CN114848527A - Collagen composite solution for injection and preparation method thereof - Google Patents
Collagen composite solution for injection and preparation method thereof Download PDFInfo
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
- A61K8/65—Collagen; Gelatin; Keratin; Derivatives or degradation products thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/19—Cosmetics or similar toiletry preparations characterised by the composition containing inorganic ingredients
- A61K8/24—Phosphorous; Compounds thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/40—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing nitrogen
- A61K8/41—Amines
- A61K8/416—Quaternary ammonium compounds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/40—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing nitrogen
- A61K8/42—Amides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/40—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing nitrogen
- A61K8/44—Aminocarboxylic acids or derivatives thereof, e.g. aminocarboxylic acids containing sulfur; Salts; Esters or N-acylated derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/40—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing nitrogen
- A61K8/44—Aminocarboxylic acids or derivatives thereof, e.g. aminocarboxylic acids containing sulfur; Salts; Esters or N-acylated derivatives thereof
- A61K8/442—Aminocarboxylic acids or derivatives thereof, e.g. aminocarboxylic acids containing sulfur; Salts; Esters or N-acylated derivatives thereof substituted by amido group(s)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
- A61K8/494—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with more than one nitrogen as the only hetero atom
- A61K8/4946—Imidazoles or their condensed derivatives, e.g. benzimidazoles
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/60—Sugars; Derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
- A61K2800/52—Stabilizers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/91—Injection
Abstract
The invention provides a collagen composite solution for injection and a preparation method thereof, wherein the collagen composite solution for injection comprises the following components in percentage by mass: 0.1-0.6wt% of active collagen, 0.15-0.35wt% of acid-base amino acid, 0.0005-0.2wt% of compound nutrient, 0.01-0.2wt% of collagen stabilizer, 0.8-1wt% of sodium chloride and the balance of water. The collagen composite solution for injection disclosed by the invention has the advantages that on the basis of maintaining the structure of a natural collagen molecule, the pH value of a system is controlled through special pH amino acid, and a collagen stabilizer is introduced to improve the hydrophilicity of the collagen molecule, so that the solubility and the stability of the collagen molecule in the compounding process with micromolecular nutrients under a neutral condition are improved.
Description
Technical Field
The invention belongs to the technical field of skin care products, and particularly relates to a collagen composite solution for injection and a preparation method thereof.
Background
With the economic development and the increasing living standard, the pursuit of the beauty of the people is gradually increased. People who love beauty are eager to seek an efficient, safe and comfortable way to improve the skin condition of the face, reduce wrinkles and realize facial rejuvenation. The injection beauty treatment is to utilize the injection method to position, fix the layer and quantitatively deliver the nutrient to the required part of the skin, thus solving the defect that the traditional beauty treatment nutrient composition can only permeate into the cuticle of the epidermis and has insignificant effect. The advantages of convenient operation, no obvious discomfort, quick effect and the like are favored by the masses of beauty-loving people.
At present, products for injection beauty treatment on the market mainly comprise hyaluronic acid and various nutrient substances. Hyaluronic acid is a recognized moisturizing material, and has the effects of moisturizing, locking water and storing water. However, as a polysaccharide in extracellular matrix, after being injected into dermis, the polysaccharide is rapidly degraded by human body and cannot maintain the injection effect for a long time; the cross-linked hyaluronic acid on the market has the problems of incomplete degradation and the like, and can bring negative effects to the skin. In addition, the sources of various composite nutrient substances are complex, and potential safety hazards exist after the composite nutrient substances are injected into the skin.
Collagen is the most prominent structural protein in the body. The special triple-helix structure of the collagen endows the collagen with excellent mechanical property, low antigenicity and good biocompatibility. The natural collagen can be used as a scaffold for cell growth and attachment after being injected into the skin, so as to induce the proliferation of fibroblasts, accelerate the repair of the skin at the mouth of the beauty injection needle and play a role in improving the state of the facial skin. Meanwhile, collagen as a natural biological macromolecule can load micromolecular nutrient substances, and the micromolecular nutrient substances are slowly released along with the degradation process of the collagen to nourish the skin. However, most small molecule additives disrupt the hydration layer around the collagen molecule under neutral conditions resulting in less soluble collagen precipitation or structural denaturation. In order to solve the problem of solubility of the collagen complex solution under neutral conditions, chinese patent application 20111313770.8 discloses a method for preparing an acylated type I collagen hydro-acupuncture, wherein the acylated collagen is collagen modified by succinic anhydride or phthalic anhydride. According to the invention, the collagen is subjected to acylation modification and then is compounded with hyaluronic acid, so that the problem of poor water solubility of natural collagen is solved. However, the collagen molecule after cross-linking modification has greatly reduced bioactivity due to the steric hindrance of the side chain modification group; and the residual cross-linking agent in the modification process also has great potential safety hazard. At present, no composite collagen solution product for injection, which takes natural collagen as a main raw material, exists.
Disclosure of Invention
The invention mainly aims to provide a collagen composite solution for injection and a preparation method thereof, and aims to solve the technical problem of low bioactivity of the existing collagen solution for injection beauty after crosslinking modification.
In order to achieve the purpose, the collagen composite solution for injection provided by the invention comprises the following components in percentage by mass: 0.1-0.6wt% of active collagen, 0.15-0.35wt% of acid-base amino acid, 0.0005-0.2wt% of compound nutrient, 0.01-0.2wt% of collagen stabilizing agent, 0.8-1wt% of sodium chloride and the balance of water.
Preferably, the acidic and basic amino acid comprises one or more of aspartic acid, glutamic acid, lysine, arginine and histidine.
Preferably, the composite nutrient substance comprises one or more of vitamins, small molecule peptides, phospholipids, beta-mannitol, chondroitin sulfate and lipoic acid.
Preferably, the collagen stabilizer comprises one or more of non-polar amino acids or short peptides thereof, trehalose, urea, gelatin, phosphate salts, quaternary ammonium salts.
Preferably, the collagen stabilizing agent comprises one or more of a non-polar amino acid or a short peptide thereof and urea.
Preferably, the collagen stabilizer is a non-polar amino acid or a short peptide thereof.
Preferably, the non-polar amino acid comprises one or more of proline, glycine, alanine, leucine, and phenylalanine.
Preferably, the composition comprises the following components in percentage by mass: 0.4wt% of active collagen, 0.1wt% of lysine, 0.15wt% of arginine, 0.15wt% of carnosine and vitamin B 2 0.0005wt%, proline 0.02wt%, sodium chloride 0.9wt%, and water for the rest.
Preferably, the composition comprises the following components in percentage by mass: 0.6wt% of active collagen, 0.12wt% of glutamic acid, 0.12wt% of histidine, 0.001wt% of vitamin C, 0.1wt% of urea, 0.05wt% of glycine, 0.9wt% of sodium chloride and the balance of water.
Preferably, the active collagen has a molecular weight of 300 kDa.
The invention also provides a preparation method of the collagen composite solution for injection, which comprises the following steps:
s1, adding sodium chloride into the collagen solution, and stirring at low temperature to obtain collagen solution;
s2, slowly adding acidic and basic amino acids into the collagen dissolving solution, stirring in a low-temperature environment, and raising the pH value to be neutral to obtain a collagen neutralizing solution;
s3, synchronously adding composite nutrient substances and a collagen stabilizer into the collagen neutralizing solution, and uniformly stirring at low temperature and in a dark environment to obtain a collagen composite solution;
s4, filling the collagen composite solution into a pre-filled and sealed injector under the conditions of negative pressure and sterility to obtain the collagen composite solution for injection.
Preferably, the pH of the collagen neutralizing solution is 7 to 7.4.
According to the technical scheme, on the basis of maintaining the structure of natural collagen molecules, the pH value of the system is controlled by special pH amino acid, and a collagen stabilizer is introduced to improve the hydrophilicity of the collagen molecules, so that the solubility and the stability of the collagen molecules in the compounding process of micromolecular nutrients under a neutral condition are improved.
Drawings
FIG. 1 is a schematic flow chart of an embodiment of a method for preparing a collagen composite solution for injection according to the present invention;
FIG. 2 is a graph showing the effect of the pushing force of the composite collagen solution for injection in example 1 of the present invention;
FIG. 3 is a graph showing the effect of the pushing force of the composite collagen solution for injection in example 4 of the present invention;
FIG. 4 is a graph showing the effect of the pushing force of the composite collagen solution for injection in comparative example 1 of the present invention;
FIG. 5 is a graph showing the effect of the pushing force of the composite collagen solution for injection in comparative example 2 of the present invention;
FIG. 6 is a graph comparing the thermal denaturation temperatures of the composite collagen solutions for injection in example 1 of the present invention and comparative example 1;
FIG. 7 is a graph comparing the results of electrophoresis of the composite collagen solutions for injection in example 1 of the present invention and comparative example 1.
Detailed Description
The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
For a better description and illustration of embodiments of the application, reference may be made to one or more of the drawings, but additional details or examples used in describing the drawings should not be construed as limiting the scope of any of the inventive concepts of the present application, the presently described embodiments, or the preferred versions.
In the description of the present invention, it should be noted that the terms "length", "width", "upper", "lower", "front", "rear", "left", "right", "top", "bottom", "inner", "outer", etc. indicate orientations or positional relationships based on the positional relationships shown in the drawings, and are only for convenience of describing the present invention, but do not indicate that the device referred to must have a specific orientation or be operated in a specific orientation, and thus, should not be construed as limiting the present invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the description of the present application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.
Collagen is a natural biological macromolecule, can load micromolecular nutrient substances, is slowly released along with the degradation process of the collagen, and nourishes the skin. However, most small molecule additives disrupt the hydration layer around the collagen molecule under neutral conditions resulting in less soluble collagen precipitation or structural denaturation.
In view of the above, the invention provides a collagen composite solution for injection, which comprises the following components in percentage by mass: 0.1-0.6wt% of active collagen, 0.15-0.35wt% of acid-base amino acid, 0.0005-0.2wt% of compound nutrient, 0.01-0.2wt% of collagen stabilizer, 0.8-1wt% of sodium chloride and the balance of water. Wherein, the sodium chloride is used for adjusting the concentration of the solution to be consistent with the osmotic pressure under physiological conditions, and PBS buffer solution can also be selected. It should be noted that all the raw materials in the invention are medicinal grade, and all the additives are dissolved by injection water and filtered twice by 0.22 μm organic filter membrane. The compound nutrient substances are various nutrient substances commonly used in injection beauty products, and the collagen stabilizer is used for improving the hydrophilicity and stability of collagen.
According to the technical scheme, on the basis of maintaining the structure of a natural collagen molecule, the pH value of the system is controlled by special pH amino acid, and a collagen stabilizer is introduced to improve the hydrophilicity of the collagen molecule, so that the solubility and the stability of the collagen molecule in the compounding process with micromolecular nutrients under a neutral condition are improved.
In order to keep the concentration of the normal saline of the system unchanged in the pH adjusting process, the pH value of the system is controlled by selecting the pH amino acid as a pH adjusting agent. The acidic and basic amino acids includeOne or more of aspartic acid, glutamic acid, lysine, arginine and histidine. It should be noted that amino acids have an amino group and a carboxyl group bonded to the same carbon atom, the amino group exhibits basicity and the carboxyl group exhibits acidity, and the amino acids differ in basicity and basicity, mainly depending on the structure and polarity of the side chain R group of the amino acid. Usually based on the amino group (-NH) contained in the amino acid molecule 2 ) And the number of carboxyl groups (-COOH), which are classified into neutral, acidic and basic amino acids. In addition, the invention selects the acidic and basic amino acid to adjust the system to be neutral, can maintain the stability of the concentration of the physiological saline of the system, and simultaneously has the effects of adjusting the moisture of the skin, balancing the grease, removing the excessive free radicals of skin cells, effectively delaying the skin aging and the like.
Further, the compound nutrient substance comprises one or more of vitamins, small molecular peptides, phospholipids, beta-mannitol, chondroitin sulfate and lipoic acid. In this embodiment, the small molecule nutrients added are all pharmaceutical grade, and can be loaded among the collagen molecular network structures and released along with the degradation of collagen. Wherein the small molecule peptide comprises carnosine and glutathione.
In order to improve the stability of the system after the small molecular nutrient substances are added into the collagen solution, the collagen stabilizer is synchronously added when the nutrient substances are added. The collagen stabilizer comprises one or more of nonpolar amino acid or short peptide thereof, trehalose, urea, gelatin, phosphate and quaternary ammonium salt. The small molecular substances can be uniformly dispersed around collagen molecules after being dissolved in a solvent system, so that the hydrophilicity and the stability of the collagen are improved, and the collagen is dissolved favorably. Preferably, the collagen stabilizer is a non-polar amino acid or a short peptide thereof and urea.
Wherein the nonpolar amino acid comprises one or more of proline, glycine, alanine, leucine and phenylalanine.
Preferably, the composition comprises the following components in percentage by mass: 0.4wt% of active collagen, 0.1wt% of lysine, 0.15wt% of arginine, 0.15wt% of carnosine and vitamin B 2 0.0005wt% proline 0.02wt%0.9wt% of sodium chloride and the balance of water.
Preferably, the composition comprises the following components in percentage by mass: 0.6wt% of active collagen, 0.12wt% of glutamic acid, 0.12wt% of histidine, 0.001wt% of vitamin C, 0.1wt% of urea, 0.05wt% of glycine, 0.9wt% of sodium chloride and the balance of water.
Preferably, the active collagen has a molecular weight of 300 kDa.
The present invention also provides a preparation method of the collagen composite solution for injection, please refer to fig. 1, fig. 1 is an embodiment of the preparation method of the collagen composite solution for injection, which includes the following steps:
and S1, adding sodium chloride into the collagen solution, and stirring at low temperature to obtain a collagen dissolving solution.
Specifically, the collagen is obtained after the extraction and purification of animal tissues and is dissolved in an acidic solution, then the pharmaceutical grade sodium chloride is added into the acidic collagen solution to ensure that the concentration of the sodium chloride in the system reaches 0.9 percent of the concentration of normal saline, and the collagen is completely dissolved by stirring and dissolving at the temperature of 4-15 ℃ to obtain a collagen dissolving solution. In this example, all additives were dissolved in water for injection and then filtered twice through a 0.22 μm organic filter; all vessels used were oven dried at 180 ℃ for 4h to remove endotoxin. In addition, the added micromolecular nutrient substances are all medicinal grade, and the safety is high.
Among them, the reason why collagen is dissolved in an acidic solution is that collagen is most soluble under acidic conditions, and a uniform solution can be obtained at the fastest speed.
And S2, slowly adding the acidic and basic amino acids into the collagen dissolving solution, stirring the solution in a low-temperature environment, and raising the pH value to be neutral to obtain the collagen neutralizing solution.
In specific implementation, the basic amino acid is slowly added into the collagen neutralizing solution to increase the pH value of the system to 7-7.4, and the solution temperature is controlled below 4 ℃ to be uniformly stirred to obtain the collagen neutralizing solution. The pH amino acid is selected to adjust the system to be neutral, so that the stability of the concentration of the physiological saline of the system can be maintained, and the pH amino acid has the effects of adjusting skin moisture, balancing grease, removing excessive free radicals of skin cells, effectively delaying skin aging and the like. If the pH is adjusted by using conventional hydrochloric acid or sodium hydroxide solution, additional salt is generated by neutralization, so that the concentration of the physiological saline of the system is changed.
And S3, synchronously adding composite nutrient substances and a collagen stabilizer into the collagen neutralizing solution, and uniformly stirring at low temperature and in a dark environment to obtain the collagen complexing solution.
In the specific implementation, the stirring time is 0.5-2 hours, and the stirring temperature is 4-10 ℃. The collagen stabilizer comprises nonpolar amino acid or its short peptide (such as proline, glycine, alanine, leucine, phenylalanine, etc.), urea, trehalose, gelatin, phosphate, quaternary ammonium salt, etc. Among them, collagen has an isoelectric point close to neutral, so that the collagen solubility is relatively low under neutral conditions; therefore, during the pH up-regulation, a collagen stabilizer needs to be added to improve the stability and solubility of collagen under neutral conditions.
S4, filling the collagen composite solution into a pre-filled and sealed injector under the conditions of negative pressure and sterility to obtain the collagen composite solution for injection.
Preferably, the pH of the collagen neutralizing solution is 7 to 7.4.
In the technical scheme of the invention, compared with the modified collagen, the prepared composite collagen solution for injection has thick jelly-like appearance; compared with the collagen injection sold in the market for filling, the collagen injection can be stably stored for two years in the environment below 15 ℃, the transparent appearance and the fluidity of the solution are kept, the extrusion force is at the pushing speed of 20mm/min, and the extrusion force can be kept below 15N by adopting a 30G needle. In addition, the collagen composite solution for injection prepared by the invention has osmotic pressure regulated by a sodium chloride or phosphate regulating system, endotoxin is less than 20 EU/piece, and the solution is sterile and can be directly used for human tissue injection.
The technical solutions of the present invention are further described in detail with reference to the following specific examples, which should be understood as merely illustrative and not limitative.
Example 1
9g of sodium chloride is weighed into 1L of acidic collagen solution with the concentration of 4mg/mL, and the solution is stirred for 2h at the temperature of below 10 ℃ until the solution is completely dissolved. Slowly adding lysine and arginine into the completely dissolved solution to increase the pH of the system to neutral, and stirring at 3 deg.C for 1 h. Adding proline, carnosine and vitamin B into the neutralized solution in sequence 2 And uniformly stirring for 4 hours in a dark state to obtain a composite collagen solution, wherein proline is added firstly to ensure that the instability of a collagen system cannot be caused by the addition of subsequent nutrient substances. And finally, under the conditions of negative pressure and sterility, filling the prepared composite collagen solution into a 3mL pre-filled syringe to obtain the composite collagen solution for injection.
Wherein the collagen composite solution for injection comprises 0.4wt% of active collagen with the molecular weight of 300kDa, 0.15wt% of arginine, 0.1wt% of lysine, 0.15wt% of carnosine and vitamin B in percentage by mass 2 0.0005wt%, proline 0.02wt%, sodium chloride 0.9wt% and water for the rest, wherein the collagen solution is prepared by extracting from cow hide, purifying, filtering, and sterilizing.
Example 2
9g of sodium chloride is weighed into 1L of acidic collagen solution with the concentration of 6mg/mL, and the solution is stirred for 2h at the temperature of below 10 ℃ until the solution is completely dissolved. Slowly adding lysine and arginine into the completely dissolved solution to increase the pH of the system to neutral, and stirring at 2 deg.C for 1 h. Adding proline, carnosine and vitamin B into the neutralized solution in sequence 2 Uniformly stirring for 4 hours in a dark state to obtain a composite collagen solution; the proline is added firstly, so that the instability of a collagen system cannot be caused by the addition of subsequent nutrients. And finally, under the conditions of negative pressure and sterility, filling the prepared composite collagen solution into a 3mL pre-filled syringe to obtain the composite collagen solution for injection.
Wherein the collagen composite solution for injection comprises 0.6wt% of active collagen with the molecular weight of 300kDa, 0.15wt% of arginine, 0.1wt% of lysine, 0.15wt% of carnosine and vitamin B in percentage by mass 2 0.0005wt%, proline 0.02wt%, sodium chloride 0.9wt%, and water in balance, wherein the collagen solution is extracted from cow hide and purifiedAnd (4) melting, filtering and sterilizing to obtain the product.
Example 3
9g of sodium chloride is weighed into 1L of acidic collagen solution with the concentration of 1mg/mL, and the solution is stirred for 2h at the temperature of below 10 ℃ until the solution is completely dissolved. Slowly adding lysine and arginine into the completely dissolved solution to increase the pH of the system to neutral, and stirring at 2 deg.C for 1 h. Adding proline, carnosine and vitamin B into the neutralized solution in sequence 2 Uniformly stirring for 4 hours in a dark state to obtain a composite collagen solution; the proline is added firstly, so that the instability of a collagen system cannot be caused by the addition of subsequent nutrients. And finally, under the conditions of negative pressure and sterility, filling the prepared composite collagen solution into a 3mL pre-filled syringe to obtain the composite collagen solution for injection.
Wherein, the collagen composite solution for injection comprises the following components in percentage by mass: 0.1wt% of active collagen with molecular weight of 300kDa, 0.15wt% of arginine, 0.1wt% of lysine, 0.15wt% of carnosine, vitamin B 2 0.0005wt%, proline 0.02wt%, sodium chloride 0.9wt% and water for the rest, wherein the active collagen is prepared by extracting from cow hide, purifying, filtering, and sterilizing.
Example 4
9g of sodium chloride was weighed and dissolved in 1L of water for injection, then 0.01M of phosphate was added to the solution to prepare a buffer solution system, glutamic acid was slowly added to the solution to lower the pH of the system to 3 or less, and the prepared solvent system was stirred for 0.5 hour and then filtered twice with a 0.22 μ M organic filter. Weighing 6g of lyophilized active collagen, dissolving in the above acidic solvent system, and slowly stirring at below 10 deg.C for 2 hr until collagen is completely dissolved. Slowly adding histidine into the completely dissolved acidic collagen solution to raise the pH of the system to neutral, sequentially adding glycine, urea and vitamin C into the neutral collagen solution system, maintaining the pH to be neutral by adopting histidine and glutamic acid, and uniformly stirring for 4 hours in a dark state to obtain a composite collagen solution; the glycine and the urea are added firstly, so that the instability of a collagen system cannot be caused by the addition of subsequent nutrient substances. And finally, under the conditions of negative pressure and sterility, filling the prepared composite collagen solution into a 3mL pre-filled syringe to obtain the composite collagen solution for injection.
Wherein, the collagen composite solution for injection comprises the following components in percentage by mass: 0.6wt% of active collagen with the molecular weight of 300kDa, 0.12wt% of glutamic acid, 0.12wt% of histidine, 0.05wt% of glycine, 0.1wt% of urea, 0.001wt% of vitamin C, 0.9wt% of sodium chloride and the balance of water, wherein the active collagen is prepared by extracting from cow leather, purifying, filtering, sterilizing and freeze-drying.
Example 5
9g of sodium chloride was weighed and dissolved in 1L of water for injection, then 0.01M of phosphate was added to the solution to prepare a buffer solution system, glutamic acid was slowly added to the solution to lower the pH of the system to 3 or less, and the prepared solvent system was stirred for 0.5 hour and then filtered twice with a 0.22 μ M organic filter. Weighing 4g of lyophilized active collagen, dissolving in the above acidic solvent system, and slowly stirring at below 10 deg.C for 2 hr until collagen is completely dissolved. Slowly adding histidine into the completely dissolved acidic collagen solution to raise the pH of the system to neutral, sequentially adding glycine, urea and vitamin C into the neutral collagen solution system, maintaining the pH to be neutral by adopting histidine and glutamic acid, and uniformly stirring for 4 hours in a dark state to obtain a composite collagen solution; the glycine and the urea are added firstly, so that the instability of a collagen system cannot be caused by the addition of subsequent nutrient substances. And finally, under the conditions of negative pressure and sterility, filling the prepared composite collagen solution into a 3mL pre-filled syringe to obtain the composite collagen solution for injection.
Wherein, the collagen composite solution for injection comprises the following components in percentage by mass: 0.4wt% of active collagen with the molecular weight of 300kDa, 0.12wt% of glutamic acid, 0.12wt% of histidine, 0.05wt% of glycine, 0.1wt% of urea, 0.001wt% of vitamin C, 0.9wt% of sodium chloride and the balance of water, wherein the active collagen is prepared by extracting from cow leather, purifying, filtering, sterilizing and freeze-drying.
Example 6
9g of sodium chloride was weighed and dissolved in 1L of water for injection, then 0.01M of phosphate was added to the solution to prepare a buffer solution system, glutamic acid was slowly added to the solution to lower the pH of the system to 3 or less, and the prepared solvent system was stirred for 0.5 hour and then filtered twice with a 0.22 μ M organic filter. Weighing 1g of lyophilized active collagen, dissolving in the above acidic solvent system, and slowly stirring at below 10 deg.C for 2 hr until collagen is completely dissolved. Slowly adding histidine into the completely dissolved acidic collagen solution to raise the pH of the system to neutral, sequentially adding glycine, urea and vitamin C into the neutral collagen solution system, maintaining the pH to be neutral by adopting histidine and glutamic acid, and uniformly stirring for 4 hours in a dark state to obtain a composite collagen solution; the glycine and the urea are added firstly, so that the instability of a collagen system cannot be caused by the addition of subsequent nutrient substances. And finally, under the conditions of negative pressure and sterility, filling the prepared composite collagen solution into a 3mL pre-filled syringe to obtain the composite collagen solution for injection.
Wherein, the collagen composite solution for injection comprises the following components in percentage by mass: 0.1wt% of active collagen with the molecular weight of 300kDa, 0.12wt% of glutamic acid, 0.12wt% of histidine, 0.05wt% of glycine, 0.1wt% of urea, 0.001wt% of vitamin C, 0.9wt% of sodium chloride and the balance of water, wherein the active collagen is prepared by extracting from cow leather, purifying, filtering, sterilizing and freeze-drying.
Comparative example 1
9g of sodium chloride is weighed into 1L of acidic collagen solution with the concentration of 4mg/mL, and the solution is stirred for 2h at the temperature of below 10 ℃ until the solution is completely dissolved. Slowly adding lysine and arginine into the completely dissolved solution to increase the pH of the system to neutral, and stirring at 3 deg.C for 1 h. And adding carnosine and vitamin B2 into the neutralized solution in sequence, and uniformly stirring for 4 hours in a dark state to obtain a composite collagen solution. And finally, under the conditions of negative pressure and sterility, filling the prepared composite collagen solution into a 3mL pre-filled syringe to obtain the composite collagen solution for injection.
The collagen composite solution for injection comprises, by mass, 0.4wt% of active collagen with the molecular weight of 300kDa, 0.15wt% of arginine, 0.1wt% of lysine, 0.15wt% of carnosine, 0.0005wt% of vitamin B, 0.9wt% of sodium chloride and the balance of water, and is prepared by extracting, purifying, filtering and sterilizing cow hide.
Comparative example 2
9g of sodium chloride is weighed into 1L of acidic collagen solution with the concentration of 10mg/mL, and the solution is stirred for 2h at the temperature of below 10 ℃ until the solution is completely dissolved. Slowly adding lysine and arginine into the completely dissolved solution to increase the pH of the system to neutral, and stirring at 3 deg.C for 1 h. And sequentially adding proline, carnosine and vitamin B2 into the neutralized solution, uniformly stirring for 4 hours in a dark state to obtain a composite collagen solution, and adding proline first to ensure that the subsequent nutrient substances are not added to cause instability of a collagen system. And finally, under the conditions of negative pressure and sterility, filling the prepared composite collagen solution into a 3mL pre-filled syringe to obtain the composite collagen solution for injection.
The collagen composite solution for injection comprises, by mass, 1.0wt% of active collagen with the molecular weight of 300kDa, 0.15wt% of arginine, 0.1wt% of lysine, 0.15wt% of carnosine, 0.0005wt% of vitamin B, 0.02wt% of proline, 0.9wt% of sodium chloride and the balance of water, and is prepared by extracting, purifying, filtering and sterilizing cow leather.
It should be noted that the procedure of comparative example 1 is slightly different from that of example 1, and proline is not added as a stabilizer of the system in comparative example 1; the procedure of comparative example 2 was slightly different from that of example 1, and the final collagen concentration was 10 mg/ml.
Pushing force test
After 1 to 4 groups of samples prepared in examples 1 and 4 and comparative examples 1 and 2, respectively, were centrifuged to remove bubbles, the resulting samples were filled in a 3mL syringe, and the pushing force of the samples at a pushing speed of 20mm/min was measured by a tensile machine using a 30G needle (equipped with a kit for measuring pushing force). The 5 second time in the sample testing process is intercepted as an object to be inspected, the change curve of the sample extrusion force in the extrusion process is drawn, and the extrusion force results of 4 groups of samples are shown in the following figures 2-5.
As a result, the average extrusion force of the group 1 sample was 5.18N, and the maximum extrusion force was 6.95N; the average extrusion force of the group 2 sample was 7.82N, and the maximum extrusion force was 8.52N;
the two groups of samples meet the requirements that the maximum extrusion force is below 20N and the average extrusion force is below 15N.
The group 3 samples had an average push force of 21.2N and a maximum push force of 44.83N. From the curve results, there was a particulate precipitate inside the sample that caused clogging of the pores, resulting in clogging of the pores during injection. The reason is mainly because the solubility of collagen molecules is changed and precipitated without adding a collagen stabilizer in the sample preparation process. The group 4 samples had an average push force of 18.64N and a maximum push force of 42.6N. The sample is mainly characterized in that the collagen concentration is 10mg/ml, and the excessive collagen concentration can cause the system to be uneven in the dissolving process and separate out in the storage process, so that the sample extrusion force has obvious fluctuation.
Since the group 3 samples are not added with the collagen stabilizer during the preparation process and may affect the triple-helical structure of the collagen during the sample preparation process, the thermal denaturation temperature and the electrophoresis result of the two groups of samples are measured as compared with the group 1 samples, and the results are shown in fig. 6-7:
from the DSC results, the group 1 sample denaturation temperature was about 39.7 ℃, consistent with the literature reported denaturation temperature for bovine dermal collagen; while the denaturation temperature of the group 3 samples was reduced to 34.1%. This is due to the fact that no collagen stabilizer was added during the preparation process.
From the electrophoresis results, the group 1 sample showed a triple helix structure band characteristic to collagen, with two α 1 chains and 1 α 2 chain at about 100kDa, and the total molecular weight was about 300 kDa. While the group 3 samples have a large number of small molecule bands below 100kDa, indicating that more collagen molecules in the samples are denatured and the chain structure is broken. This is due to the fact that the group 3 samples were not added with collagen stabilizer during the preparation process.
The contrast experiment can show that the solubility and the stability of the injection composite collagen solution added with the collagen stabilizer are greatly improved within a reasonable collagen concentration range.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present application, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the concept of the present application, which falls within the scope of protection of the present application. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. The collagen composite solution for injection is characterized by comprising the following components in percentage by mass: 0.1-0.6wt% of active collagen, 0.15-0.35wt% of acid-base amino acid, 0.0005-0.2wt% of compound nutrient, 0.01-0.2wt% of collagen stabilizer, 0.8-1wt% of sodium chloride and the balance of water.
2. The injectable collagen complex solution of claim 1, wherein said acidic and basic amino acids comprise one or more of aspartic acid, glutamic acid, lysine, arginine, and histidine.
3. The injectable collagen complex solution of claim 2, wherein said complex nutrients comprise one or more of vitamins, small peptides, phospholipids, β -mannitol, chondroitin sulfate, lipoic acid.
4. The injectable collagen complex solution of claim 3, wherein the collagen stabilizer comprises one or more of a non-polar amino acid or a short peptide thereof, trehalose, urea, gelatin, phosphate, quaternary ammonium salt.
5. The injectable collagen complex solution of claim 4, wherein said collagen stabilizing agent comprises one or more of a non-polar amino acid or a short peptide thereof and urea.
6. The injectable collagen complex solution of claim 4, wherein said non-polar amino acids comprise one or more of proline, glycine, alanine, leucine, phenylalanine.
7. The injectable collagen composite solution according to claim 4, comprising the following components in percentage by mass: 0.4wt% of active collagen, 0.1wt% of lysine, 0.15wt% of arginine, 0.15wt% of carnosine and vitamin B 2 0.0005wt%, proline 0.02wt%, sodium chloride 0.9wt%, and water for the rest.
8. The injectable collagen complex solution of claim 1, wherein said active collagen has a molecular weight of 300 kDa.
9. A method for preparing the collagen complex solution for injection according to any one of claims 1 to 8, comprising the steps of:
s1, adding sodium chloride into the collagen solution, and stirring at low temperature to obtain a collagen solution;
s2, slowly adding acid and basic amino acids into the collagen dissolving solution, stirring in a low-temperature environment, and raising the pH value to be neutral to obtain a collagen neutralizing solution;
s3, synchronously adding composite nutrient substances and a collagen stabilizer into the collagen neutralizing solution, and uniformly stirring at low temperature and in a dark environment to obtain a collagen composite solution;
s4, filling the collagen composite solution into a pre-filled and sealed injector under the conditions of negative pressure and sterility to obtain the collagen composite solution for injection.
10. The method for preparing a collagen complex solution for injection according to claim 9, wherein the pH of the collagen neutralizing solution is 7 to 7.4.
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