Preparation method of bone tendon mucosa multi-target composite preparation based on cold extraction enzymolysis high-temperature transduction technology

Abstract

The invention discloses a preparation method of a bone tendon mucosa multi-target composite preparation based on a cold extraction enzymolysis high-temperature transduction technology, and relates to the technical field of medicines. The method comprises the following steps: step 1, raw material pretreatment: selecting fresh and pollution-free cod skin and deer sinew as main raw materials, washing the cod skin, removing impurities and fat on the surface, cutting the cod skin into small blocks with the side length of about 0.5-1cm, and soaking the deer sinew in warm water with the temperature of 30-40 DEG C for 12-15 hours for soaking; through the unique low-temperature stage of 4-15 DEG C, the stepped heating stage of 15-37 DEG C and pulse heat treatment, damage of traditional heat extraction to heat-sensitive components such as collagen is effectively avoided, the natural activity of the raw materials is reserved, heat-sensitive substances in the raw materials can be completely reserved in the low-temperature stage under mild enzymolysis conditions, and the product quality is improved. The conformation conversion rate of the collagen peptide is improved through pulse heat treatment, the bioavailability and activity of the collagen peptide are remarkably improved, and absorption and utilization of the collagen peptide by the human body are facilitated.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to a method for preparing a bone tendon mucosa multi-target composite preparation based on cold extraction enzymatic hydrolysis and high temperature energy conversion technology. Background Art

[0002] Traditional thermal extraction technology is widely used to extract active ingredients from biological raw materials, but this method severely damages key components such as collagen. Collagen is a vital component of bone and tendon tissue, and its unique triple-helix structure imparts excellent biomechanical properties and biological functions to the tissue. During traditional thermal extraction, high temperatures cause the collagen triple-helix structure to disintegrate, significantly reducing the effectiveness of products prepared using this raw material in promoting bone and tendon repair and maintaining tissue strength, thus failing to meet clinical and market demand for highly effective products.

[0003] Many bone-targeted formulations on the market contain only a single active ingredient, resulting in suboptimal bioavailability. The body has natural limitations in its absorption and utilization of a single ingredient, making it difficult to fully promote bone health. This makes the expected effects less effective when addressing complex bone metabolism and bone disease treatments.

[0004] Vitamins K2 and D3 play a key role in maintaining bone health and promoting calcium absorption. They are often formulated into micelles to enhance bioavailability. However, existing technologies for micellar vitamins K2 and D3 suffer from poor stability. During storage, transportation, and use, the micelles are prone to aggregation and rupture, resulting in reduced vitamin activity and an inability to stably and effectively deliver the vitamins, compromising product efficacy.

[0005] Current treatments for bone, tendon, and mucosal diseases, such as conventional medications and physical therapy, suffer from long treatment cycles, significant side effects, and incomplete therapeutic effects. For example, in cases of tendon calcification and rotator cuff injuries, traditional treatments often fail to fundamentally repair damaged tissue, leading to recurrences and prolonged pain and functional limitations for patients.

[0006] To this end, we provide a preparation method for a multi-target composite preparation of bone tendon and mucosa based on cold extraction enzymatic hydrolysis and high temperature energy conversion technology to solve the above problems. Summary of the Invention

[0007] The purpose of the present invention is to provide a method for preparing a multi-target composite preparation of bone tendon and mucosa based on cold extraction enzymatic hydrolysis and high temperature energy conversion technology, which solves the problems of heat extraction used in the prior art leading to destruction of the triple helix structure of collagen, low bioavailability and poor micelle stability.

[0008] To solve the above technical problems, the present invention is implemented through the following technical solutions.

[0009] The present invention is a method for preparing a multi-target composite preparation of bone tendon and mucosa based on cold extraction enzymatic hydrolysis and high temperature energy conversion technology, comprising the following steps:

[0010] Step 1: Raw material pretreatment:

[0011] Select fresh, unpolluted cod skin and deer tendon as the main ingredients. Wash the cod skin, remove impurities and fat from the surface, and cut it into small pieces with a side length of approximately 0.5-1 cm. Soak the deer tendon in 30-40°C warm water for 12-15 hours to allow it to expand, changing the water every 3-4 hours. After expansion, remove any remaining fascia and impurities, and cut it into 1-2 cm small pieces for later use.

[0012] Step 2: Cold extraction-variable temperature coupled enzymatic hydrolysis process:

[0013] Low temperature stage: the pretreated cod skin and deer tendon raw materials are placed in a reaction vessel with a temperature control function, deionized water 3-5 times the weight of the raw materials is added to completely immerse the raw materials, and enzyme preparations are added in a ratio of 1:1 between papain and bromelain, with a total amount of 2-3g of enzyme preparation added per kilogram of raw materials. At the same time, the pH value of the reaction system is adjusted to 3.5-4.0 using citric acid and disodium hydrogen phosphate. The reaction vessel is placed in a low temperature environment of 4-15°C and the enzymatic hydrolysis is continued for 48 hours. During the enzymatic hydrolysis of the cod skin, fresh green Pu'er tea accounting for 0.1% of the total weight of the raw materials is added. The fresh green Pu'er tea needs to be crushed into 100-120 mesh fine powder in advance. During this stage, the pH value of the reaction system is tested every 6 hours. If the pH value deviates from the range, the above-mentioned regulator is used for fine adjustment to ensure that the pH value is always maintained within the specified range;

[0014] Step heating stage: After the low-temperature stage, the reaction system is heated at a slow heating rate of 1°C / 30min. As the temperature gradually rises from 15°C to 37°C, β-glucosidase is activated. During the heating process, a magnetic stirrer is used to continuously stir the reaction mixture at a speed of 100-150r / min to ensure uniform temperature distribution and promote full contact between the enzyme and the substrate.

[0015] Pulse heat treatment: The product after the step-by-step enzymatic hydrolysis is transferred to a dedicated pulse heat treatment device for pulse heat treatment. The specific conditions are: after treating at 85°C for 5 seconds, the temperature is quickly cooled to 40°C within 1-2 seconds through the built-in cooling device, and this cycle is repeated three times;

[0016] Step 3: Nanocomplex construction:

[0017] Preparation of EGCG-curcumin nanoparticles: EGCG-curcumin nanoparticles were prepared by a solvent evaporation method. EGCG and curcumin were dissolved in ethanol at a mass ratio of 3:2, with a total concentration of 5-8 mg / mL to form a uniform solution. The solution was then slowly added dropwise at a rate of 1-2 drops / second to an aqueous phase containing 1-2% polysorbate 80. Under high-speed stirring, the ethanol gradually evaporated, and EGCG and curcumin aggregated in the aqueous phase to form nanoparticles. The particle size of the nanoparticles was monitored in real time by a dynamic light scattering instrument. When the particle size reached 80-120 nm, stirring was stopped, and the zeta potential of the nanoparticles was detected using a zeta potential analyzer. The zeta potential was adjusted to -35 mV by adjusting the concentration of polysorbate 80.

[0018] Preparation of vitamin K2 / D3 micelles: Using Brazil nut lipid as a carrier, vitamin K2 / D3 micelles were constructed using a thin film dispersion method. First, Brazil nut lipid, vitamin K2, and vitamin D3 were dissolved in an appropriate amount of chloroform at a mass ratio of 10:2:1. The chloroform was completely evaporated on a rotary evaporator at 40-45°C and a speed of 100-120 r / min to form a uniform lipid film on the container wall. Then, a phosphate buffer solution with a pH value of 7.2-7.4 was added at a liquid-to-solid ratio of 8-10 mL / g. Ultrasonic treatment was performed in an ultrasonicator at a power of 200-300 W for 15-20 minutes to disperse the lipid film to form micelles. The encapsulation efficiency of vitamin K2 and D3 was detected by high-performance liquid chromatography. The encapsulation efficiency was ensured to be ≥92% by adjusting the preparation process parameters.

[0019] Step 4: Mixing:

[0020] The products obtained by the above enzymatic hydrolysis and nanocomplex construction steps are mixed in a mass ratio of cod collagen peptide: deer tendon oligopeptide: mulberry anthocyanin of 5:3:2. During the mixing process, a high-speed stirrer is used to fully stir at a speed of 500-600 r / min for 30-40 minutes to ensure that the components are evenly mixed. At the same time, the quality of the mixed product is tested by high-performance liquid chromatography to ensure that Gly-Pro-Hyp is ≥15% and ginsenoside Rb1 / Rg1 is 2:1. If the test results do not meet the requirements, the mixing ratio needs to be fine-tuned or the components need to be further purified.

[0021] Step 5: High-pressure homogenization:

[0022] The mixed product was transferred to a high-pressure homogenizer and homogenized 5 times under a pressure of 150 MPa. During the homogenization process, the particle size of the emulsion was monitored in real time by a laser particle size analyzer to ensure that the particle size D90 of the emulsion after treatment was less than 200 nm.

[0023] Step 6: Post-processing and packaging:

[0024] The product after high-pressure homogenization is sterile filtered using a 0.22μm microporous filter membrane to remove existing microorganisms and impurities, and is packaged according to the dosage form requirements of the product. The packaging process must be carried out in a sterile environment that complies with pharmaceutical production quality management standards.

[0025] The present invention is further configured such that, in said step one, the selected cod skin and deer tendon are subjected to microbial testing and heavy metal content testing, the microbial limits must comply with the prescribed standards for oral and topical preparations, and the heavy metal lead, mercury, cadmium, and arsenic contents shall not exceed 0.5 mg / kg, 0.01 mg / kg, 0.3 mg / kg, and 2 mg / kg, respectively.

[0026] The present invention is further configured such that, in the step three, the prepared EGCG-curcumin nanoparticles and vitamin K2 / D3 micelles are respectively subjected to transmission electron microscopy to observe their microscopic morphology and structure. The EGCG-curcumin nanoparticles should be spherical or approximately spherical, and the vitamin K2 / D3 micelles should have a uniform vesicle-like structure.

[0027] The present invention is further configured such that a step-by-step dissolution and stirring method is adopted when mixing the ingredients in step four, firstly dissolving the deer tendon oligopeptide in part of the water, and then sequentially adding the cod collagen peptide and the mulberry anthocyanin. During mixing, the temperature is controlled at 20-25°C, and the stirring speed is gradually increased. After mixing, in addition to conventional testing, the stability of the mulberry anthocyanin is also tested.

[0028] The present invention is further configured that, after the preparation of the EGCG-curcumin nanoparticles in step three is completed, they are dialyzed to remove impurities and freeze-dried, and the vitamin K2 / D3 micelles are ultrasonically filtered through 0.45 μm and 0.22 μm filter membranes in sequence.

[0029] The present invention is further configured as follows: after adding the enzyme and fresh Pu'er green tea in step 1, ultrasonic assisted treatment is performed, with an ultrasonic power of 100-150W, a frequency of 20-30kHz, and intermittent operation, and bioactive factors are added 2-3 hours before the end.

[0030] The present invention is further configured such that, in the steps 5 and 6, the powder is spray-dried after homogenization, and when the oral preparation is packaged in capsules, the composite preparation powder is microencapsulated with sodium alginate and chitosan and then packed into enteric-coated capsules.

[0031] The present invention is further configured such that, in step 1, before processing the cod skin and deer tendons, a component pre-analysis is performed on them, and the basic nutritional components and potential active ingredient contents therein are determined by high performance liquid chromatography-mass spectrometry.

[0032] The present invention is further configured as follows: in step 1, the cod skin and deer tendon are soaked and cleaned for 15-20 minutes using a cleaning solution containing 0.1%-0.3% tea polyphenols to remove surface impurities and utilize the antioxidant properties of tea polyphenols to protect the active ingredients in the raw materials.

[0033] A multi-target composite preparation for bone, tendon and mucosa based on cold-extraction enzymatic hydrolysis and high-temperature energy conversion technology. The composite preparation includes:

[0034] Cod collagen peptide, deer tendon oligopeptide, and mulberry anthocyanin mixed in a mass ratio of 5:3:2;

[0035] EGCG-curcumin nanoparticles, with a particle size of 80-120 nm and a zeta potential of -35 mV;

[0036] Vitamin K2 / D3 micelles, encapsulation efficiency ≥92%, and key activity indicators Gly-Pro-Hyp ≥15%, ginsenoside Rb1 / Rg1=2:1

[0037] The present invention has the following beneficial effects.

[0038] 1. The present invention effectively avoids the destruction of heat-sensitive components such as collagen by traditional heat extraction through a unique 4-15°C low temperature stage, a 15-37°C step-by-step heating stage and pulse heat treatment, thereby retaining the natural activity of the raw materials. In the low temperature stage, mild enzymatic hydrolysis conditions can completely retain the heat-sensitive substances in the raw materials, and pulse heat treatment improves the conformational conversion rate of collagen peptides, significantly improving their bioavailability and activity, making them more conducive to human absorption and utilization. The prepared EGCG-curcumin nanoparticles and vitamin K2 / D3 micelles have good stability and dispersibility, nanometer-level particle size and suitable potential characteristics, so that the EGCG-curcumin nanoparticles can be better absorbed and transported by the human body. The vitamin K2 / D3 micelles with high encapsulation efficiency can effectively protect the vitamins and improve their delivery efficiency and stability in the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments.

[0040] Figure 1 The present invention is a process flow chart for the preparation of a multi-target composite preparation of bone tendon and mucosa based on cold extraction enzymatic hydrolysis and high temperature energy conversion technology.

[0041] Figure 2 The present invention is a flow chart of the cold extraction-variable temperature coupled enzymatic hydrolysis process in the preparation method of the bone tendon mucosa multi-target composite preparation based on the cold extraction enzymatic hydrolysis high temperature energy conversion technology.

[0042] Figure 3The present invention is a flow chart of the construction of nanocomplexes in the preparation method of multi-target composite preparations for bone tendon and mucosa based on cold extraction enzymatic hydrolysis and high temperature energy conversion technology.

[0043] Figure 4 The present invention is a flow chart of the low-temperature enzymatic hydrolysis stage in the preparation method of bone tendon mucosa multi-target composite preparation based on cold extraction enzymatic hydrolysis and high-temperature energy conversion technology.

[0044] Figure 5 The present invention is a flow chart of the preparation of EGCG-curcumin nanoparticles in the preparation method of bone tendon mucosa multi-target composite preparation based on cold extraction enzymatic hydrolysis and high temperature energy conversion technology.

[0045] Figure 6 The present invention is a flow chart of the preparation of vitamin K2 / D3 micelles in the preparation method of bone tendon mucosa multi-target composite preparation based on cold extraction enzymatic hydrolysis and high temperature energy conversion technology. DETAILED DESCRIPTION

[0046] The technical solutions in the embodiments of the present invention will be described below in conjunction with the drawings in the embodiments of the present invention. The described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0047] Example 1

[0048] See also Figure 1-6 The present invention is a method for preparing a multi-target composite preparation of bone tendon and mucosa based on cold extraction enzymatic hydrolysis and high temperature energy conversion technology, comprising the following steps:

[0049] Step 1: Raw material pretreatment:

[0050] Select fresh, unpolluted cod skin and deer tendon as the main ingredients. Wash the cod skin, remove impurities and fat from the surface, and cut it into small pieces with a side length of approximately 0.5-1 cm. Soak the deer tendon in 30-40°C warm water for 12-15 hours to allow it to expand, changing the water every 3-4 hours. After expansion, remove any remaining fascia and impurities, and cut it into 1-2 cm small pieces for later use.

[0051] Step 2: Cold extraction-variable temperature coupled enzymatic hydrolysis process:

[0052] Low temperature stage: the pretreated cod skin and deer tendon raw materials are placed in a reaction vessel with a temperature control function, deionized water 3-5 times the weight of the raw materials is added to completely immerse the raw materials, and enzyme preparations are added according to a ratio of 1:1 between papain and bromelain, with a total amount of 2-3g of enzyme preparation added per kilogram of raw materials. At the same time, the pH value of the reaction system is adjusted to 3.5-4.0 using citric acid and disodium hydrogen phosphate. The reaction vessel is placed in a low temperature environment of 4-15°C and the enzymatic hydrolysis is continued for 48 hours. During the enzymatic hydrolysis of the cod skin, 0.1% of the total weight of the raw materials is added to fresh green tea Pu'er (polyphenol content ≥98%). The fresh green tea Pu'er needs to be crushed into 100-120 mesh fine powder in advance. During this stage, the pH value of the reaction system is tested every 6 hours. If the pH value deviates from the range, the above-mentioned regulator is used for fine adjustment to ensure that the pH value is always maintained within the specified range.

[0053] Step heating stage: After the low-temperature stage, the reaction system is heated at a slow heating rate of 1°C / 30min. As the temperature gradually rises from 15°C to 37°C, β-glucosidase is activated. During the heating process, a magnetic stirrer is used to continuously stir the reaction mixture at a speed of 100-150r / min to ensure uniform temperature distribution and promote full contact between the enzyme and the substrate.

[0054] Pulse heat treatment: The product after the step-by-step enzymatic hydrolysis is transferred to a dedicated pulse heat treatment device for pulse heat treatment. The specific conditions are: after treating at 85°C for 5 seconds, the temperature is quickly cooled to 40°C within 1-2 seconds through the built-in cooling device, and this cycle is repeated three times;

[0055] Step 3: Nanocomplex construction:

[0056] Preparation of EGCG-curcumin nanoparticles: EGCG-curcumin nanoparticles were prepared by a solvent evaporation method. EGCG and curcumin were dissolved in ethanol at a mass ratio of 3:2, with a total concentration of 5-8 mg / mL to form a uniform solution. The solution was then slowly added dropwise at a rate of 1-2 drops / second to an aqueous phase containing 1-2% polysorbate 80. Under high-speed stirring, the ethanol gradually evaporated, and EGCG and curcumin aggregated in the aqueous phase to form nanoparticles. The particle size of the nanoparticles was monitored in real time by a dynamic light scattering instrument. When the particle size reached 80-120 nm, stirring was stopped, and the zeta potential of the nanoparticles was detected using a zeta potential analyzer. The zeta potential was adjusted to -35 mV by adjusting the concentration of polysorbate 80.

[0057] Preparation of vitamin K2 / D3 micelles: Using Brazil nut lipid as a carrier, vitamin K2 / D3 micelles were constructed using a thin film dispersion method. First, Brazil nut lipid, vitamin K2, and vitamin D3 were dissolved in an appropriate amount of chloroform at a mass ratio of 10:2:1. The chloroform was completely evaporated on a rotary evaporator at 40-45°C and a speed of 100-120 r / min to form a uniform lipid film on the container wall. Then, a phosphate buffer solution with a pH value of 7.2-7.4 was added at a liquid-to-solid ratio of 8-10 mL / g. Ultrasonic treatment was performed in an ultrasonicator at a power of 200-300 W for 15-20 minutes to disperse the lipid film to form micelles. The encapsulation efficiency of vitamin K2 and D3 was detected by high-performance liquid chromatography. The encapsulation efficiency was ensured to be ≥92% by adjusting the preparation process parameters.

[0058] Step 4: Mixing:

[0059] The products obtained by the above enzymatic hydrolysis and nanocomplex construction steps are mixed in a mass ratio of cod collagen peptide: deer tendon oligopeptide: mulberry anthocyanin of 5:3:2. During the mixing process, a high-speed stirrer is used to fully stir at a speed of 500-600 r / min for 30-40 minutes to ensure that the components are evenly mixed. At the same time, the quality of the mixed product is tested by high-performance liquid chromatography to ensure that Gly-Pro-Hyp is ≥15% and ginsenoside Rb1 / Rg1 is 2:1. If the test results do not meet the requirements, the mixing ratio needs to be fine-tuned or the components need to be further purified.

[0060] Step 5: High-pressure homogenization:

[0061] The mixed product was transferred to a high-pressure homogenizer and homogenized 5 times under a pressure of 150 MPa. During the homogenization process, the particle size of the emulsion was monitored in real time by a laser particle size analyzer to ensure that the particle size D90 of the emulsion after treatment was less than 200 nm.

[0062] Step 6: Post-processing and packaging:

[0063] The product after high-pressure homogenization is sterile filtered using a 0.22μm microporous filter membrane to remove existing microorganisms and impurities, and is packaged according to the dosage form requirements of the product. The packaging process must be carried out in a sterile environment that complies with pharmaceutical production quality management standards.

[0064] Example 2

[0065] Raw material pretreatment: Fresh cod skin and healthy deer tendons from unpolluted waters were selected. The cod skin was washed, impurities removed, and cut into 0.5cm small pieces. The deer tendons were soaked in 30℃ warm water for 12 hours, with the water changed every 3 hours. After soaking, the fascia impurities were removed and the tendons were cut into 1cm small pieces. After testing, the microbial limits and heavy metal content were found to meet the standards. At the same time, high-performance liquid chromatography-mass spectrometry technology was used to pre-analyze the ingredients of the raw materials.

[0066] Cold extraction-variable temperature coupled enzymatic hydrolysis process: In the low temperature stage, the pretreated raw materials are placed in a reaction vessel, 3 times the mass of deionized water is added, 2g of enzyme preparation (papain and bromelain 1:1) is added per kilogram of raw materials, and the pH is adjusted to 3.5 with citric acid and disodium hydrogen phosphate. It is placed in a 4°C environment for enzymatic hydrolysis for 48 hours. When enzymatic hydrolysis of cod skin, 0.1% of fresh green tea Pu'er powder crushed to 100 mesh fine powder is added. The pH value is tested and fine-tuned every 6 hours. After adding enzymes and fresh green tea Pu'er, ultrasonic assisted treatment is carried out at 100W power and 20kHz frequency for 30 minutes, intermittently. Bioactive factors are added 2 hours before the end. In the step heating stage, the temperature is increased to 37°C at 1°C / 30min, and stirred with a magnetic stirrer at 100r / min. In the pulse heat treatment stage, it is treated at 85°C for 5s and then cooled to 40°C within 1s, and the cycle is repeated 3 times.

[0067] Nanocomplex construction: EGCG-curcumin nanoparticles were prepared by dissolving EGCG and curcumin in ethanol at a mass ratio of 3:2 (total concentration 5 mg / mL), dropping the mixture into an aqueous phase containing 1% polysorbate 80, and stirring at high speed until the particle size reached 80 nm. After preparation, the mixture was dialyzed for impurity removal and freeze-dried. For the preparation of vitamin K2 / D3 micelles, Brazil nut lipids, vitamin K2, and vitamin D3 were dissolved in chloroform at a mass ratio of 10:2:1, and rotary evaporated at 40°C and 100 r / min to form a film. A pH 7.2 phosphate buffer solution (liquid-to-solid ratio 8 mL / g) was added, and the mixture was ultrasonicated at 200 W for 15 minutes. After ultrasonication, the mixture was filtered through 0.45 μm and 0.22 μm filter membranes.

[0068] Mixing and preparation: Prepare a mixed product with a mass ratio of cod collagen peptide: deer tendon oligopeptide: mulberry anthocyanin of 5:3:2. First dissolve the deer tendon oligopeptide in part of the water, and then add other ingredients in turn. Control the temperature at 20°C, gradually increase the stirring speed from 300r / min to 500r / min, and stir for 30 minutes. HPLC detection showed that the Gly-Pro-Hyp content was 16%, the ginsenoside Rb1 / Rg1 ratio was 2:1, and the stability of mulberry anthocyanin was good.

[0069] High-pressure homogenization treatment: The product was cyclically homogenized 5 times at a pressure of 150 MPa, and the emulsion particle size D90 was 180 nm.

[0070] Post-processing and packaging: After sterile filtration, spray dry and powder. If it is an oral preparation, the composite preparation powder is microencapsulated with sodium alginate and chitosan and then filled into enteric-coated capsules; if it is an external preparation or injection, it is packaged according to the requirements of the corresponding dosage form.

[0071] Example 3

[0072] Raw material pretreatment: fresh cod skin and deer tendon were selected and processed in the same manner as in Example 2. After the microbial and heavy metal tests were qualified and the components were pre-analyzed, they were soaked and cleaned in a cleaning solution containing 0.2% tea polyphenols for 15 minutes.

[0073] Cold extraction-variable temperature coupled enzymatic hydrolysis process: in the low temperature stage, 4 times the mass of deionized water was added, 2.5 g of enzyme preparation was added per kilogram of raw material, the pH was adjusted to 3.8, and enzymatic hydrolysis was carried out at 10°C for 48 hours. The ultrasonic assisted treatment power was 120 W and the frequency was 25 kHz. The bioactive factor was added 2.5 hours before the end. In the step heating stage, the heating rate was 1°C / 30 min, the stirring speed was 120 r / min, and the pulse heat treatment was the same as in Example 2.

[0074] Nanocomplex construction: EGCG-curcumin nanoparticles were prepared at a total concentration of 6 mg / mL, resulting in a particle size of 100 nm after dropwise addition. For the preparation of vitamin K2 / D3 micelles, the rotary evaporation temperature was 42°C, the speed was 110 rpm, the ultrasonic power was 250 W, and the time was 18 minutes. Subsequent filtration treatment was the same.

[0075] Mixing and blending: The temperature was controlled at 22°C during mixing, the stirring speed was increased from 350 r / min to 550 r / min, and stirring was carried out for 35 minutes. The detection showed that the Gly-Pro-Hyp content was 17%, the ginsenoside Rb1 / Rg1 ratio was 2:1, and the stability of mulberry anthocyanins met the standards.

[0076] High-pressure homogenization treatment: homogenization pressure 150 MPa, after 5 cycles, the emulsion particle size D90 is 190 nm.

[0077] Post-processing and packaging: After sterile filtration, spray dry and powder. If it is an oral preparation, the composite preparation powder is microencapsulated with sodium alginate and chitosan and then filled into enteric-coated capsules; if it is an external preparation or injection, it is packaged according to the requirements of the corresponding dosage form.

[0078] Example 4

[0079] Raw material pretreatment: Process cod skin and deer tendon, detect microorganisms and heavy metals, pre-analyze ingredients, and soak in 0.3% tea polyphenols cleaning solution for 20 minutes.

[0080] Cold extraction-variable temperature coupled enzymatic hydrolysis process: in the low temperature stage, add 5 times the mass of deionized water, add 3g enzyme preparation per kilogram of raw material, pH 4.0, enzymatic hydrolysis at 15℃ for 48h, ultrasonic power 150W, frequency 30kHz, add bioactive factors 3 hours before the end, in the step-by-step heating stage, the stirring speed is 150r / min, and the pulse heat treatment parameters remain unchanged.

[0081] Nanocomplex construction: EGCG-curcumin nanoparticles were prepared at a total concentration of 8 mg / mL and a particle size of 120 nm. Vitamin K2 / D3 micelles were prepared using rotary evaporation at 45°C, 120 rpm, and ultrasonication at 300 W for 20 minutes. Filtration was performed as before.

[0082] Mixing and blending: The temperature was controlled at 25°C during mixing, the stirring speed was increased from 400 r / min to 600 r / min, and stirring was carried out for 40 minutes. The Gly-Pro-Hyp content was detected to be 18%, the ginsenoside Rb1 / Rg1 ratio was 2:1, and the stability of mulberry anthocyanins met the requirements.

[0083] High-pressure homogenization treatment: 150 MPa pressure cycle homogenization 5 times, the emulsion particle size D90 is 195 nm.

[0084] Post-processing and packaging: After sterile filtration, spray dry and powder. If it is an oral preparation, the composite preparation powder is microencapsulated with sodium alginate and chitosan and then filled into enteric-coated capsules; if it is an external preparation or injection, it is packaged according to the requirements of the corresponding dosage form.

[0085] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all details in detail, nor do they limit the invention to only the specific implementation methods described. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can better understand and utilize the present invention.

Claims

1. A method for preparing a multi-target composite preparation for bone, tendon and mucosa based on cold extraction enzymatic hydrolysis and high temperature energy conversion technology, characterized by: The following steps are involved: Step 1: Raw material pretreatment: Select fresh, unpolluted cod skin and deer tendon as the main ingredients. Wash the cod skin, remove impurities and fat from the surface, and cut it into small pieces with a side length of approximately 0.5-1 cm. Soak the deer tendon in 30-40°C warm water for 12-15 hours to allow it to expand, changing the water every 3-4 hours. After expansion, remove any remaining fascia and impurities, and cut it into 1-2 cm small pieces for later use. Step 2: Cold extraction-variable temperature coupled enzymatic hydrolysis process: Low temperature stage: the pretreated cod skin and deer tendon raw materials are placed in a reaction vessel with a temperature control function, deionized water 3-5 times the weight of the raw materials is added to completely immerse the raw materials, and enzyme preparations are added in a ratio of 1:1 between papain and bromelain, with a total amount of 2-3g of enzyme preparation added per kilogram of raw materials. At the same time, the pH value of the reaction system is adjusted to 3.5-4.0 using citric acid and disodium hydrogen phosphate. The reaction vessel is placed in a low temperature environment of 4-15°C and the enzymatic hydrolysis is continued for 48 hours. During the enzymatic hydrolysis of the cod skin, fresh green Pu'er tea accounting for 0.1% of the total weight of the raw materials is added. The fresh green Pu'er tea needs to be crushed into 100-120 mesh fine powder in advance. During this stage, the pH value of the reaction system is tested every 6 hours. If the pH value deviates from the range, the above-mentioned regulator is used for fine adjustment to ensure that the pH value is always maintained within the specified range; Step heating stage: After the low-temperature stage, the reaction system is heated at a slow heating rate of 1°C / 30min. As the temperature gradually rises from 15°C to 37°C, β-glucosidase is activated. During the heating process, a magnetic stirrer is used to continuously stir the reaction mixture at a speed of 100-150r / min to ensure uniform temperature distribution and promote full contact between the enzyme and the substrate. Pulse heat treatment: The product after the step-by-step enzymatic hydrolysis is transferred to a dedicated pulse heat treatment device for pulse heat treatment. The specific conditions are: after treating at 85°C for 5 seconds, the temperature is quickly cooled to 40°C within 1-2 seconds through the built-in cooling device, and this cycle is repeated three times; Step 3: Nanocomplex construction: Preparation of EGCG-curcumin nanoparticles: EGCG-curcumin nanoparticles were prepared by a solvent evaporation method. EGCG and curcumin were dissolved in ethanol at a mass ratio of 3:2, with a total concentration of 5-8 mg / mL to form a uniform solution. The solution was then slowly added dropwise at a rate of 1-2 drops / second to an aqueous phase containing 1-2% polysorbate 80. Under high-speed stirring, the ethanol gradually evaporated, and EGCG and curcumin aggregated in the aqueous phase to form nanoparticles. The particle size of the nanoparticles was monitored in real time by a dynamic light scattering instrument. When the particle size reached 80-120 nm, stirring was stopped, and the zeta potential of the nanoparticles was detected using a zeta potential analyzer. The zeta potential was adjusted to -35 mV by adjusting the concentration of polysorbate 80. Preparation of vitamin K2 / D3 micelles: Using Brazil nut lipid as a carrier, vitamin K2 / D3 micelles were constructed using a thin film dispersion method. First, Brazil nut lipid, vitamin K2, and vitamin D3 were dissolved in an appropriate amount of chloroform at a mass ratio of 10:2:

1. The chloroform was completely evaporated on a rotary evaporator at 40-45°C and a speed of 100-120 r / min to form a uniform lipid film on the container wall. Then, a phosphate buffer solution with a pH value of 7.2-7.4 was added at a liquid-to-solid ratio of 8-10 mL / g. Ultrasonic treatment was performed in an ultrasonicator at a power of 200-300 W for 15-20 minutes to disperse the lipid film to form micelles. The encapsulation efficiency of vitamin K2 and D3 was detected by high-performance liquid chromatography. The encapsulation efficiency was ensured to be ≥92% by adjusting the preparation process parameters. Step 4: Mixing: The products obtained by the above enzymatic hydrolysis and nanocomplex construction steps are mixed in a mass ratio of cod collagen peptide: deer tendon oligopeptide: mulberry anthocyanin of 5:3:

2. During the mixing process, a high-speed stirrer is used to fully stir at a speed of 500-600 r / min for 30-40 minutes to ensure that the components are evenly mixed. At the same time, the quality of the mixed product is tested by high-performance liquid chromatography to ensure that Gly-Pro-Hyp is ≥15% and ginsenoside Rb1 / Rg1 is 2:

1. If the test results do not meet the requirements, the mixing ratio needs to be fine-tuned or the components need to be further purified. Step 5: High-pressure homogenization: The mixed product was transferred to a high-pressure homogenizer and homogenized 5 times under a pressure of 150 MPa. During the homogenization process, the particle size of the emulsion was monitored in real time by a laser particle size analyzer to ensure that the particle size D90 of the emulsion after treatment was less than 200 nm. Step 6: Post-processing and packaging: The product after high-pressure homogenization is sterile filtered using a 0.22μm microporous filter membrane to remove existing microorganisms and impurities, and is packaged according to the dosage form requirements of the product. The packaging process must be carried out in a sterile environment that complies with pharmaceutical production quality management standards.

2. The method for preparing a multi-target composite preparation for bone tendon and mucosa based on cold extraction enzymatic hydrolysis and high temperature energy conversion technology according to claim 1, characterized in that: In the step 1, the selected cod skin and deer tendon are subjected to microbial testing and heavy metal content testing. The microbial limits must comply with the prescribed standards for oral and topical preparations, and the heavy metal lead, mercury, cadmium, and arsenic contents must not exceed 0.5 mg / kg, 0.01 mg / kg, 0.3 mg / kg, and 2 mg / kg, respectively.

3. The method for preparing a multi-target composite preparation for bone tendon and mucosa based on cold extraction enzymatic hydrolysis and high temperature energy conversion technology according to claim 1, characterized in that: In the step 3, the prepared EGCG-curcumin nanoparticles and vitamin K2 / D3 micelles are respectively subjected to transmission electron microscopy to observe their microscopic morphology and structure. The EGCG-curcumin nanoparticles should be spherical or approximately spherical, and the vitamin K2 / D3 micelles should have a uniform vesicle-like structure.

4. The method for preparing a multi-target composite preparation for bone tendon and mucosa based on cold extraction enzymatic hydrolysis and high temperature energy conversion technology according to claim 1, characterized in that: In step 4, the ingredients are mixed using a step-by-step dissolution and stirring method. Deer tendon oligopeptide is first dissolved in part of the water, and then cod collagen peptide and mulberry anthocyanin are added in sequence. The temperature is controlled at 20-25°C during mixing, and the stirring speed is gradually increased. After mixing, in addition to conventional testing, the stability of mulberry anthocyanin is also tested.

5. The method for preparing a multi-target composite preparation for bone tendon and mucosa based on cold extraction enzymatic hydrolysis and high temperature energy conversion technology according to claim 1, characterized in that: In the step 3, the EGCG-curcumin nanoparticles are dialyzed to remove impurities and freeze-dried after preparation, and the vitamin K2 / D3 micelles are filtered through 0.45 μm and 0.22 μm filter membranes in sequence after sonication.

6. The method for preparing a multi-target composite preparation for bone tendon and mucosa based on cold extraction enzymatic hydrolysis and high temperature energy conversion technology according to claim 1, characterized in that: After adding the enzyme and fresh Pu'er green tea in step 1, ultrasonic-assisted treatment is performed with an ultrasonic power of 100-150 W and a frequency of 20-30 kHz, and the treatment is performed intermittently. Bioactive factors are added 2-3 hours before the end.

7. The method for preparing a multi-target composite preparation for bone tendon and mucosa based on cold extraction enzymatic hydrolysis and high temperature energy conversion technology according to claim 1, characterized in that: After homogenization in step five and step six, spray drying is performed to prepare powder. When the oral preparation is packaged in capsules, the composite preparation powder is microencapsulated with sodium alginate and chitosan and then packed into enteric-coated capsules.

8. The method for preparing a multi-target composite preparation for bone tendon and mucosa based on cold extraction enzymatic hydrolysis and high temperature energy conversion technology according to claim 1, characterized in that: In the first step, before processing the cod skin and deer tendons, a component pre-analysis is performed on them, and the basic nutritional components and potential active ingredient contents therein are determined by high performance liquid chromatography-mass spectrometry.

9. The method for preparing a multi-target composite preparation for bone tendon and mucosa based on cold extraction enzymatic hydrolysis and high temperature energy conversion technology according to claim 1, characterized in that: In the step 1, the cod skin and deer tendon are soaked and cleaned for 15-20 minutes using a cleaning solution containing 0.1%-0.3% tea polyphenols to remove surface impurities and utilize the antioxidant properties of tea polyphenols to protect the active ingredients in the raw materials.

10. A multi-target composite preparation for bone, tendon and mucosa based on cold-extraction enzymatic hydrolysis and high-temperature energy conversion technology, characterized in that: Prepared by the preparation method according to any one of claims 1 to 9, the composite preparation comprises: Cod collagen peptide, deer tendon oligopeptide, and mulberry anthocyanin mixed in a mass ratio of 5:3:2; EGCG-curcumin nanoparticles, with a particle size of 80-120 nm and a zeta potential of -35 mV; Vitamin K2 / D3 micelles have an encapsulation rate of ≥92%, and have key activity indicators Gly-Pro-Hyp ≥15%, and ginsenoside Rb1 / Rg1 = 2:1.

Images