CN111100200B - Fibronectin stabilizer and fibronectin preparation added with corresponding stabilizer - Google Patents
Fibronectin stabilizer and fibronectin preparation added with corresponding stabilizer Download PDFInfo
- Publication number
- CN111100200B CN111100200B CN201910841974.5A CN201910841974A CN111100200B CN 111100200 B CN111100200 B CN 111100200B CN 201910841974 A CN201910841974 A CN 201910841974A CN 111100200 B CN111100200 B CN 111100200B
- Authority
- CN
- China
- Prior art keywords
- fibronectin
- stabilizer
- preparation
- sodium alginate
- aqueous solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 102000016359 Fibronectins Human genes 0.000 title claims abstract description 182
- 108010067306 Fibronectins Proteins 0.000 title claims abstract description 182
- 238000002360 preparation method Methods 0.000 title claims abstract description 76
- 239000003381 stabilizer Substances 0.000 title claims abstract description 70
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 74
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical group CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims abstract description 49
- 235000010413 sodium alginate Nutrition 0.000 claims abstract description 49
- 239000000661 sodium alginate Substances 0.000 claims abstract description 49
- 229940005550 sodium alginate Drugs 0.000 claims abstract description 49
- 239000007864 aqueous solution Substances 0.000 claims abstract description 46
- 102000008186 Collagen Human genes 0.000 claims abstract description 40
- 108010035532 Collagen Proteins 0.000 claims abstract description 40
- 229920001436 collagen Polymers 0.000 claims abstract description 40
- 239000000413 hydrolysate Substances 0.000 claims abstract description 32
- 235000011187 glycerol Nutrition 0.000 claims abstract description 25
- 239000000203 mixture Substances 0.000 claims abstract description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 21
- 108010009736 Protein Hydrolysates Proteins 0.000 claims description 7
- 230000002776 aggregation Effects 0.000 abstract description 20
- 238000004220 aggregation Methods 0.000 abstract description 18
- 238000000034 method Methods 0.000 abstract description 14
- 239000006185 dispersion Substances 0.000 abstract description 12
- 102000004169 proteins and genes Human genes 0.000 abstract description 11
- 108090000623 proteins and genes Proteins 0.000 abstract description 11
- 230000009286 beneficial effect Effects 0.000 abstract description 5
- 238000013329 compounding Methods 0.000 abstract description 3
- 239000000243 solution Substances 0.000 description 40
- 239000000126 substance Substances 0.000 description 20
- 238000002474 experimental method Methods 0.000 description 16
- 238000002835 absorbance Methods 0.000 description 15
- 230000015271 coagulation Effects 0.000 description 13
- 238000005345 coagulation Methods 0.000 description 13
- 239000002244 precipitate Substances 0.000 description 12
- 230000008859 change Effects 0.000 description 10
- 230000000087 stabilizing effect Effects 0.000 description 10
- 238000003756 stirring Methods 0.000 description 10
- 238000003860 storage Methods 0.000 description 10
- 235000010418 carrageenan Nutrition 0.000 description 7
- 239000000679 carrageenan Substances 0.000 description 7
- 229920001525 carrageenan Polymers 0.000 description 7
- 229940113118 carrageenan Drugs 0.000 description 7
- 238000009472 formulation Methods 0.000 description 7
- 229920001223 polyethylene glycol Polymers 0.000 description 7
- UHVMMEOXYDMDKI-JKYCWFKZSA-L zinc;1-(5-cyanopyridin-2-yl)-3-[(1s,2s)-2-(6-fluoro-2-hydroxy-3-propanoylphenyl)cyclopropyl]urea;diacetate Chemical compound [Zn+2].CC([O-])=O.CC([O-])=O.CCC(=O)C1=CC=C(F)C([C@H]2[C@H](C2)NC(=O)NC=2N=CC(=CC=2)C#N)=C1O UHVMMEOXYDMDKI-JKYCWFKZSA-L 0.000 description 7
- 239000002202 Polyethylene glycol Substances 0.000 description 6
- 239000008367 deionised water Substances 0.000 description 6
- 229910021641 deionized water Inorganic materials 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 230000008569 process Effects 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- LUEWUZLMQUOBSB-FSKGGBMCSA-N (2s,3s,4s,5s,6r)-2-[(2r,3s,4r,5r,6s)-6-[(2r,3s,4r,5s,6s)-4,5-dihydroxy-2-(hydroxymethyl)-6-[(2r,4r,5s,6r)-4,5,6-trihydroxy-2-(hydroxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-4,5-dihydroxy-2-(hydroxymethyl)oxan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol Chemical compound O[C@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@@H](O[C@@H]2[C@H](O[C@@H](OC3[C@H](O[C@@H](O)[C@@H](O)[C@H]3O)CO)[C@@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O LUEWUZLMQUOBSB-FSKGGBMCSA-N 0.000 description 5
- 244000247812 Amorphophallus rivieri Species 0.000 description 5
- 235000001206 Amorphophallus rivieri Nutrition 0.000 description 5
- 229920002581 Glucomannan Polymers 0.000 description 5
- 235000010469 Glycine max Nutrition 0.000 description 5
- 244000068988 Glycine max Species 0.000 description 5
- 229920002752 Konjac Polymers 0.000 description 5
- 229940046240 glucomannan Drugs 0.000 description 5
- 150000004676 glycans Chemical class 0.000 description 5
- 239000000252 konjac Substances 0.000 description 5
- 235000010485 konjac Nutrition 0.000 description 5
- 229920001282 polysaccharide Polymers 0.000 description 5
- 239000005017 polysaccharide Substances 0.000 description 5
- 239000000654 additive Substances 0.000 description 4
- 230000007774 longterm Effects 0.000 description 4
- 239000011550 stock solution Substances 0.000 description 4
- 229920001503 Glucan Polymers 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000003796 beauty Effects 0.000 description 3
- 238000004925 denaturation Methods 0.000 description 3
- 230000036425 denaturation Effects 0.000 description 3
- 238000007710 freezing Methods 0.000 description 3
- 230000008014 freezing Effects 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 230000001954 sterilising effect Effects 0.000 description 3
- 229920002307 Dextran Polymers 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 239000004480 active ingredient Substances 0.000 description 2
- 238000005054 agglomeration Methods 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 238000007820 coagulation assay Methods 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 230000010355 oscillation Effects 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000007921 spray Substances 0.000 description 2
- 230000006641 stabilisation Effects 0.000 description 2
- 238000011105 stabilization Methods 0.000 description 2
- 238000010257 thawing Methods 0.000 description 2
- 238000002371 ultraviolet--visible spectrum Methods 0.000 description 2
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 238000011481 absorbance measurement Methods 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 210000004381 amniotic fluid Anatomy 0.000 description 1
- 238000000149 argon plasma sintering Methods 0.000 description 1
- 239000000022 bacteriostatic agent Substances 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- -1 plasma Substances 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 239000006076 specific stabilizer Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Cosmetics (AREA)
- Medicinal Preparation (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention belongs to the field of macromolecular protein application, and particularly discloses a fibronectin stabilizer and a fibronectin preparation added with the same. The effective component of the fibronectin stabilizer is sodium alginate or a mixture of sodium alginate and collagen hydrolysate or a mixture formed by compounding the sodium alginate, the collagen hydrolysate and glycerol. The fibronectin preparation comprises fibronectin and the fibronectin stabilizer. The fibronectin stabilizer provided by the invention can improve the aggregation stability and the dispersion stability of the fibronectin aqueous solution, and is beneficial to maintaining the stability of the fibronectin aqueous solution for a long time. The fibronectin preparation prepared by the invention has excellent aggregation stability and dispersion stability, and can be stored for a long time at room temperature; moreover, the application range is wide, and the method has important popularization and application values.
Description
Technical Field
The invention belongs to the application field of macromolecular proteins, and particularly relates to a plurality of fibronectin aqueous solution system stabilizers, and also relates to a fibronectin preparation added with corresponding stabilizers.
Background
Fibronectin (also known as Fibronectin, abbreviated as FN) is a macromolecular condensation glycoprotein with a molecular weight of 470-500kDa, and is widely present in animal tissues and tissue fluids, such as plasma, milk, amniotic fluid, saliva, etc. Fibronectin is a growth factor with important biological functions in animals and has a variety of biological properties due to its structural multiple domains. Currently, fibronectin has found wide application in the fields of cell culture, nerve repair, and clinical infusion. Wound dressings made from modified porcine plasma fibronectin have also been patented. Fibronectin also has great potential in the field of cosmetic skin care.
Fibronectin is generally prepared by isolation from plasma, which must be stored at-20 ℃ in order to prevent degradation of fibronectin, and all purification steps must be performed below 4 ℃. The separated fibronectin is dispersed in water, has certain dynamic stability, and can keep the solution stable in a certain time. However, the system is a thermodynamically unstable system in nature and is susceptible to coagulation in a short time due to a change in ambient temperature. For long-term storage and transportation, proteins are stored in lyophilized formulations in industrial production, and research on aggregation stability of ready-to-use fibronectin aqueous solutions is still blank, and reagents and systems for improving the stability of fibronectin aqueous solutions are lacking. For other common protein preparations, various additives have been developed in the market, which are also known to provide a stabilizing effect, but it must be noted that most additives are aimed at improving the protein's resistance to denaturation, not at preventing protein coagulation or improving its dispersion stability. Meanwhile, the additive has two functions, and can stabilize the protein and induce the structural change of the protein so as to generate coagulation. Therefore, the selection of additives for fibronectin, a specific protein preparation, requires extensive trial and error feedback loop experiments.
In the early exploratory experiment, the inventor of the patent prepares fibronectin solution with the concentrations of 0.1mg/mL, 0.5mg/mL, 1mg/mL, 1.5mg/mL and 2mg/mL respectively under the aseptic condition (a ten thousand-grade aseptic laboratory), hermetically places the solution at room temperature, and observes and records the change of the properties of the solution every day. Observations showed that the fibronectin aqueous solutions at different concentrations appeared cloudy on day 4 or 5, and that a cloudy precipitate was observed on day 7, which was visually discernable. The precipitated fibronectin is not easily redispersed, resulting in a reduction in the actual effective dose in the system. In the field of beauty and skin care, the existence of protein precipitates can affect the uniformity of coating and weaken the repairing effect of the coating. In addition, if the fibronectin aqueous solution is used as a spray, the flocculent precipitate is easy to block the spray head, which directly results in that the product cannot be used continuously. In view of these problems, there is a need in the art to develop a fibronectin preparation that has improved aggregation stability in an aqueous dispersion thereof.
Disclosure of Invention
In view of the problems and deficiencies of the prior art, it is an object of the present invention to provide a fibronectin stabilizer and a fibronectin preparation.
In order to realize the purpose of the invention, the technical scheme adopted by the invention is as follows:
the application of sodium alginate in preparing fibronectin stabilizer.
A fibronectin stabilizer comprises the following effective components in percentage by weight: 1-5 parts of sodium alginate and 1-5 parts of collagen hydrolysate. The collagen hydrolysates in the present invention are all calculated as solids.
According to the fibronectin stabilizer, the dissolving process before use is saved for further convenience of use, and the stabilizer is an aqueous solution formed by dispersing sodium alginate and collagen hydrolysate in water.
A fibronectin stabilizer comprises sodium alginate, collagen hydrolysate and glycerol as effective components.
According to the above fibronectin stabilizer, the effective components preferably comprise, by weight: 1-5 parts of sodium alginate, 1-5 parts of collagen hydrolysate and 10-50 parts of glycerol; the stabilizer is an aqueous solution formed by dispersing sodium alginate, collagen hydrolysate and glycerol into water.
A fibronectin preparation comprises fibronectin and sodium alginate.
According to the fibronectin preparation, in order to further greatly prolong the storage life, the preparation is freeze-dried powder, and the freeze-dried powder contains 1-5 parts of fibronectin and 1-5 parts of sodium alginate in parts by weight.
Preferably, the fibronectin preparation further comprises 1 to 5 parts of a collagen hydrolysate.
According to the fibronectin preparation, the preparation is preferably an aqueous solution preparation, the concentration of fibronectin in the aqueous solution is 0.4-4.0 mg/mL, and the concentration of sodium alginate is 1.0-5.0 mg/mL.
According to the fibronectin preparation, the aqueous solution preparation preferably further contains a collagen hydrolysate, and the concentration of the collagen hydrolysate in the aqueous solution preparation is preferably 1.0 to 5.0 mg/mL.
According to the fibronectin preparation, the aqueous solution preparation preferably further contains glycerol, and the concentration of the glycerol in the aqueous solution preparation is 10 to 50 mg/mL.
Preferably, the fibronectin preparation also comprises a pharmaceutically acceptable preservative bacteriostatic agent.
Preferably, the molecular weight of the collagen hydrolysate is 2000 to 100000 according to the fibronectin preparation.
According to the fibronectin preparation, the molecular weight of the collagen hydrolysate is preferably in a range of 2000 to 20000.
According to the fibronectin preparation, the molecular weight of the sodium alginate is preferably 2000-200000.
Compared with the prior art, the invention has the following positive beneficial effects:
(1) the invention adopts sodium alginate or a mixture of sodium alginate and collagen hydrolysate or a mixture of sodium alginate, collagen hydrolysate and glycerol as a stabilizer of fibronectin, and the three stabilizers can improve the aggregation stability and dispersion stability of fibronectin water solution, which is proved by high-temperature accelerated coagulation experiment and room-temperature storage experiment, and is beneficial to maintaining the stability of fibronectin water solution for a long time and realizing long-term storage of fibronectin water solution at room temperature; moreover, after the stabilizer provided by the invention is added into the fibronectin aqueous solution, even if a small amount of flocculent precipitate is generated after long-time storage, the precipitate is in a loose state, and the precipitate can be dispersed again by simple oscillation or stirring; the redispersed fibronectin aqueous solution is still uniformly and stably used and stored for a longer period of time.
(2) The components (sodium alginate, collagen hydrolysate and glycerol) in the fibronectin stabilizer interact with fibronectin through hydrogen bonds and electrostatic effect, so that irreversible denaturation of fibronectin is avoided, and the structure and performance of fibronectin are not obviously influenced; therefore, when the fibronectin solution added with the stabilizer is directly used or used for preparing other products, the stabilizer does not need to be separated and removed, namely the stabilizer provided by the invention does not influence the direct application of the fibronectin solution in the fields of beauty treatment, health care, biomedicine and the like, and the fibronectin stabilizer plays a positive role in promoting the research, development, production and application of fibronectin products.
(3) The fibronectin stabilizer has the advantages of wide raw material source, low price and simple preparation method, and the liquid stabilizer is prepared by dispersing the effective components of the stabilizer into water, so the liquid stabilizer can be directly mixed with the fibronectin solution in proportion when in use, the use is convenient, the dissolving process when in use is omitted, the possible problems of agglomeration, sedimentation, uneven dispersion and the like when the solid stabilizer component is directly added are also avoided, the use convenience of the stabilizer is greatly improved, the process of producing and storing fibronectin is not required to be improved, and the cost is saved.
(4) At present, the research on specific stabilizers of fibronectin or the successful formulation on the market is not abundant, and Kharrasasov and the like carry out some researches on the influence of plasma stabilizers on the performance of fibronectin in the storage process, but do not relate to the research on the solution of the stability of the aqueous solution of the plasma stabilizers; the invention utilizes sodium alginate or a mixture of sodium alginate and collagen hydrolysate or a mixture of sodium alginate, collagen hydrolysate and glycerol as effective components to develop a stabilizer which is beneficial to stable storage of fibronectin, and the stabilizer plays a positive role in improving the application range of fibronectin and reducing the storage cost while improving the aggregation stability of fibronectin. Therefore, the fibronectin stabilizer has wide application prospect and important popularization value.
(5) The fibronectin preparation prepared by the invention has excellent aggregation stability and dispersion stability, and can be stored for a long time at room temperature; moreover, even if a small amount of flocculent precipitate is generated after the fibronectin preparation is stored for a long time, the precipitate is in a loose state, and the precipitate can be dispersed again by simple oscillation or stirring; the re-dispersed fibronectin preparation can be used and stored uniformly and stably for a longer period of time.
(6) The fibronectin preparation prepared by the invention can be directly used without removing a stabilizer component in the preparation when in use, so the fibronectin preparation is convenient to use and wide in application range, and has important popularization and application values.
(7) Compared with the prior fibronectin solution which needs long-term freezing preservation, the fibronectin preparation prepared by the invention not only reduces the requirements on related freezing equipment, but also does not need to be repeatedly frozen and thawed, avoids protein denaturation possibly caused by repeated freezing and thawing, and is favorable for flexibly and conveniently applying fibronectin to the fields of beauty treatment, health care and biomedicine.
Drawings
FIG. 1 is a graph of the Δ Abs corresponding to various possible stabilizer concentrations in a fibronectin preparationmaxA histogram; wherein, (a) is collagen hydrolysate, (b) is carrageenan, (c) is soybean polysaccharide, (d) is sodium alginate, (e) is dextran, (f) is PEG, (g) is glycerol, and (h) is konjac glucomannan;
FIG. 2 is a graph of the UV-VIS absorption spectrum of a control fibronectin aqueous solution at 37 ℃ as a function of time;
FIG. 3 is a graph of the UV-VIS absorption spectrum of a fibronectin aqueous solution with sodium alginate added, as a function of time, when placed at 37 ℃;
fig. 4 is a photograph of the appearance of different fibronectin preparations stored at room temperature for 7 days, left: adding stabilizer sodium alginate (5 mg/mL); and (3) right: only deionized water was added (control).
Detailed Description
The present invention will be described in detail with reference to specific embodiments, but the scope of the present invention is not limited thereto.
Method for evaluating stabilization effect of fibronectin stabilizer
The influence of different substances on the aggregation stability of the fibronectin aqueous solution is evaluated by adopting a high-temperature accelerated coagulation experiment, the possibility of using the substances as a stabilizer is discussed, and a direct reference is provided for the room-temperature stabilizing effect of the substances. The specific method of the high-temperature accelerated coagulation experiment comprises the following steps:
(1) preparation of fibronectin aqueous solution: a fibronectin stock solution was prepared according to the method described in patent publication CN105950576, and the concentration of the fibronectin stock solution was adjusted to 0.5 to 5.0mg/mL by concentration or by adding deionized water, and the solution was stored at-20 ℃ for future use.
(2) Taking a fibronectin solution stored at the temperature of minus 20 ℃, thawing the fibronectin solution to be used as an experimental sample of a high-temperature accelerated coagulation experiment, setting an experimental group and a control group for the experiment, adding a corresponding substance possibly having a stable effect into the fibronectin solution of the experimental group, adding an equal amount of deionized water into the fibronectin solution of the control group, uniformly mixing, and respectively sterilizing the fibronectin solutions of the control group and the experimental group; then, an ultraviolet-visible spectrophotometer is adopted to respectively detect the absorbance values of the experimental group fibronectin white water solution and the control group fibronectin white water solution at the wavelength of 278nm, wherein the absorbance values are initial absorbance values of all groups and are recorded as Abs0。
(3) The fibronectin water solutions of the experimental group and the control group are respectively sealed in glass sample bottles and then are respectively placed in a constant-temperature cell culture box at 37 DEG CDetermination of the absorbance values at a wavelength of 278nm after the test and control fibronectin aqueous solutions have been left in the thermostated cell incubator for different times t (1d, 2d, 3d, 4d, 5d, 6d and 7d) (the glass vials were gently shaken before the determination) and recorded as Abst。
(4) According to the formula Δ Abs ═ Abst-Abs0(Δ Abs means absorbance change rate, and Δ Abs is positive value) the absorbance change rate was calculated for the experimental group sample and the control group sample at different times. In the long-time high-temperature accelerated coagulation experiment process, the precipitation caused by the aggregation of fibronectin influences the light scattering behavior, and the absorbance of a sample may be increased and then decreased, so that the maximum value (delta Abs) of the absorbance change rate after 7d of continuous measurement is takenmax) As a basis for assessing the stability of fibronectin aqueous solutions. Furthermore, since the fibronectin aqueous solution is liable to aggregate and precipitate after being left for a long time, and the fibronectin in the solution aggregates to form a large amount of flocculent or flaky precipitates and precipitates on the bottom of the sample bottle, the absorbance of the solution may be decreased even by a slight shaking before the absorbance measurement, and therefore, if Δ Abs is calculated to be a negative value according to the above formula, the result is not considered.
The change rate of absorbance of solution Δ Abs intuitively reflects the degree of fibronectin agglomeration and can be used as an index for evaluating the stability of fibronectin aqueous solutions. The more stable the fibronectin aqueous system, the smaller its Δ Abs, and vice versa the larger the value. The high-temperature accelerated coagulation experiment can compare the stabilizing effect of different substances on fibronectin aqueous solution, so that the stabilizer with better stabilizing effect can be screened out.
(di) fibronectin stabilizer discovery experiments
Example 1: single component stabilizer screening experiment
In the experiment, substances which can be used as stabilizers such as sodium alginate, collagen hydrolysate, carrageenan, soybean polysaccharide, glucan, polyethylene glycol, glycerol, konjac glucomannan and the like are taken as research objects respectively, and the influence of the substances on the aggregation stability of the fibronectin aqueous solution is evaluated through a high-temperature accelerated coagulation experiment so as to screen the stabilizer with good stabilizing effect.
The experimental process is as follows:
(1) preparation of fibronectin aqueous solution: a fibronectin stock solution was prepared according to the method described in patent publication CN105950576, and the prepared fibronectin stock solution was concentrated or deionized water was added to adjust the concentration of the fibronectin aqueous solution to 0.5mg/mL, and the solution was stored at-20 ℃ for further use.
(2) Preparing different substance solutions:
according to the physicochemical characteristics of various substances such as sodium alginate, collagen hydrolysate, carrageenan, soybean polysaccharide, glucan, polyethylene glycol, glycerol, konjac glucomannan and the like, the substances are respectively fully stirred and dissolved in deionized water at different temperatures (30-80 ℃) to prepare solutions with different concentrations of various substances (wherein the concentration range of the sodium alginate solution is 25 mg/mL-250 mg/mL, the concentration range of the collagen hydrolysate is 25 mg/mL-250 mg/mL, the concentration range of the carrageenan is 5 mg/mL-25 mg/mL, the concentration range of the soybean polysaccharide is 50 mg/mL-250 mg/mL, the concentration range of the glucan is 1 mg/mL-5 mg/mL, the concentration range of the polyethylene glycol is 250 mg/mL-1000 mg/mL, the concentration range of the glycerol is 250 mg/mL-1000 mg/mL, the concentration range of the konjac glucomannan is 5 mg/mL-25 mg/mL), and solutions with different concentrations of different substances are obtained and are used for preparing experimental fibronectin preparations.
(3) Preparation of fibronectin preparations: and (3) taking 10mL of each solution prepared in the step (2), adding the solution into 40mL of unfrozen fibronectin aqueous solution under slow stirring, continuously stirring for 10 minutes after the addition is finished, sterilizing, and sealing and storing to obtain the experimental fibronectin preparation.
And adding 10mL of deionized water into 40mL of unfrozen fibronectin aqueous solution under slow stirring, continuously stirring for 10 minutes after the addition is finished, sterilizing, and sealing for storage to obtain a control fibronectin preparation.
(4) Determining the delta Abs of each fibronectin preparation prepared in step (3) in a constant temperature cell incubator for different periods of time according to the procedure of the accelerated coagulation assay at high temperature as described abovemaxDetermining the optimal concentration of different substances (same substance as Δ Abs)maxMinimum corresponding concentration of the substance in the fibronectin preparationDegree is the optimal concentration of the substance), see fig. 1 for specific results; at the same time, by comparing the delta Abs of different substances at optimal concentrationsmaxAnd Δ Abs max, the stabilization effect of each substance on the fibronectin solution was analyzed, and the results are shown in table 1, fig. 2 to fig. 4.
TABLE 1 Effect of different substances on the stability of fibronectin solutions
FIG. 1 is a graph of the Δ Abs corresponding to different concentrations of various possible stabilizing substances (possible stabilizers) in a fibronectin preparation of the inventionmaxA histogram. As is clear from FIG. 1, the optimum concentration of sodium alginate was 5mg/mL, as evaluated from the absorbance point of view; for collagen hydrolysate, the optimal concentration is 5 mg/mL; for carrageenan, the optimal concentration is 1 mg/mL; for soybean polysaccharide, the optimal concentration is 10 mg/mL; for dextran, the optimal concentration is 0.4 mg/mL; for polyethylene glycol, the optimal concentration is 200 mg/mL; for glycerol, the optimal concentration is 50 mg/mL; for konjac glucomannan, the optimal concentration is 1 mg/mL.
Fig. 2 is a graph showing a change in absorbance with time of a control fibronectin preparation in a high-temperature accelerated coagulation experiment, and fig. 3 is a graph showing a change in absorbance with time of a fibronectin preparation to which sodium alginate was added in a high-temperature accelerated coagulation experiment (the concentration of sodium alginate in the fibronectin preparation is 5mg/mL), and it can be seen from fig. 2 and 3 that a fibronectin aqueous solution to which a proper amount of sodium alginate was added exhibited an excellent aggregation stability with a small change in absorbance after being left at 37 ℃ for 7 days, as compared with the control. As can also be seen in fig. 4, the fibronectin formulation with sodium alginate added was clear after 7 days at room temperature, with no visible floc; the solution of the control fibronectin preparation turns turbid after being stored for 7 days at room temperature, and obvious floccules appear in the solution, which shows that the sodium alginate has a good stabilizing effect on fibronectin, can improve the aggregation stability and the dispersion stability of the fibronectin solution, is beneficial to long-term storage of the fibronectin solution at room temperature, and can be used as a stabilizer of a fibronectin solution system.
As is clear from Table 1, the Δ Abs of each fibronectin preparation obtained by adding sodium alginate, collagen hydrolysate, carrageenan, polyethylene glycol and glycerin to each fibronectin preparation from the viewpoint of absorbance alonemaxAre all smaller and far smaller than the control group; however, in the fibronectin preparations containing carrageenan or polyethylene glycol, many loose pieces (i.e., large pieces formed by aggregation or aggregation of fibronectin) appeared in the fibronectin preparation when Δ Abs was maximized, indicating that a large amount of fibronectin was aggregated in the fibronectin preparation and the stability was poor, and therefore, Δ Abs was used to increase the amount of fibronectinmaxAnd the comprehensive analysis of two aspects of the appearance of the solution, the sodium alginate, the collagen hydrolysate and the glycerol have good stabilizing effect on the fibronectin, so the sodium alginate, the collagen hydrolysate and the glycerol are selected as the raw materials of the fibronectin stabilizer.
Example 2: preparation of fibronectin stabilizer by compounding sodium alginate and collagen hydrolysate
(1) Preparation of fibronectin stabilizer:
according to the dosage of the sodium alginate and the collagen hydrolysate in the examples 2-1 to 2-12 in the table 2, 1 to 5g of the sodium alginate and 1 to 5g of the collagen hydrolysate are fully dissolved in water to prepare 200mL of water solution, and the fibronectin stabilizer required by each example is obtained.
(2) Preparation of fibronectin preparations:
dissolving and unfreezing fibronectin frozen at-20 ℃ at the concentration of 0.5-5.0 mg/mL, and taking 40 mL; 10mL of the fibronectin stabilizer prepared in step (1) and corresponding to each example in Table 2 was added to the thawed fibronectin aqueous solution while stirring slowly, and after the addition, stirring was continued for 10 minutes, and the mixture was sterilized and stored in a sealed state.
(3) Determining the Δ Abs of the fibronectin preparation prepared in step (2) according to the procedure of the aforementioned high temperature accelerated coagulation assay, based on the Δ AbsmaxResults the stabilizing effect of the different stabilizers was evaluated. See table 2 for specific experimental results.
TABLE 2 stability analysis of two-component compositely prepared stabilizers
As is clear from Table 2, the Δ Abs of each of the fibronectin preparations prepared in examples 2-1 to 2-12maxThis is much smaller than the control, which indicates that each of the fibronectin formulations prepared in examples 2-1 to 2-12 of the present invention has excellent aggregation and dispersion stability, which means that the shelf life of these fibronectin formulations can be extended to a limited extent relative to the control. As is clear from examples 2 to 5 and examples 2 to 10 and examples 2 to 12, the Δ Abs of fibronectin was found to be significantly higher (from 0.4mg/mL to 4.0mg/mL) at the same stabilizer concentrationmaxAnd only slightly increased. These results fully demonstrate that stabilizers prepared with sodium alginate (1-5 parts by weight) and collagen hydrolysate (1-5 parts by weight) as the active ingredients both improve the aggregation stability and dispersion stability of fibronectin aqueous solutions, facilitating the fibronectin aqueous solutions to maintain their stability over a longer period of time.
Example 3: preparation of fibronectin stabilizer by compounding three components of sodium alginate, collagen hydrolysate and glycerol
(1) Preparation of fibronectin stabilizer:
according to the dosage of the sodium alginate, the collagen hydrolysate and the glycerol in the examples 3-1 to 3-25 in the table 3, 1-5 g of the sodium alginate, 1-5 g of the collagen hydrolysate and 10-50 g of the glycerol are respectively dispersed in water to prepare 200mL of aqueous solution, and the fibronectin stabilizer required by each example is obtained.
(2) Preparation of fibronectin preparations:
10mL of the fibronectin stabilizer prepared in step (1) and corresponding to each example in Table 2 was added to the thawed fibronectin aqueous solution while stirring slowly, and after the addition, stirring was continued for 10 minutes, and the mixture was sterilized and stored in a sealed state.
(3) Measuring the fibronectin preparation prepared in step (2) according to the procedure of the high temperature accelerated coagulation testΔ Abs of (A)maxAccording to Δ AbsmaxResults the stabilizing effect of the different stabilizers was evaluated. See table 3 for specific experimental results.
TABLE 3 stability analysis of three-component composite prepared stabilizers
As is clear from Table 3, the Δ Abs of each of the fibronectin preparations prepared in examples 3-1 to 3-28maxThis is much smaller than the control group, which indicates that each of the fibronectin formulations prepared in examples 3-1 to 3-28 of the present invention has excellent aggregation and dispersion stability, which means that the shelf life of these fibronectin formulations can be extended to a limited extent relative to the control group. As is clear from examples 3 to 7 and examples 3 to 26 to 3 to 28, the Δ Abs of the fibronectin concentration was significantly increased (from 0.4mg/mL to 4.0mg/mL) at the same stabilizer concentrationmaxAnd also does not vary much. These results fully demonstrate that stabilizers prepared with sodium alginate (1-5 parts by weight), collagen hydrolysate (1-5 parts by weight) and glycerol (10-50 parts by weight) as the active ingredients all improve the aggregation stability and dispersion stability of the fibronectin aqueous solution, facilitating the fibronectin aqueous solution to maintain its stability over a longer period of time.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the present invention, but rather as the following description is intended to cover all modifications, equivalents and improvements falling within the spirit and scope of the present invention.
Claims (4)
1. The use of a composition for the preparation of a fibronectin stabilizer, wherein the composition comprises, by weight: 1-5 parts of sodium alginate and 1-5 parts of collagen hydrolysate, wherein the fibronectin stabilizer is an aqueous solution formed by dispersing the sodium alginate and the collagen hydrolysate in water.
2. The use of a composition for the preparation of a fibronectin stabilizer, wherein the composition comprises, by weight: 1-5 parts of sodium alginate, 1-5 parts of collagen hydrolysate and 10-50 parts of glycerol; the fibronectin stabilizer is an aqueous solution formed by dispersing sodium alginate, collagen hydrolysate and glycerol into water.
3. A fibronectin preparation comprising fibronectin, sodium alginate, and a collagen hydrolysate; the fibronectin preparation is an aqueous solution preparation, the concentration of fibronectin in the aqueous solution preparation is 0.4-4.0 mg/mL, the concentration of sodium alginate is 1.0-5.0 mg/mL, and the concentration of collagen hydrolysate is 1.0-5.0 mg/mL.
4. The fibronectin preparation of claim 3, wherein the aqueous solution preparation further comprises glycerol, and the concentration of glycerol in the aqueous solution preparation is 10-50 mg/mL.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910841974.5A CN111100200B (en) | 2019-09-06 | 2019-09-06 | Fibronectin stabilizer and fibronectin preparation added with corresponding stabilizer |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910841974.5A CN111100200B (en) | 2019-09-06 | 2019-09-06 | Fibronectin stabilizer and fibronectin preparation added with corresponding stabilizer |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111100200A CN111100200A (en) | 2020-05-05 |
| CN111100200B true CN111100200B (en) | 2022-02-11 |
Family
ID=70421414
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910841974.5A Active CN111100200B (en) | 2019-09-06 | 2019-09-06 | Fibronectin stabilizer and fibronectin preparation added with corresponding stabilizer |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111100200B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113577378A (en) * | 2021-08-28 | 2021-11-02 | 上海纤瑞生物技术有限公司 | Fibronectin liquid wound spray dressing and preparation method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1923296A (en) * | 2006-09-15 | 2007-03-07 | 郑州福恩生物工程技术有限公司 | Fibronectin cicatrisation agent for wounds and ulcers |
| CN102250390A (en) * | 2011-05-25 | 2011-11-23 | 天津大学 | Alginate hydrogel microcarrier and preparation method thereof |
| WO2012027827A1 (en) * | 2010-08-30 | 2012-03-08 | Oral Delivery Technology Ltd. | Nitric oxide amino acid esters for skin rejuvenation |
-
2019
- 2019-09-06 CN CN201910841974.5A patent/CN111100200B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1923296A (en) * | 2006-09-15 | 2007-03-07 | 郑州福恩生物工程技术有限公司 | Fibronectin cicatrisation agent for wounds and ulcers |
| WO2012027827A1 (en) * | 2010-08-30 | 2012-03-08 | Oral Delivery Technology Ltd. | Nitric oxide amino acid esters for skin rejuvenation |
| CN102250390A (en) * | 2011-05-25 | 2011-11-23 | 天津大学 | Alginate hydrogel microcarrier and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| 明胶与海藻酸钠的相互作用及其应用;赵谋明等;《食品与发酵工业》;20000620;第26卷(第3期);第46页右栏第2段，第3.1-3.2节 * |
| 海藻酸钠对植物蛋白饮料稳定性影响研究;范素琴等;《粮食与油脂》;20090210(第2期);第2.1.1节，表1 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111100200A (en) | 2020-05-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Wan et al. | The formation and disaggregation of soy protein isolate fibril: Effects of pH | |
| CN110200287B (en) | Oyster polypeptide-nano selenium particle compound with effects of dispelling effects of alcohol and protecting liver as well as preparation method and application thereof | |
| Pokrowiecki et al. | Nanoparticles and human saliva: a step towards drug delivery systems for dental and craniofacial biomaterials | |
| CN111100200B (en) | Fibronectin stabilizer and fibronectin preparation added with corresponding stabilizer | |
| CN113367997A (en) | Preparation method of oil-in-water Pickering emulsion containing ethanol, product and application thereof | |
| Kim et al. | Thermo-responsive human α-elastin self-assembled nanoparticles for protein delivery | |
| CN105054073A (en) | Water-soluble vitamin D3 nanometer particles and preparation method thereof | |
| CN109054054A (en) | A kind of fibroin nanoparticles taking and preparation method thereof | |
| EP1711511B1 (en) | Method for crystallization of proteins using polysaccharides | |
| CN110810399B (en) | Cell transportation protective solution, preparation method and application | |
| CN116286371B (en) | Freeze-drying protective agent formula for improving freeze-drying effect of strain | |
| JP3100058B2 (en) | Stable pharmaceutical composition containing fibroblast growth factor | |
| CN115073768B (en) | Preparation method of functional component loaded double-network hydrogel | |
| CN106076193B (en) | Using ionic liquid as undenatured collagen base biological surfactant of reaction medium and preparation method thereof | |
| KR20210052451A (en) | Compositions for transport and/or cryopreservation of cells and/or tissues | |
| CN107362352B (en) | Protein or polypeptide composition and preparation method and application thereof | |
| CN115227597A (en) | Active collagen freeze-dried preparation and preparation method and application thereof | |
| CN115005199A (en) | Cryopreservation solution and cryopreservation method for natural killer cells and application of cryopreservation solution and cryopreservation method | |
| CN106310221A (en) | Pharmaceutical composition containing carfilzomib and preparation method thereof | |
| CN102145001A (en) | Stable aztreonam composition and preparation method thereof | |
| WO2004050121A1 (en) | Pharmaceutical compositions of cell lysate and processes for the production and use thereof | |
| US20230312763A1 (en) | Method for preparing chitin highly dispersible in aqueous phase and chitin prepared thereby | |
| CN114099648B (en) | Cosolvent, hemoglobin-oxygen carrier freeze-dried powder and injection containing cosolvent and preparation method of hemoglobin-oxygen carrier freeze-dried powder and injection | |
| CN103271879B (en) | Stable fructose composition preparation and preparation method thereof | |
| Ahmed et al. | Preparation of lysozyme enzyme coated with chitosan nanoparticles |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |