for 1 h to 1.5 h, inactivate the enzyme, cool to room temperature, filter with a 3 kDa ultrafiltration membrane, take the filtrate below 3 kDa, and concentrate it to 10% to 15% by volume to obtain an enzymolysis solution;

[0010] S2: According to the mass ratio of soybean lecithin: cholesterol: anhydrous ethanol = (8-10): (2-2.5): (25-35), soybean lecithin and cholesterol are placed in anhydrous ethanol, stirred and mixed for more than 10 minutes, and then ultrasonically mixed for 1-3 minutes. After being uniformly dissolved, they are placed in an evaporation container and evaporated at 60-65°C to remove the anhydrous ethanol, and a thin film is formed on the inner wall of the evaporation container;

[0011] S3: According to the mass ratio of film: enzyme hydrolysate: phosphate buffer = (0.5-1): (10-15): (80-100), place the film and enzyme hydrolysate in phosphate buffer, stir and mix for more than 30 minutes, let stand for 30-40 minutes, and mix by ultrasonic in a water bath at 4℃-5℃ for 10-15 minutes to obtain an emulsion;

[0012] S4: The emulsion is placed in a syringe, and is repeatedly pushed out through a 0.38 μm polycarbonate filter membrane for 3 to 5 times, freeze-dried, and broken up to obtain calf bone marrow hydrolyzate liposome freeze-dried powder.

[0013] In S1 of the above technical solution, the pancreatic lipase has an enzymatic activity of 50,000 U / g to 100,000 U / g and can be derived from cattle.

[0014] In S1 of the above technical solution, the alkaline phosphatase has an enzyme activity of 5,000 U / g to 10,000 U / g and can be derived from cattle.

[0015] In S1 of the above technical solution, the trypsin has an enzymatic activity of 2000U / g to 4000U / g and can be derived from cattle.

[0016] In S1 of the above technical solution, the elastase has an enzyme activity of 100,000 U / g to 500,000 U / g and can be derived from pigs.

[0017] In S1 of the above technical solution, the enzymatic activity of the ficin is 600,000 U / g to 800,000 U / g, and can be derived from figs.

[0018] In S1 of the above technical solution, the calf bone marrow is the bone marrow of a yak under 1 year old.

[0019] In S1 of the above technical solution, the temperature for inactivating the enzyme is 100° C. to 120° C., and the time is 10 min to 20 min.

[0020] In S2 of the above technical solution, the frequency of the ultrasound is 20kHz to 30kHz.

[0021] In S3 of the above technical solution, the phosphate buffer is a 0.2M phosphate buffer solution with a pH of 6.5 to 7.0.

[0022] In S3 of the above technical solution, the frequency of the ultrasound is 40kHz to 50kHz.

[0023] The invention discloses an application of the calf bone marrow hydrolyzate liposome freeze-dried powder in preparing a medicine having the effects of promoting hematopoiesis and resisting aging.

[0024] The calf bone marrow hydrolyzate liposome freeze-dried powder provided by the present invention and its application in hematopoiesis promotion and anti-aging products have the following beneficial effects compared with the prior art:

[0025] 1. The present invention uses the bone marrow of young yaks under 1 year old as raw material, and the product obtained through multi-stage enzymatic hydrolysis is rich in multiple small and medium-molecule nutritional active substances, growth factors, and small-molecule peptide components, and the combination of each component can enhance the efficacy. The calf is in a vigorous stage of growth and development, and the content of growth factors in its bone marrow is relatively high. The small-molecule peptide products obtained by specific graded enzymatic hydrolysis have good biological activity and play an important role in stimulating the proliferation and differentiation of hematopoietic cells, so it can more effectively promote hematopoiesis.

[0026] Second, calves have active metabolism, and the cells in their bone marrow are renewed quickly, so the products after enzymatic hydrolysis have stronger hematopoietic support capabilities. Compared with adult cattle bone marrow, the fat content in calf bone marrow is usually lower. This means that the effective ingredients for hematopoiesis are relatively more concentrated, thereby improving the efficiency of promoting hematopoiesis.

[0027] 3. Bovine bone marrow peptide can also improve the bone marrow hematopoietic microenvironment, which plays an important supporting and regulatory role in the survival, proliferation and differentiation of hematopoietic cells. It can enhance the nutrient supply of the bone marrow, improve the blood circulation of the bone marrow, create more favorable living conditions for hematopoietic cells, and thus indirectly promote the improvement of hematopoietic function.

[0028] Fourth, the liposome-encapsulated enzymatic hydrolysate can retain better therapeutic activity, and the liposomes also have nutritional components to promote therapeutic effects.

[0029] 5. The enzymatic hydrolysis product of calf bone marrow is rich in small molecule peptide components and has antioxidant capacity. It can effectively inhibit DPPH free radicals, eliminate free radicals in the body, reduce the damage of oxidation reactions to cells and tissues, and delay the aging process. DETAILED DESCRIPTION

[0030] The present invention is further described below in conjunction with specific implementation cases, but the present invention is not limited to these embodiments.

[0031] Example 1

[0032] A calf bone marrow hydrolysate liposome freeze-dried powder, wherein the calf bone marrow hydrolysate liposome freeze-dried powder is prepared by using calf bone marrow as a raw material, performing step-by-step enzymolysis to obtain an enzymolysate below 3 kDa, and then coating the enzymolysate with liposomes to obtain a product; the specific preparation method comprises the following steps:

[0033] S1: Take calf bone marrow, add 7 times the weight of water, stir to make a slurry, adjust pH8.0, add 1.0% weight of pancreatic lipase from calf bone marrow, enzymolyze at 37℃ for 2.5h, then adjust pH8.8, add 0.5% weight of alkaline phosphatase from calf bone marrow, enzymolyze at 42℃ for 1.5h, then adjust pH8.2, add 1.4% weight of trypsin from calf bone marrow, enzymolyze at 37℃ for 1h, inactivate enzyme at 110℃ for 15min, cool to room temperature, add 1.8% weight of elastase from calf bone marrow, enzymolyze at 26℃ for 1h, inactivate enzyme at 110℃ for 15min, cool to room temperature, adjust pH7.0, add 1.8% weight of ficin from calf bone marrow, enzymolyze at 62℃ for 1h, inactivate enzyme at 110℃ for 15min, cool to room temperature, filter with 3kDa ultrafiltration membrane, take the filtrate below 3kDa, concentrate to 12% volume, and obtain enzymolysis solution;

[0034] The activity of pancreatic lipase is 100,000 U / g, which is derived from cattle. The activity of alkaline phosphatase is 10,000 U / g, which is derived from cattle. The activity of trypsin is 4,000 U / g, which is derived from cattle; the activity of elastase is 500,000 U / g, which is derived from pigs; the activity of ficin is 800,000 U / g, which is derived from figs. Calf bone marrow is the bone marrow of a young yak about half a year old.

[0035] S2: According to the mass ratio of soy lecithin: cholesterol: anhydrous ethanol = 9:2.2:30, soy lecithin and cholesterol are placed in anhydrous ethanol, stirred and mixed for 15 minutes, and then ultrasonically mixed at 25kHz for 2 minutes. After uniform dissolution, they are placed in an evaporation container and anhydrous ethanol is evaporated at 62°C to form a thin film on the inner wall of the evaporation container.

[0036] S3: According to the mass ratio of film: enzymatic hydrolysate: phosphate buffer = 0.8:12:92, the film and enzymatic hydrolysate were placed in a pH 6.8, 0.2 M phosphate buffer solution, stirred and mixed for 35 min, allowed to stand for 35 min, and ultrasonically mixed for 12 min at 5°C in a water bath at 45 kHz to obtain an emulsion.

[0037] S4: The emulsion was placed in a syringe, and pushed out through a 0.38 μm polycarbonate filter membrane for 4 times, freeze-dried, and broken up to obtain calf bone marrow hydrolyzate liposome freeze-dried powder.

[0038] The invention discloses an application of the calf bone marrow hydrolyzate liposome freeze-dried powder in preparing a medicine having the effects of promoting hematopoiesis and resisting aging.

[0039] Example 2

[0040] A calf bone marrow hydrolysate liposome freeze-dried powder, wherein the calf bone marrow hydrolysate liposome freeze-dried powder is prepared by using calf bone marrow as a raw material, performing step-by-step enzymolysis to obtain an enzymolysate below 3 kDa, and then coating the enzymolysate with liposomes to obtain a product; the specific preparation method comprises the following steps:

[0041] S1: Take calf bone marrow, add 6 times the weight of water, stir to make a slurry, adjust pH7.5, add 0.5% weight of pancreatic lipase from calf bone marrow, enzymolyze at 35℃ for 2h, then adjust pH8.5, add 0.3% weight of alkaline phosphatase from calf bone marrow, enzymolyze at 40℃ for 1.5h, then adjust pH8.0, add 0.8% weight of trypsin from calf bone marrow, enzymolyze at 35℃ for 1h, inactivate enzyme at 100℃ for 10min, cool to room temperature, add 1% weight of elastase from calf bone marrow, enzymolyze at 25℃ for 1h, inactivate enzyme at 100℃ for 10min, cool to room temperature, adjust pH6.8, add 1.5% weight of ficin from calf bone marrow, enzymolyze at 60℃ for 1h, inactivate enzyme at 100℃ for 10min, cool to room temperature, filter with 3kDa ultrafiltration membrane, take filtrate below 3kDa, concentrate to 10% volume, and obtain enzymolysis solution;

[0042] The activity of pancreatic lipase is 50,000 U / g, which is derived from cattle. The activity of alkaline phosphatase is 5,000 U / g, which is derived from cattle. The activity of trypsin is 2000 U / g, which is derived from cattle; the activity of elastase is 100,000 U / g, which is derived from pigs; the activity of ficin is 600,000 U / g, which is derived from figs. Calf bone marrow is the bone marrow of a young yak of about half a year old.

[0043] S2: According to the mass ratio of soy lecithin: cholesterol: anhydrous ethanol = 8:2:25, soy lecithin and cholesterol are placed in anhydrous ethanol, stirred and mixed for 10 minutes, and then ultrasonically mixed at 20kHz for 1 minute. After uniform dissolution, they are placed in an evaporation container and evaporated at 60°C to remove the anhydrous ethanol, and a thin film is formed on the inner wall of the evaporation container.

[0044] S3: According to the mass ratio of film: enzyme hydrolysate: phosphate buffer = 0.5:10:80, the film and enzyme hydrolysate were placed in a pH 6.5, 0.2 M phosphate buffer solution, stirred and mixed for 30 minutes, allowed to stand for 30 minutes, and ultrasonically mixed for 10 minutes at 40 kHz in a 4°C water bath to obtain an emulsion.

[0045] S4: The emulsion was placed in a syringe, and was repeatedly pushed out through a 0.38 μm polycarbonate filter membrane for three times, freeze-dried, and broken up to obtain calf bone marrow hydrolysate liposome freeze-dried powder.

[0046] The invention discloses an application of the calf bone marrow hydrolyzate liposome freeze-dried powder in preparing a medicine having the effects of promoting hematopoiesis and resisting aging.

[0047] Example 3

[0048] A calf bone marrow hydrolysate liposome freeze-dried powder, wherein the calf bone marrow hydrolysate liposome freeze-dried powder is prepared by using calf bone marrow as a raw material, performing step-by-step enzymolysis to obtain an enzymolysate below 3 kDa, and then coating the enzymolysate with liposomes to obtain a product; the specific preparation method comprises the following steps:

[0049] S1: Take calf bone marrow, add 6 times the weight of water, stir to make a slurry, adjust pH8.5, add 0.5% weight of pancreatic lipase from calf bone marrow, enzymolyze at 40℃ for 2h, then adjust pH9.0, add 0.3% weight of alkaline phosphatase from calf bone marrow, enzymolyze at 45℃ for 1.5h, then adjust pH8.5, add 0.8% weight of trypsin from calf bone marrow, enzymolyze at 40℃ for 1h, inactivate enzyme at 120℃ for 10min, cool to room temperature, add 2.5% weight of elastase from calf bone marrow, enzymolyze at 25℃ for 1.5h, inactivate enzyme at 100℃ for 20min, cool to room temperature, adjust pH6.8, add 2.0% weight of ficin from calf bone marrow, enzymolyze at 60℃ for 1.5h, inactivate enzyme at 100℃ for 20min, cool to room temperature, filter with 3kDa ultrafiltration membrane, take filtrate below 3kDa, concentrate to 10% volume, and obtain enzymolysis solution;

[0050] The activity of pancreatic lipase is 100,000 U / g, which is derived from cattle. The activity of alkaline phosphatase is 5,000 U / g, which is derived from cattle. The activity of trypsin is 4,000 U / g, which is derived from cattle; the activity of elastase is 100,000 U / g, which is derived from pigs; the activity of ficin is 800,000 U / g, which is derived from figs. Calf bone marrow is the bone marrow of a young yak of about half a year old.

[0051] S2: According to the mass ratio of soy lecithin: cholesterol: anhydrous ethanol = 8:2.5:25, soy lecithin and cholesterol are placed in anhydrous ethanol, stirred and mixed for 20 minutes, and then ultrasonically mixed at 20kHz for 3 minutes. After uniform dissolution, they are placed in an evaporation container and anhydrous ethanol is evaporated at 60°C to form a thin film on the inner wall of the evaporation container.

[0052] S3: According to the mass ratio of film: enzyme hydrolysate: phosphate buffer = 1:10:100, the film and enzyme hydrolysate were placed in a pH 6.5, 0.2 M phosphate buffer solution, stirred and mixed for 40 min, allowed to stand for 30 min, and ultrasonically mixed for 15 min at 40 kHz in a 5°C water bath to obtain an emulsion.

[0053] S4: The emulsion was placed in a syringe, and was repeatedly pushed out through a 0.38 μm polycarbonate filter membrane for three times, freeze-dried, and broken up to obtain calf bone marrow hydrolysate liposome freeze-dried powder.

[0054] The invention discloses an application of the calf bone marrow hydrolyzate liposome freeze-dried powder in preparing a medicine having the effects of promoting hematopoiesis and resisting aging.

[0055] Example 4

[0056] A calf bone marrow hydrolysate liposome freeze-dried powder, wherein the calf bone marrow hydrolysate liposome freeze-dried powder is prepared by using calf bone marrow as a raw material, performing step-by-step enzymolysis to obtain an enzymolysate below 3 kDa, and then coating the enzymolysate with liposomes to obtain a product; the specific preparation method comprises the following steps:

[0057] S1: Take calf bone marrow, add 8 times the weight of water, stir to make a slurry, adjust pH8.5, add 1.5% weight of pancreatic lipase from calf bone marrow, enzymolyze at 40℃ for 3h, then adjust pH9.0, add 0.8% weight of alkaline phosphatase from calf bone marrow, enzymolyze at 45℃ for 2h, then adjust pH8.5, add 2% weight of trypsin from calf bone marrow, enzymolyze at 40℃ for 1.5h, inactivate enzyme at 120℃ for 20min, cool to room temperature, add 2.5% weight of elastase from calf bone marrow, enzymolyze at 28℃ for 1.5h, inactivate enzyme at 120℃ for 20min, cool to room temperature, adjust pH7.2, add 2.0% weight of ficin from calf bone marrow, enzymolyze at 65℃ for 1.5h, inactivate enzyme at 120℃ for 20min, cool to room temperature, filter with 3kDa ultrafiltration membrane, take the filtrate below 3kDa, concentrate to 15% volume, and obtain enzymolysis solution;

[0058] The activity of pancreatic lipase is 100,000 U / g, which is derived from cattle. The activity of alkaline phosphatase is 10,000 U / g, which is derived from cattle. The activity of trypsin is 4,000 U / g, which is derived from cattle; the activity of elastase is 500,000 U / g, which is derived from pigs; the activity of ficin is 800,000 U / g, which is derived from figs. Calf bone marrow is the bone marrow of a young yak about half a year old.

[0059] S2: According to the mass ratio of soy lecithin: cholesterol: anhydrous ethanol = 10:2.5:35, soy lecithin and cholesterol are placed in anhydrous ethanol, stirred and mixed for 20 minutes, and then ultrasonically mixed at 30kHz for 3 minutes. After uniform dissolution, they are placed in an evaporation container and anhydrous ethanol is evaporated at 65°C to form a thin film on the inner wall of the evaporation container.

[0060] S3: According to the mass ratio of film: enzyme hydrolysate: phosphate buffer = 1:15:100, the film and enzyme hydrolysate were placed in a pH 7.0, 0.2 M phosphate buffer solution, stirred and mixed for 40 min, allowed to stand for 40 min, and ultrasonically mixed for 15 min at 5°C in a water bath at 50 kHz to obtain an emulsion.

[0061] S4: The emulsion was placed in a syringe, and was repeatedly pushed out through a 0.38 μm polycarbonate filter membrane for 5 times, freeze-dried, and broken up to obtain calf bone marrow hydrolyzate liposome freeze-dried powder.

[0062] The invention discloses an application of the calf bone marrow hydrolyzate liposome freeze-dried powder in preparing a medicine having the effects of promoting hematopoiesis and resisting aging.

[0063] Example 5

[0064] A calf bone marrow hydrolysate liposome freeze-dried powder, wherein the calf bone marrow hydrolysate liposome freeze-dried powder is prepared by using calf bone marrow as a raw material, performing step-by-step enzymolysis to obtain an enzymolysate below 3 kDa, and then coating the enzymolysate with liposomes to obtain a product; the specific preparation method comprises the following steps:

[0065] S1: Take calf bone marrow, add 8 times the weight of water, stir to make a slurry, adjust pH7.5, add 1.5% weight of pancreatic lipase from calf bone marrow, enzymolyze at 35℃ for 3h, then adjust pH8.5, add 0.8% weight of alkaline phosphatase from calf bone marrow, enzymolyze at 40℃ for 2h, then adjust pH8.0, add 2% weight of trypsin from calf bone marrow, enzymolyze at 35℃ for 1.5h, inactivate enzyme at 100℃ for 20min, cool to room temperature, add 1% weight of elastase from calf bone marrow, enzymolyze at 28℃ for 1h, inactivate enzyme at 120℃ for 10min, cool to room temperature, adjust pH7.2, add 1.5% weight of ficin from calf bone marrow, enzymolyze at 65℃ for 1h, inactivate enzyme at 120℃ for 10min, cool to room temperature, filter with 3kDa ultrafiltration membrane, take filtrate below 3kDa, concentrate to 15% volume, and obtain enzymolysis solution;

[0066] The activity of pancreatic lipase is 50,000 U / g, which is derived from cattle. The activity of alkaline phosphatase is 10,000 U / g, which is derived from cattle. The activity of trypsin is 2000 U / g, which is derived from cattle; the activity of elastase is 500,000 U / g, which is derived from pigs; the activity of ficin is 600,000 U / g, which is derived from figs. Calf bone marrow is the bone marrow of a young yak about half a year old.

[0067] S2: According to the mass ratio of soy lecithin: cholesterol: anhydrous ethanol = 10:2:35, soy lecithin and cholesterol are placed in anhydrous ethanol, stirred and mixed for 10 minutes, and then ultrasonically mixed at 30kHz for 1 minute. After uniform dissolution, they are placed in an evaporation container and anhydrous ethanol is evaporated at 65°C to form a thin film on the inner wall of the evaporation container.

[0068] S3: According to the mass ratio of film: enzyme hydrolysate: phosphate buffer = 0.5:15:80, the film and enzyme hydrolysate were placed in a pH 7.0, 0.2 M phosphate buffer solution, stirred for 30 min, allowed to stand for 40 min, and ultrasonically mixed for 10 min at 50 kHz in a 4°C water bath to obtain an emulsion.

[0069] S4: The emulsion was placed in a syringe, and was repeatedly pushed out through a 0.38 μm polycarbonate filter membrane for 5 times, freeze-dried, and broken up to obtain calf bone marrow hydrolyzate liposome freeze-dried powder.

[0070] The invention discloses an application of the calf bone marrow hydrolyzate liposome freeze-dried powder in preparing a medicine having the effects of promoting hematopoiesis and resisting aging.

[0071] Comparative Example 1

[0072] Calf bone marrow was not subjected to pancreatic lipase and alkaline phosphatase enzymatic hydrolysis, but directly subjected to trypsin enzymatic hydrolysis, followed by elastase enzymatic hydrolysis, and finally to ficin enzymatic hydrolysis; other parameters and methods were the same as those in Example 1.

[0073] Comparative Example 2

[0074] Calf bone marrow was not subjected to elastase and ficin enzymatic hydrolysis; other parameters and methods were the same as in Example 1.

[0075] Comparative Example 3

[0076] Elastase and trypsin were not subjected to graded enzymatic hydrolysis. Elastase and trypsin were added simultaneously for co-enzymatic hydrolysis, followed by ficin enzymatic hydrolysis. Other parameters and methods were the same as in Example 1.

[0077] Comparative Example 4

[0078] Calf bone marrow was not subjected to trypsin and elastase enzymatic hydrolysis, but was directly subjected to ficin enzymatic hydrolysis after alkaline phosphatase enzymatic hydrolysis; other parameters and methods were the same as those in Example 1.

[0079] Comparative Example 5

[0080] The enzymatic hydrolysate obtained in S1 of Example 1 is directly freeze-dried and broken up to obtain calf bone marrow hydrolysate.

[0081] Comparative Example 6

[0082] The bone marrow of 5-year-old yaks was used as the raw material; other parameters and methods were the same as those in Example 1.

[0083] The products of the above examples and comparative examples were tested.

[0084] 1. Toxicity test:

[0085] 88 healthy mice were selected and divided into 11 groups (4 males and 4 females in each group), each group corresponding to the product samples of each embodiment and comparative example, and an acute oral toxicity test was performed on mice. The oral administration of the sample was 0.03 g / kg body weight, and the reaction was observed within 48 hours. The mice had no adverse reactions. The mice were fed with 0.03 g / kg body weight of the sample every day for 30 consecutive days, and no abnormalities were found in the organs, and the results were safe.

[0086] 2. Hematopoietic test:

[0087] Anemia rat modeling: A number of 3-week-old weaned rats (half male and half female) were taken, and 6 rats (half male and half female) were randomly selected as the blank control group and fed with normal feed throughout the experiment; the remaining rats were fed with low-iron feed and deionized water until the hemoglobin content was about 60 g / L, and the anemia modeling was completed.

[0088] Treatment: The rats after modeling were divided into 12 groups, with 6 rats in each group (half male and half female), of which 11 groups were gavaged with 80 mg / kg / day of the product samples of each embodiment and each comparative example; the remaining group was not treated and served as the anemia control group; the 12 groups of modeling rats continued to be fed with low-iron feed and deionized water for 6 weeks.

[0089] Test: The rats were forbidden to eat for 12 hours after the last oral gavage. After anesthesia, blood was collected from the abdominal aorta to measure the rat blood hemoglobin (HGB) and red blood cell count (RBC). The rats were killed, the right tibia was stripped, and the bone density was tested using a bone density tester. The results are shown in Table 1 below:

[0090] Table 1 Average hematopoietic test results (average value)

[0091] Sample Hemoglobin g / L <![CDATA[Red blood cell count 10 12 cells / L]]> <![CDATA[Bone density g / cm 2 > Example 1 114.32 2.34 0.19 Example 2 108.65 2.25 0.18 Example 3 110.21 2.27 0.18 Example 4 117.42 2.38 0.19 Example 5 113.65 2.35 0.18 Comparative Example 1 96.23 2.10 0.17 Comparative Example 2 74.61 1.86 0.16 Comparative Example 3 83.54 1.95 0.17 Comparative Example 4 71.86 1.83 0.16 Comparative Example 5 101.21 2.18 0.18 Comparative Example 6 93.42 2.04 0.17 Blank control group 121.63 2.45 0.20 Anemia control group 64.58 1.22 0.15

[0092] From the above results, it can be seen that the products of Examples 1 to 5 (calf bone marrow hydrolyzate liposome freeze-dried powder) have a good effect of promoting hematopoiesis and increasing bone density, and can be close to normal after taking for 6 weeks. From the test results of Comparative Examples 1 to 4, it can be seen that different enzymatic hydrolysis steps and omitted enzymatic hydrolysis steps have different product components, and the effect of promoting hematopoiesis and increasing bone density is poor. From the test results of Comparative Example 5, it can be seen that the liposome-coated enzymatic hydrolysis product can retain better therapeutic activity, and the liposome also has nutritional components to promote the therapeutic effect. From the test results of Comparative Example 6, it can be seen that the product prepared by enzymatic hydrolysis using adult bovine bone marrow as raw material is poor in promoting hematopoiesis and increasing bone density.

[0093] 3. Anti-aging test:

[0094] 1. Prepare a product sample solution with a concentration of 0.5 mg / mL using anhydrous ethanol as solvent.

[0095] 2. Add 2.0 mL of sample solution and 2.0 mL of 1.0 × 10 4 mol / L DPPH solution was mixed evenly and allowed to stand for 30 min. A 95% volume concentration ethanol aqueous solution was used as a reference solution, and the absorbance value A at 517 nm was measured.

[0096] 3. Evenly mix 2.0 mL of the sample solution and 2.0 mL of 95% ethanol aqueous solution, let stand for 30 min, and use the 95% ethanol aqueous solution as the reference solution to measure the absorbance A0 at 517 nm.

[0097] 4. Add 2.0mL, 1.0×10 4 mol / L DPPH solution was mixed evenly with 2.0 mL of 95% volume concentration ethanol aqueous solution, and allowed to stand for 30 min. The 95% volume concentration ethanol aqueous solution was used as a reference solution, and the absorbance value A1 at 517 nm was measured.

[0098] The DPPH clearance rate was calculated according to the formula: Clearance rate (%) = [1-(A-A0) / A1]×100.

[0099] Table 1 DPPH removal rate

[0100] Sample DPPH removal rate% Sample DPPH removal rate% Example 1 47.6 Comparative Example 1 38.1 Example 2 43.9 Comparative Example 2 29.5 Example 3 45.7 Comparative Example 3 35.4 Example 4 48.3 Comparative Example 4 26.7 Example 5 46.5 Comparative Example 5 47.5 Comparative Example 6 40.2

[0101] From the above results, it can be seen that the products of Examples 1 to 5 (calf bone marrow hydrolyzate liposome freeze-dried powder) have good DPPH free radical inhibition and anti-aging effects. From the test results of Comparative Examples 1 to 4, it can be seen that different enzymatic hydrolysis steps and omitted enzymatic hydrolysis steps result in different product components, which are relatively poor in inhibiting DPPH free radicals and anti-aging effects. From the test results of Comparative Example 6, it can be seen that the product prepared by enzymatic hydrolysis using adult bovine bone marrow as raw material is poor in scavenging free radicals and anti-aging.

Claims

1. A calf bone marrow hydrolysate liposome freeze-dried powder, characterized in that: Calf bone marrow hydrolysate liposome freeze-dried powder is a product obtained by using calf bone marrow as raw material, performing step-by-step enzymatic hydrolysis to obtain an enzymatic hydrolysate below 3 kDa, and then coating the product with liposomes; the specific preparation method comprises the following steps: S1: Take calf bone marrow from yaks under 1 year old, add 6 to 8 times the weight of water, stir to make a slurry, adjust the pH to 7.5 to 8.5, add 0.5% to 1.5% of the weight of pancreatic lipase from calf bone marrow, enzymolyze at 35℃ to 40℃ for 2h to 3h, then adjust the pH to 8.5 to 9.0, add 0.3% to 0.8% of the weight of alkaline phosphatase from calf bone marrow, enzymolyze at 40℃ to 45℃ for 1.5h to 2h, then adjust the pH to 8.0 to 8.5, add 0.8% to 2% of the weight of trypsin from calf bone marrow, and enzymolyze at 35℃. ~40℃ for 1h~1.5h, inactivate the enzyme, cool to room temperature, add 1%~2.5% by weight of elastase from calf bone marrow, enzymolyze at 25℃~28℃ for 1h~1.5h, inactivate the enzyme, cool to room temperature, adjust pH to 6.8~7.2, add 1.5%~2.0% by weight of ficin from calf bone marrow, enzymolyze at 60℃~65℃ for 1h~1.5h, inactivate the enzyme, cool to room temperature, filter with 3kDa ultrafiltration membrane, take the filtrate below 3kDa, concentrate to 10%~15% by volume, and obtain enzymolysis solution; S2: According to the mass ratio of soybean lecithin: cholesterol: anhydrous ethanol = (8-10): (2-2.5): (25-35), soybean lecithin and cholesterol are placed in anhydrous ethanol, stirred and mixed for more than 10 minutes, and then ultrasonically mixed for 1-3 minutes. After being uniformly dissolved, they are placed in an evaporation container and evaporated at 60-65°C to remove the anhydrous ethanol, and a thin film is formed on the inner wall of the evaporation container; S3: According to the mass ratio of film: enzyme hydrolysate: phosphate buffer = (0.5-1): (10-15): (80-100), place the film and enzyme hydrolysate in phosphate buffer, stir and mix for more than 30 minutes, let stand for 30-40 minutes, and mix by ultrasonic in a water bath at 4℃-5℃ for 10-15 minutes to obtain an emulsion; S4: The emulsion is placed in a syringe, and is repeatedly pushed out through a 0.38 μm polycarbonate filter membrane for 3 to 5 times, freeze-dried, and broken up to obtain calf bone marrow hydrolyzate liposome freeze-dried powder.

2. The calf bone marrow hydrolysate liposome freeze-dried powder according to claim 1, characterized in that: In S1, the enzymatic activity of the pancreatic lipase is 50,000 U / g to 100,000 U / g.

3. The calf bone marrow hydrolysate liposome freeze-dried powder according to claim 1, characterized in that: In S1, the enzyme activity of the alkaline phosphatase is 5,000 U / g to 10,000 U / g.

4. The calf bone marrow hydrolysate liposome freeze-dried powder according to claim 1, characterized in that: In S1, the enzymatic activity of the trypsin is 2000U / g to 4000U / g; the enzymatic activity of the elastase is 100,000U / g to 500,000U / g; and the enzymatic activity of the ficin is 600,000U / g to 800,000U / g.

5. The calf bone marrow hydrolysate liposome freeze-dried powder according to claim 1, characterized in that: In S1, the enzyme inactivation temperature is 100°C to 120°C, and the time is 10 min to 20 min.

6. The calf bone marrow hydrolysate liposome freeze-dried powder according to claim 1, characterized in that: In S2, the frequency of the ultrasound is 20kHz to 30kHz.

7. The calf bone marrow hydrolysate liposome freeze-dried powder according to claim 1, characterized in that: In S3, the phosphate buffer is a 0.2 M phosphate buffer solution with a pH of 6.5 to 7.

0.

8. The calf bone marrow hydrolysate liposome freeze-dried powder according to claim 1, characterized in that: In S3, the frequency of the ultrasound is 40kHz to 50kHz.

9. Use of the calf bone marrow hydrolyzate liposome freeze-dried powder according to claim 1 in the preparation of a drug having hematopoiesis-promoting and anti-aging effects.