CN118480588A - Preparation method and application of unsaturated sodium hyaluronate disaccharide - Google Patents
Preparation method and application of unsaturated sodium hyaluronate disaccharide Download PDFInfo
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- CN118480588A CN118480588A CN202410812661.8A CN202410812661A CN118480588A CN 118480588 A CN118480588 A CN 118480588A CN 202410812661 A CN202410812661 A CN 202410812661A CN 118480588 A CN118480588 A CN 118480588A
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- sodium hyaluronate
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- -1 unsaturated sodium hyaluronate disaccharide Chemical class 0.000 title claims abstract description 94
- 238000002360 preparation method Methods 0.000 title claims abstract description 23
- 239000000047 product Substances 0.000 claims abstract description 82
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- 239000012528 membrane Substances 0.000 claims abstract description 32
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- 229940010747 sodium hyaluronate Drugs 0.000 claims abstract description 30
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- 238000000034 method Methods 0.000 claims abstract description 29
- YWIVKILSMZOHHF-QJZPQSOGSA-N sodium;(2s,3s,4s,5r,6r)-6-[(2s,3r,4r,5s,6r)-3-acetamido-2-[(2s,3s,4r,5r,6r)-6-[(2r,3r,4r,5s,6r)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2- Chemical compound [Na+].CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 YWIVKILSMZOHHF-QJZPQSOGSA-N 0.000 claims abstract description 27
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7016—Disaccharides, e.g. lactose, lactulose
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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
- A61K8/60—Sugars; Derivatives thereof
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- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
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- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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Abstract
The invention discloses a preparation method and application of unsaturated sodium hyaluronate disaccharide, belonging to the technical field of preparation of oligomeric sodium hyaluronate, wherein the preparation method comprises the following steps: enzymolysis, separation and drying; the invention has no organic solvent in the preparation of the unsaturated sodium hyaluronate disaccharide, no environmental pollution, only one-step hyaluronidase enzymolysis is adopted, and only one-step organic membrane ultrafiltration technology is needed after the enzymolysis, so that the technology is simple, and the desalination concentration, filtration sterilization and active carbon adsorption are not needed again, and the filtrate is directly dried; the method has high yield and low cost, and is suitable for large-scale industrial production; the prepared unsaturated sodium hyaluronate disaccharide has high purity, less impurities and no cytotoxicity, has good effects on in-vitro compaction, wrinkle resistance, repair and the like, and can be applied to the fields of cosmetics, foods, health care products, medicines and medical appliances.
Description
Technical Field
The invention relates to the technical field of preparation of oligomeric sodium hyaluronate, in particular to a preparation method and application of unsaturated sodium hyaluronate disaccharide.
Background
Hyaluronic Acid (HA) is an acid mucopolysaccharide, which is a unbranched polymeric glycosaminoglycan composed of N-acetylglucosamine and D-glucuronic acid disaccharide repeating units through β (1→4) glycosidic bonds and β (1→3) glycosidic bonds, present in the interstitial tissue of animals and in the capsule of certain bacteria. Hyaluronic acid is widely used in the fields of medicine, cosmetics, food, and has a molecular weight of typically 10 5-107 daltons (Da).
The oligomeric sodium hyaluronate refers to sodium hyaluronate with molecular weight less than 10kDa, and research shows that hyaluronic acid with different molecular weights has different biological activities, even completely opposite biological activities, and high molecular weight hyaluronic acid is often applied to the fields of eye drops, beauty filling, orthopaedics filling and the like due to the moisture retention, viscoelasticity and lubricity, but when the molecular weight of the hyaluronic acid is reduced to the level of oligosaccharide (below 10 kDa), the hyaluronic acid oligosaccharide shows completely different performances from the high molecular weight hyaluronic acid and has the effects of tightening, anti-wrinkle and repairing.
The molecular weight (delta DiHA) of the unsaturated hyaluronic acid disaccharide is 383Da, and the unsaturated hyaluronic acid disaccharide is prepared by the enzymolysis of hyaluronic acid by bacterial hyaluronidase, and is different from the structure of the hyaluronic acid disaccharide, and the unsaturated hyaluronic acid disaccharide forms an unsaturated bond at the 4, 5-glycosidic bond of glucuronic acid. The early-stage research shows that the unsaturated hyaluronic acid disaccharide has stronger oxidation resistance, can obviously promote proliferation of human umbilical vein endothelial cells and human cornea epithelial cells, and has wide application prospect in the field of medicines. In addition, the high-purity unsaturated hyaluronic acid disaccharide can also be used as a standard substance for detecting the content and purity of hyaluronic acid and related products thereof.
In the prior art, the biological enzyme method has the advantages of mild condition, simple operation, higher efficiency and the like, so the sodium hyaluronate disaccharide is mainly prepared by utilizing HAase to carry out enzymolysis on macromolecular sodium hyaluronate. Because of different sources and enzyme activities of the hyaluronate lyase, the molecular weight and concentration of the sodium hyaluronate in the enzymolysis reaction are different, so that the treatment and purification processes of the sodium hyaluronate solution after the enzymolysis are different. At present, the traditional industrialized methods such as organic solvent sedimentation assisting, active carbon adsorption, plate-frame filtration, ultrafiltration membrane separation, nanofiltration membrane desalination concentration, freeze drying and spray drying are mostly adopted, but the method can lead to low yield of the small molecule sodium hyaluronate disaccharide, complicated process, high production cost and low efficiency, and is not suitable for industrialized continuous production of the small molecule sodium hyaluronate disaccharide.
Chinese patent CN110982862B discloses a method for preparing high-purity unsaturated hyaluronic acid disaccharide in a large scale, which comprises the steps of enzymolysis and purification in two steps, wherein the high-molecular hyaluronic acid or salt thereof is subjected to enzymolysis by hyaluronidase, the obtained product is purified by low organic solvent multiple, the low-molecular hyaluronic acid or salt thereof with higher purity is obtained, and then the low-molecular hyaluronic acid or salt thereof is subjected to thorough enzymolysis, and the obtained product is purified by high organic solvent multiple, so that the high-purity unsaturated hyaluronic acid disaccharide is obtained. However, the invention adopts two-step enzymolysis, and the process is tedious and difficult to control; the two steps of organic solvent purification are easy to pollute the environment; activated carbon is adopted for adsorption and impurity removal after enzymolysis, so that the risk of carbon pollution to operators is increased.
Chinese patent CN116179629a discloses a process for preparing unsaturated sodium hyaluronate disaccharide, the process of the invention comprises: enzymolysis, specifically, carrying out enzymolysis reaction on macromolecular sodium hyaluronate by adopting hyaluronidase to obtain an enzymolysis solution containing unsaturated sodium hyaluronate disaccharide; separating the enzymatic hydrolysate by using an ultrafiltration membrane with the molecular weight cut-off of 1-5kDa to obtain ultrafiltration permeate; desalting and concentrating, namely concentrating and desalting the ultrafiltration permeate by adopting a nanofiltration membrane to obtain nanofiltration concentrate; and (3) degerming and pulverizing, namely sequentially degerming and drying the nanofiltration concentrated solution to obtain the unsaturated sodium hyaluronate disaccharide. The mass concentration of the enzymolysis macromolecular sodium hyaluronate in the invention is 0.5-2%, the mass production yield is low, the enzyme addition amount is high, the enzymolysis time is long, and the subsequent processes of desalting, concentrating, degerming and the like are needed, so that the process is complex, the energy consumption is high, and the cost is high.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a preparation method and application of unsaturated sodium hyaluronate disaccharide, no organic solvent is used in the preparation of the unsaturated sodium hyaluronate disaccharide, no environmental pollution is caused, only one step of hyaluronidase enzymolysis is adopted, only one step of organic membrane ultrafiltration technology is needed after the enzymolysis, the technology is simple, the secondary desalination concentration, filtration sterilization and active carbon adsorption are not needed, and the filtrate is directly dried; the method has high yield and low cost, and is suitable for large-scale industrial production; the prepared unsaturated sodium hyaluronate disaccharide has high purity, less impurities and no cytotoxicity, has good effects on in-vitro compaction, wrinkle resistance, repair and the like, and can be applied to the fields of cosmetics, foods, health care products, medicines and medical appliances.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a method for preparing unsaturated sodium hyaluronate disaccharide, comprising: enzymolysis, separation and drying;
The enzymolysis is carried out, pure water is controlled to 36-38 ℃, purified hyaluronidase is added, macromolecular sodium hyaluronate is added, and after sealing, the enzymolysis is carried out for 4.5-5.5 hours at 36-38 ℃ to obtain enzymolysis liquid;
In the enzymolysis, the mass ratio of the macromolecular sodium hyaluronate to the pure water is 10-15:100;
the ratio of the total activity of the macromolecular sodium hyaluronate to the purified hyaluronidase is 0.8-1.2g:2×10 5 IU;
the molecular weight of the macromolecular sodium hyaluronate is 100wDa-200wDa;
separating the enzymolysis liquid by using an ultrafiltration membrane, and collecting the permeate liquid to obtain the separated enzymolysis liquid;
in the separation, the filtering precision of the ultrafiltration membrane is 0.5-10kDa;
the temperature of the materials is 30-35 ℃ and the pressure is 0.4-0.5MPa;
preferably, the ultrafiltration membrane is an organic ultrafiltration membrane;
preferably, the solid content of the permeate is tested in the separation, and when the solid content of the permeate is more than 5%, the permeate is collected again;
And (3) drying the separated enzymolysis liquid to obtain the unsaturated sodium hyaluronate disaccharide.
The drying mode in the drying is one of freeze drying or spray drying;
when the drying mode is freeze drying, the freeze drying temperature is between-60 ℃ and-50 ℃ and the time is 45-50 hours;
when the drying mode is spray drying, the air inlet temperature of the spray drying is 170-180 ℃ and the air outlet temperature is 70-80 ℃.
The application of the unsaturated sodium hyaluronate disaccharide prepared by the preparation method in cosmetics, foods, health products, medicines and medical appliance products comprises the following three application modes:
The first is to directly apply the separated enzymolysis liquid obtained in the separation to cosmetics, foods, health products, medicines and medical appliance products;
the second is to apply the unsaturated hyaluronate disaccharide obtained in the drying to cosmetics, foods, health products, medicines and medical appliance products;
The second is to prepare the unsaturated sodium hyaluronate disaccharide obtained in the drying into aqueous solution according to the requirements of customers and then apply the aqueous solution to cosmetics, foods, health-care products, medicines and medical appliance products.
Compared with the prior art, the invention has the beneficial effects that:
(1) The preparation method of the unsaturated sodium hyaluronate disaccharide comprises an enzymolysis reaction, specifically, the enzymolysis reaction is carried out on macromolecular sodium hyaluronate by adopting hyaluronidase to obtain an enzymolysis solution containing the unsaturated sodium hyaluronate disaccharide, the enzyme addition amount is as low as 1 per mill, and the substrate concentration is as high as 15%; the method also comprises ultrafiltration separation by an organic membrane, specifically adopting the organic membrane with the molecular weight cutoff of 0.5-10kDa to carry out ultrafiltration separation on the enzymolysis liquid to obtain ultrafiltration permeate; and further comprises post-treatment, in particular freeze drying or spray drying, and finally obtaining the unsaturated sodium hyaluronate disaccharide product. The preparation method has the advantages of no organic solvent use, no environmental pollution, only one-step hyaluronidase enzymolysis, only one-step organic membrane ultrafiltration process after enzymolysis, no need of desalination and concentration again, filtration and sterilization and active carbon adsorption, short enzymolysis time, simple process and low cost, and is suitable for large-scale industrial production, and the prepared unsaturated sodium hyaluronate disaccharide has high yield, high purity and few impurities, shows good efficacy in the aspects of in vitro compaction, crease resistance, repair and the like, and can be applied to the fields of cosmetics, foods, health products, medicines and medical appliances;
(2) The unsaturated sodium hyaluronate disaccharide is white or light yellow granule or powder, the sodium hyaluronate content is 98-100%, the average relative molecular weight meets the requirement, the solution light transmittance is 99.75-100%, the drying weight loss is 1.57-7.11%, the pH value is 5.55-6.85, and the protein content is 0.02-0.04%.
Drawings
FIG. 1 is a liquid chromatogram of the first group of the resulting unsaturated sodium hyaluronate disaccharide samples of example 1;
FIG. 2 is a liquid chromatogram of the unsaturated sodium hyaluronate disaccharide sample obtained in the third group of example 2;
FIG. 3 is a liquid chromatogram of an unsaturated sodium hyaluronate disaccharide sample obtained in example 3;
FIG. 4 is a liquid chromatogram of an unsaturated sodium hyaluronate disaccharide sample obtained in example 4;
FIG. 5 is a graph showing the concentration gradient and activity of the unsaturated sodium hyaluronate disaccharide sample obtained in test example 1;
FIG. 6 is a graph showing cell morphology of the unsaturated sodium hyaluronate disaccharide sample obtained in test example 1 under different concentration gradients;
FIG. 7 is a bar graph showing the results of Elastin (Elastin) test obtained in test example 2;
FIG. 8 is a bar graph of the type I Collagen (Collagen I) test results obtained in test example 2;
FIG. 9 is a graph of the results of the AQP3 immunofluorescence in test example 3;
FIG. 10 is a bar graph of AQP3 relative Integrated Optical Density (IOD) versus cell number average for test example 3;
FIG. 11 is a bar graph showing the results of the detection of the FLG gene in test example 3;
FIG. 12 is a bar graph showing the results of CD44 gene detection in test example 3;
FIG. 13 is a graph of the results of the AQP3 immunofluorescence in test example 4;
FIG. 14 is a bar graph of the relative Integrated Optical Density (IOD) of AQP3 versus the cell number average for test example 4.
Detailed Description
Specific embodiments of the present invention will now be described in order to provide a clearer understanding of the technical features, objects and effects of the present invention.
The purified hyaluronidases used in examples 1 to 4 were prepared according to the preparation method of the recombinant hyaluronidase and the application thereof in example 1, and the patent publication No. CN118086255A was used.
Example 1
The preparation method of the unsaturated sodium hyaluronate disaccharide specifically comprises the following steps:
1. and (3) enzymolysis reaction:
3L of pure water is added into a 5L beaker, after the water bath is heated to 37 ℃, 3mL of purified hyaluronidase (the enzyme activity is 2X 10 7 IU/mL) is added, and 300g of macromolecular sodium hyaluronate (the molecular weight is 100 wDa) is added again;
Remarks: enzyme dosage is added according to (substrate concentration 10%, enzyme dosage is 2X 10 5 IU purified hyaluronidase added into every 1g macromolecular sodium hyaluronate solution);
Then sealing the beaker by using a preservative film, carrying out enzymolysis reaction for 5 hours at 37 ℃, and detecting the kinematic viscosity every half hour in the process;
2. Pretreatment of enzymolysis liquid:
dividing the enzymolysis liquid obtained in the step 1 into three groups for pretreatment to respectively obtain pretreated enzymolysis liquid, wherein the specific pretreatment method is as follows:
a first group: no treatment is performed;
Second group: terminating the enzyme reaction, specifically heating at 80deg.C for 30min;
Third group: stopping enzyme reaction, specifically heating at 80deg.C for 30min, and charcoal absorbing at 50deg.C for 30min;
3. organic ultrafiltration membrane separation:
Separating the pretreated enzymolysis liquid obtained in the first group, the second group and the third group in the step 2 by using 1wDa organic ultrafiltration membranes respectively, controlling the temperature of materials at 30-35 ℃ and the pressure at 0.4-0.5MPa, testing the solid content by using a handheld content meter, and collecting the permeate when the solid content is above 5%;
4. And (3) freeze drying:
and 3, freeze-drying the permeate collected in the step 3, specifically freeze-drying at-60 ℃ for 48 hours to obtain an unsaturated sodium hyaluronate disaccharide product.
And detecting the unsaturated sodium hyaluronate disaccharide products obtained from the first group, the second group and the third group, wherein the detection results are shown in the following table:
HPLC detection is carried out on the unsaturated sodium hyaluronate disaccharide products obtained from the first group, the second group and the third group, the HPLC chromatograms of the obtained first group are shown in figure 1, and as can be seen from figure 1, the purity of the unsaturated sodium hyaluronate disaccharide in the unsaturated sodium hyaluronate disaccharide products obtained from the first group is 99.59%.
The results show that the unsaturated sodium hyaluronate disaccharide with high purity can be prepared by only separating the enzymolysis liquid by an organic ultrafiltration membrane without heating and carbon absorption treatment.
The embodiment also provides an application of the unsaturated sodium hyaluronate disaccharide prepared by the preparation method in cosmetics, foods, health products, medicines and medical instrument products, wherein the application modes comprise the following three modes:
the first is to directly apply the permeate collected in the separation of the organic ultrafiltration membrane to cosmetics, foods, health products, medicines and medical appliance products;
The second is to apply the unsaturated sodium hyaluronate disaccharide product obtained in freeze-drying to cosmetics, foods, health products, medicines and medical appliance products;
The second is to prepare the unsaturated sodium hyaluronate disaccharide product obtained in freeze-drying into aqueous solution according to the requirements of customers and then apply the aqueous solution to cosmetics, foods, health products, medicines and medical appliance products.
Example 2
The preparation method of the unsaturated sodium hyaluronate disaccharide specifically comprises the following steps:
1. and (3) enzymolysis reaction:
3L of pure water is added into a 5L beaker, after the water bath kettle is heated to 37 ℃, 3mL of purified hyaluronidase (enzyme activity is 2 multiplied by 10 7 IU/mL) is added, and 300g of macromolecular sodium hyaluronate (molecular weight is 100 wDa) is added again;
Remarks: enzyme dosage is added according to (substrate concentration 10%, enzyme dosage is 2X 10 5 IU purified hyaluronidase added into every 1g macromolecular sodium hyaluronate solution);
Then sealing the beaker by using a preservative film, carrying out enzymolysis reaction for 5 hours at 37 ℃, and detecting the kinematic viscosity every half hour in the process;
2. Organic ultrafiltration membrane separation:
separating the enzymatic hydrolysate obtained in the step 1 by using a 1000Da organic ultrafiltration membrane, collecting the permeate liquid, taking part of the permeate liquid as a first group, separating the residual permeate liquid by using a 500Da organic ultrafiltration membrane, collecting the permeate liquid as a second group, and taking the concentrated liquid as a third group. Controlling the temperature of the material at 30-35 ℃ and the pressure at 0.4-0.5MPa, testing the solid content by using a handheld content meter when collecting the permeate, and starting to collect the permeate when the solid content is above 5%;
3. and (3) freeze drying:
And (3) freeze-drying the permeate collected in the step (2), specifically freeze-drying at-60 ℃ for 48 hours to obtain an unsaturated sodium hyaluronate disaccharide product.
And detecting the unsaturated sodium hyaluronate disaccharide products obtained from the first group, the second group and the third group, wherein the detection results are shown in the following table:
HPLC detection is carried out on the unsaturated sodium hyaluronate disaccharide products obtained from the first group, the second group and the third group, the HPLC chromatogram of the obtained third group is shown in figure 2, and as can be seen from figure 2, the purity of the unsaturated sodium hyaluronate disaccharide in the unsaturated sodium hyaluronate disaccharide products obtained from the third group is 100%.
The results show that the size of the organic ultrafiltration membrane has little influence on the purity of disaccharide, and the 0.5-10kDa can prepare the high-purity unsaturated sodium hyaluronate disaccharide.
The embodiment also provides an application of the unsaturated sodium hyaluronate disaccharide prepared by the preparation method in cosmetics, foods, health products, medicines and medical instrument products, wherein the application modes comprise the following three modes:
the first is to directly apply the permeate collected in the separation of the organic ultrafiltration membrane to cosmetics, foods, health products, medicines and medical appliance products;
The second is to apply the unsaturated sodium hyaluronate disaccharide product obtained in freeze-drying to cosmetics, foods, health products, medicines and medical appliance products;
The second is to prepare the unsaturated sodium hyaluronate disaccharide product obtained in freeze-drying into aqueous solution according to the requirements of customers and then apply the aqueous solution to cosmetics, foods, health products, medicines and medical appliance products.
Example 3
The preparation method of the unsaturated sodium hyaluronate disaccharide specifically comprises the following steps:
1. and (3) enzymolysis reaction:
3L of pure water is added into a 5L beaker, after the water bath is heated to 37 ℃, 5mL of purified hyaluronidase (enzyme activity is 2 multiplied by 10 7 IU/mL) is added, and 450g of sodium hyaluronate (molecular weight is 100 wDa) is added in batches;
Remarks: enzyme dosage is added according to (substrate concentration 15%, enzyme dosage is 2X 10 5 IU purified hyaluronidase added into every 1g macromolecule sodium hyaluronate solution);
Then sealing the beaker by using a preservative film, carrying out enzymolysis reaction for 5 hours at 37 ℃, and detecting the kinematic viscosity every half hour in the process;
2. Organic ultrafiltration membrane separation:
separating the enzymolysis liquid obtained in the step 1 by using a 1wDa organic ultrafiltration membrane, collecting the permeate liquid, controlling the temperature of the material at 30-35 ℃ and the pressure at 0.4-0.5MPa, testing the solid content by using a handheld content meter, and when the solid content is above 5%, starting to collect the permeate liquid;
3. and (3) freeze drying:
And (3) freeze-drying the permeate collected in the step (2), specifically freeze-drying at-60 ℃ for 48 hours to obtain an unsaturated sodium hyaluronate disaccharide product.
The obtained unsaturated sodium hyaluronate disaccharide product is detected, and the detection results are shown in the following table:
HPLC detection is carried out on the obtained unsaturated sodium hyaluronate disaccharide product, the obtained HPLC chromatogram is shown in figure 3, and the purity of the unsaturated sodium hyaluronate disaccharide in the obtained unsaturated sodium hyaluronate disaccharide product is 99.48% as can be seen from figure 3.
The embodiment also provides an application of the unsaturated sodium hyaluronate disaccharide prepared by the preparation method in cosmetics, foods, health products, medicines and medical instrument products, wherein the application modes comprise the following three modes:
the first is to directly apply the permeate collected in the separation of the organic ultrafiltration membrane to cosmetics, foods, health products, medicines and medical appliance products;
The second is to apply the unsaturated sodium hyaluronate disaccharide product obtained in freeze-drying to cosmetics, foods, health products, medicines and medical appliance products;
The second is to prepare the unsaturated sodium hyaluronate disaccharide product obtained in freeze-drying into aqueous solution according to the requirements of customers and then apply the aqueous solution to cosmetics, foods, health products, medicines and medical appliance products.
Example 4
The preparation method of the unsaturated sodium hyaluronate disaccharide specifically comprises the following steps:
1. and (3) enzymolysis reaction:
14L of pure water is added into a stainless steel barrel, after the water bath kettle is heated to 37 ℃,14 mL of purified hyaluronidase (enzyme activity is 2 multiplied by 10 7 IU/mL) is added, and then macromolecular sodium hyaluronate (molecular weight is 100 wDa) is added in batches to total 2100g;
Remarks: enzyme dosage is added according to (substrate concentration 15%, enzyme dosage is 2X 10 5 IU purified hyaluronidase added into every 1g macromolecule sodium hyaluronate solution);
Then sealing the beaker by using a preservative film, carrying out enzymolysis reaction for 5 hours at 37 ℃, and detecting the kinematic viscosity every half hour in the process;
2. organic ultrafiltration membrane separation
Separating the enzymolysis liquid obtained in step 1 by using a 1wDa organic ultrafiltration membrane, collecting the permeate, controlling the temperature of the material at 30-35 ℃ and the pressure at 0.4-0.5MPa, testing the solid content by using a handheld content meter, and collecting the permeate when the solid content is above 5%.
3. Spray drying
And (3) spray drying the permeate liquid collected in the step (2), controlling the inlet air temperature at 175 ℃ and the outlet air temperature at 75 ℃ to obtain an unsaturated sodium hyaluronate disaccharide product.
The obtained unsaturated sodium hyaluronate disaccharide product is detected, and the detection results are shown in the following table:
HPLC detection is carried out on the obtained unsaturated sodium hyaluronate disaccharide product, the obtained HPLC chromatogram is shown in figure 4, and the purity of the unsaturated sodium hyaluronate disaccharide in the obtained unsaturated sodium hyaluronate disaccharide product is 99.25% as can be seen from figure 4.
Test examples 1 to 4 the unsaturated sodium hyaluronate disaccharide product obtained in example 4 was used as a sample to be tested.
The embodiment also provides an application of the unsaturated sodium hyaluronate disaccharide prepared by the preparation method in cosmetics, foods, health products, medicines and medical instrument products, wherein the application modes comprise the following three modes:
the first is to directly apply the permeate collected in the separation of the organic ultrafiltration membrane to cosmetics, foods, health products, medicines and medical appliance products;
The second is to apply the unsaturated sodium hyaluronate disaccharide product obtained in spray drying to cosmetics, foods, health products, medicines and medical appliance products;
The second is to prepare the unsaturated sodium hyaluronate disaccharide product obtained in spray drying into aqueous solution according to the requirement of customers and then apply the aqueous solution to cosmetics, foods, health products, medicines and medical appliance products.
Test example 1 cytotoxicity evaluation
1. Cell viability test method
(1) Cell inoculation: after resuscitating the cells, when the plating rate reached 60%, the cells were inoculated into 96-well plates and incubated overnight in a CO 2 incubator (37 ℃, 5% CO 2).
(2) Test grouping: the test set was zeroed, solvent control, positive control, and sample. In the sample group, 8 concentration gradients were set, with 3 duplicate wells set for each concentration gradient.
(3) Preparing liquid: sample working solutions with different concentrations are prepared according to a test concentration setting table.
(4) Administration: administration was performed when the cell plating rate in 96-well plates reached 50-60%.
200 Mu of culture solution is added to each hole of the solvent control group; 200. Mu.L of culture medium containing 10% DMSO was added to each well of the positive control group; 200 mu L of culture solution containing a sample to be tested with corresponding concentration is added into each hole of the sample group; the zeroed group was inoculated without cells and only 200. Mu.L of cell culture medium was added.
After completion of the administration, the 96-well plate was placed in a CO 2 incubator (37 ℃, 5% CO 2) and cultured for 24 hours.
(5) And (3) detection: after incubation of the cells for 24h, the supernatant was discarded, MTT working solution (0.5 mg/mL) was added, incubated at 37℃for 4h in the absence of light, after incubation, the supernatant was discarded, 150. Mu.L of DMSO was added per well, and the OD was read at 490 nm.
(6) Cell relative viability calculation: cell relative viability (%) =
2. Cell morphology test method
(1) Cell inoculation: after resuscitating the cells, when the plating rate reached 60%, the cells were inoculated into 24 well plates and incubated overnight in a CO 2 incubator (37 ℃, 5% CO 2).
2) Test grouping: the test sets a solvent control group and a sample group. In the sample group, 5 concentration gradients were set for each sample.
3) Administration: administration was performed when the cell plating rate in the 24-well plate reached 50-60%.
Adding 1mL of culture solution into each hole of the solvent control group; 1mL of culture solution containing the sample to be tested with the corresponding concentration is added into each hole of the sample group. After completion of the administration, the 24-well plate was placed in a CO 2 incubator (37 ℃, 5% CO 2) and cultured for 24 hours.
4) Photographing: after the incubation, the supernatant was discarded and photographed under an inverted microscope.
3. Test results
The concentration gradient and activity are plotted according to the above table, the result is shown in fig. 5, and the cell morphology at different concentration gradients is characterized, and the obtained cell morphology is shown in fig. 6.
From the above results, it can be seen that the unsaturated sodium hyaluronate disaccharide sample showed no significant cytotoxicity in the concentration range of 10mg/mL based on keratinocytes.
Test example 2 fibroblast-based test
1. Test method
(1) Cell inoculation: after resuscitating the cells, cells were inoculated into 6-well plates and incubated overnight in a CO 2 incubator (37 ℃, 5% CO 2) when the plating rate reached 60%.
(2) Preparing liquid: and preparing a test object working solution according to the test group.
(3) Administration: according to the test grouping, when the cell plating rate in the 6-hole plate reaches 40-60%, grouping drug administration is carried out, and 3 compound holes are arranged in each group.
2ML of culture solution is added to each hole of the blank control group, 2mL of culture solution containing TGF-beta 1 is added to each hole of the positive control group, and 2mL of culture solution containing samples to be tested with corresponding concentrations is added to each hole of the sample group. After completion of the administration, the 6-well plate was placed in a CO 2 incubator (37 ℃, 5% CO 2) and cultured for 24 hours.
(4) ELISA detection: cell culture supernatants were collected and assayed according to the instructions of the ELISA kit.
(5) And (3) calculating a lifting rate: improvement rate (%) =
(6) Results statistical analysis: the results are expressed as mean±sd using GRAPHPAD PRISM plots. Comparisons between groups were performed using t-test statistical analysis. Statistical analysis was double tailed. P <0.05 was considered to have significant differences and P <0.01 was considered to have very significant differences.
2. Test results
(1) Elastin (Elastin) test results
Remarks: when statistical analysis is performed using the t-test method, significance is expressed as x, P-value <0.05 is expressed as x, and P-value <0.01 is expressed as x, compared to BC group.
The bar graph is plotted according to the results of the above table, and the resulting bar graph is shown in fig. 7.
Compared with the BC group, the Elastin content of the PC group is obviously increased, which proves that the positive control of the test is effective.
Compared with the BC group, the content of Elastin of the disaccharide sample at the concentration of 2.5mg/mL is obviously increased, and the lifting rate is 5.86%. The disaccharide sample can improve the content of Elastin (Elastin) at the concentration, and has a tightening effect.
(2) Collagen I (Collagen I) test results
Remarks: when statistical analysis is performed using the t-test method, significance is expressed as x, P-value <0.05 is expressed as x, and P-value <0.01 is expressed as x, compared to BC group.
The bar graph is plotted according to the results of the above table, and the resulting bar graph is shown in fig. 8.
Compared with the BC group, the Collagen I content of the PC group is obviously increased, which proves that the positive control of the test is effective.
Compared with the BC group, the collagani content of the disaccharide sample at the concentration of 2.5mg/mL is obviously increased, and the increase rate is 13.41%. It is demonstrated that disaccharide samples at this concentration are able to increase the amount of type I Collagen (Collagen I) with anti-wrinkle efficacy.
Test example 3 keratinocyte-based test
1. Test method
(1) Immunofluorescence test method
1) Cell inoculation: after resuscitating the cells, when the plating rate reached 60%, the cells were inoculated into 24 well plates and incubated overnight in a CO 2 incubator (37 ℃, 5% CO 2).
2) Preparing liquid: and preparing a test object working solution according to the test group.
3) Administration: according to the test grouping, when the cell plating rate in the 24-well plate reaches 40-60%, grouping drug administration is carried out, and 3 compound wells are arranged in each group.
1ML of culture solution is added into each hole of a blank control group, 1mL of culture solution containing CaCl 2 is added into each hole of a positive control group, and 1mL of culture solution containing samples to be tested with corresponding concentrations is added into each hole of a sample group. After completion of the administration, the 24-well plate was placed in a CO 2 incubator (37 ℃, 5% CO 2) and cultured for 24 hours.
4) Immunofluorescence detection: fixing with 4% paraformaldehyde for 30min, immunofluorescence detecting, photographing under microscope, observing, collecting picture, and analyzing.
5) And (3) calculating a lifting rate: improvement rate (%) =
6) Results statistical analysis: the results are expressed as mean±sd using GRAPHPAD PRISM plots. Comparisons between groups were performed using t-test statistical analysis. Statistical analysis was double tailed. P <0.05 was considered to have significant differences and P <0.01 was considered to have very significant differences.
(2) Gene expression test method
1) Cell inoculation: after resuscitating the cells, when the plating rate reached around 60%, the cells were inoculated into 6-well plates and incubated overnight in a CO 2 incubator (37 ℃, 5% CO 2).
2) Preparing liquid: and preparing a test object working solution according to the test group.
3) Administration: according to the test grouping, when the cell plating rate in the 6-hole plate reaches 40-60%, grouping drug administration is carried out, and 3 compound holes are arranged in each group.
2ML of culture solution is added to each hole of the blank control group, 2mL of culture solution containing WY14643 is added to each hole of the PC group, and 2mL of culture solution containing samples to be tested with corresponding concentrations is added to each hole of the sample group. After completion of the administration, the 6-well plate was placed in a CO 2 incubator (37 ℃, 5% CO 2) and cultured for 24 hours.
4) Collecting cells: after incubation, old solution was aspirated, washed twice with PBS, 1mLRNAiso Plus was added to each well, lysed cells were blown on, and samples were collected.
5) And (3) gene expression detection: RNA was extracted, reverse transcribed to cDNA, and then subjected to fluorescent quantitative PCR detection, and the result was calculated by the method of 2 -△△CT.
6) And (5) calculating an up-regulation rate: up-regulation rate (%) =
7) Results statistical analysis: the results are expressed as mean±sd using GRAPHPAD PRISM plots. Comparisons between groups were performed using t-test statistical analysis. Statistical analysis was double tailed. P <0.05 was considered to have significant differences and P <0.01 was considered to have very significant differences.
2. Test results
(1) Aquaporin 3 (AQP 3) test results
AQP3 immunofluorescence results are shown in FIG. 9;
the summary of the results of the AQP3 immunofluorescence assay is as follows:
Remarks: integrated Optical Density (IOD), whose value reflects the AQP3 content. When statistical analysis is performed using the t-test method, significance is expressed as x, P-value <0.05 is expressed as x, and P-value <0.01 is expressed as x, compared to BC group.
According to the results of the above table, a histogram of the relative Integrated Optical Density (IOD) of AQP3 versus the average of the cell number is shown in fig. 10.
Compared with the BC group, the AQP3 content of the PC group is obviously increased, which proves that the positive control of the test is effective.
Compared with the BC group, the AQP3 content of the disaccharide sample at the concentration of 2.5mg/mL is obviously increased, and the lifting rate is 82.00%.
(2) FLG Gene test results
Remarks: and (3) carrying out result calculation by adopting a 2-delta CT method, and carrying out statistical analysis by adopting a t-test method, wherein the significance is expressed as a x, the P-value <0.05 is expressed as a x, and the P-value <0.01 is expressed as a x when the BC group mRNA amplification multiple is normalized.
The histogram of the FLG gene detection results is shown in FIG. 11.
Compared with the BC group, the FLG gene expression level of the PC group is obviously up-regulated.
Compared with the BC group, the FLG gene expression level of the disaccharide sample at the concentration of 2.5mg/mL is obviously up-regulated, and the up-regulating rate is 48.00%.
(3) CD44 Gene test results
Remarks: when the statistical analysis is performed by the method ttest, the mRNA amplification fold of the BC group is normalized, the significance is expressed as x, P-value <0.05 is expressed as x, and P-value <0.01 is expressed as x, compared with the BC group.
The histogram of the CD44 gene detection results is shown in FIG. 12.
The CD44 gene expression level was significantly up-regulated in the PC group compared to the BC group.
Compared with the BC group, the CD44 gene expression level of the disaccharide sample at the concentration of 2.5mg/mL is obviously up-regulated, and the up-regulating rate is 35.00%.
Based on keratinocytes, the content of aquaporin 3 (AQP 3) of the disaccharide sample is obviously increased at the concentration of 2.5mg/mL compared with the control group, and the improvement rate is 82.00%; the expression levels of FLG and CD44 genes are obviously up-regulated, and the up-regulation rates are respectively 48.00% and 35.00%.
Test example 4 test of keratinocytes based on UVB irradiation
1. Test method
1) Cell inoculation: after resuscitating the cells, when the plating rate reached 60%, the cells were inoculated into 24 well plates and incubated overnight in a CO 2 incubator (37 ℃, 5% CO 2).
2) Preparing liquid: and preparing a test object working solution according to the test group.
3) Administration: according to the test group of Table 17, when the cell plating rate in the 24-well plate reached 30-50%, the group administration was performed, and 3 compound wells were set for each group.
1ML of culture solution is added into each hole of the blank control group and the negative control group, 1mL of culture solution containing CaCl 2 is added into each hole of the positive control group, and 1mL of culture solution containing samples to be tested with corresponding concentrations is added into each hole of the sample group. After completion of the administration, the 24-well plate was placed in a CO 2 incubator (37 ℃, 5% CO 2) and cultured for 24 hours.
4) UVB irradiation: according to the test group in Table 14, UVB irradiation was performed on the other groups except the blank control group at a dose of 300mJ/cm 2, and after the irradiation was completed, the culture was continued in a CO 2 incubator (37 ℃ C., 5% CO 2) for 24 hours.
5) Immunofluorescence detection: fixing with 4% paraformaldehyde for 30min, immunofluorescence detecting, photographing under microscope, observing, collecting picture, and analyzing.
6) And (3) calculating a lifting rate: improvement rate (%) =
7) Results statistical analysis: the results are expressed as mean±sd using GRAPHPAD PRISM plots. Comparisons between groups were performed using t-test statistical analysis. Statistical analysis was double tailed. P <0.05 was considered to have significant differences and P <0.01 was considered to have very significant differences.
2. Test results
The results of the AQP3 immunofluorescence are shown in FIG. 13.
The summary of the results of the AQP3 immunofluorescence assay is as follows:
Remarks: integrated Optical Density (IOD), whose value reflects the AQP3 content. When the statistical analysis is performed by the t-test method, compared with the BC group, the significance is expressed as # and the P-value <0.05 is expressed as # and the P-value <0.01 is expressed as #; compared to BC group, significance is expressed as P-value <0.05
Expressed as x, P-value <0.01 is expressed as x.
The histogram of the relative Integrated Optical Density (IOD) of AQP3 versus the cell number average is shown in fig. 14.
Compared with the BC group, the AQP3 content of the NC group is obviously reduced, which indicates that the test stimulation condition is effective.
Compared with the NC group, the AQP3 content of the PC group is obviously increased, which proves that the positive control of the test is effective.
Compared with NC group, the AQP3 content of disaccharide sample at the concentration of 2.5mg/mL is obviously increased, and the improvement rate is 125.00%.
Compared with a control group, the content of aquaporin 3 (AQP 3) of a disaccharide sample at the concentration of 2.5mg/mL is obviously increased, and the increasing rate is 125.00% based on the keratinocytes irradiated by UVB;
in conclusion, the disaccharide sample can increase the content of aquaporin 3 (AQP 3) under the concentration, up-regulate the gene expression quantity of Caspase-14 and FLG, LOR, CD44, and has repairing effect.
The percentages used in the present invention are mass percentages unless otherwise indicated.
Finally, it should be noted that: the foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present invention has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A method for preparing unsaturated sodium hyaluronate disaccharide, comprising the steps of: enzymolysis, separation and drying.
2. The method for preparing unsaturated sodium hyaluronate disaccharide according to claim 1, wherein the enzymolysis is carried out by controlling pure water to 36-38 ℃, adding purified hyaluronidase, adding macromolecular sodium hyaluronate, sealing, and hydrolyzing at 36-38 ℃ for 4.5-5.5h to obtain enzymolysis liquid.
3. The method for preparing unsaturated sodium hyaluronate disaccharide according to claim 2, wherein in the enzymolysis, the mass ratio of the macromolecular sodium hyaluronate to the pure water is 10-15:100;
The ratio of the total activity of the macromolecular sodium hyaluronate to the purified hyaluronidase is 0.8-1.2g:2×10 5 IU.
4. The method for producing an unsaturated sodium hyaluronate disaccharide according to claim 2, wherein the molecular weight of the macromolecular sodium hyaluronate is 100wDa-200wDa.
5. The method for producing an unsaturated sodium hyaluronate disaccharide according to claim 1, wherein the separation is carried out by separating the enzymatic hydrolysate with an ultrafiltration membrane and collecting the permeate to obtain the separated enzymatic hydrolysate.
6. The method for producing an unsaturated hyaluronate disaccharide according to claim 5, wherein in the separation, the filtration accuracy of the ultrafiltration membrane is 0.5-10kDa;
the temperature of the materials is 30-35 ℃ and the pressure is 0.4-0.5MPa.
7. The method for producing an unsaturated sodium hyaluronate disaccharide according to claim 5, wherein in the separation, the ultrafiltration membrane is an organic ultrafiltration membrane;
the solid content of the permeate was tested during the separation, and when the solid content of the permeate was 5% or more, collection of the permeate was resumed.
8. The method for producing an unsaturated sodium hyaluronate disaccharide according to claim 1, wherein the drying is performed by drying the separated enzymatic hydrolysate to obtain an unsaturated sodium hyaluronate disaccharide.
9. The method for producing an unsaturated sodium hyaluronate disaccharide according to claim 8, wherein the drying means in the drying is one of freeze-drying or spray-drying;
when the drying mode is freeze drying, the freeze drying temperature is between-60 ℃ and-50 ℃ and the time is 45-50 hours;
when the drying mode is spray drying, the air inlet temperature of the spray drying is 170-180 ℃ and the air outlet temperature is 70-80 ℃.
10. Use of the unsaturated sodium hyaluronate disaccharide prepared by the preparation method of any one of claims 1-9 in cosmetics, foods, health products, medicines and medical appliance products, characterized in that the application modes of the unsaturated sodium hyaluronate disaccharide in cosmetics, foods, health products, medicines and medical appliance products are as follows:
The first is to directly apply the separated enzymolysis liquid obtained in the separation to cosmetics, foods, health products, medicines and medical appliance products;
the second is to apply the unsaturated hyaluronate disaccharide obtained in the drying to cosmetics, foods, health products, medicines and medical appliance products;
The second is to prepare the unsaturated sodium hyaluronate disaccharide obtained in the drying into aqueous solution according to the requirements of customers and then apply the aqueous solution to cosmetics, foods, health-care products, medicines and medical appliance products.
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