WO2023006108A1 - 一种全分子量分布的透明质酸钠及其制备方法和应用 - Google Patents
一种全分子量分布的透明质酸钠及其制备方法和应用 Download PDFInfo
- Publication number
- WO2023006108A1 WO2023006108A1 PCT/CN2022/109329 CN2022109329W WO2023006108A1 WO 2023006108 A1 WO2023006108 A1 WO 2023006108A1 CN 2022109329 W CN2022109329 W CN 2022109329W WO 2023006108 A1 WO2023006108 A1 WO 2023006108A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- sodium hyaluronate
- molecular weight
- weight distribution
- preparation
- solution
- Prior art date
Links
- 229920002385 Sodium hyaluronate Polymers 0.000 title claims abstract description 171
- 229940010747 sodium hyaluronate Drugs 0.000 title claims abstract description 171
- 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 title claims abstract description 170
- 238000009826 distribution Methods 0.000 title claims abstract description 48
- 238000002360 preparation method Methods 0.000 title claims abstract description 28
- 239000000243 solution Substances 0.000 claims abstract description 49
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims abstract description 33
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 32
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims abstract description 28
- 238000001728 nano-filtration Methods 0.000 claims abstract description 26
- 239000002994 raw material Substances 0.000 claims abstract description 26
- 239000012528 membrane Substances 0.000 claims abstract description 20
- 238000000034 method Methods 0.000 claims abstract description 18
- 239000012670 alkaline solution Substances 0.000 claims abstract description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 15
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 14
- 239000000463 material Substances 0.000 claims abstract description 14
- 239000007787 solid Substances 0.000 claims abstract description 12
- 238000001914 filtration Methods 0.000 claims abstract description 11
- 238000001179 sorption measurement Methods 0.000 claims abstract description 10
- 238000001035 drying Methods 0.000 claims abstract description 3
- 238000005507 spraying Methods 0.000 claims abstract description 3
- 238000003756 stirring Methods 0.000 claims description 22
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 14
- 238000009210 therapy by ultrasound Methods 0.000 claims description 14
- 230000015556 catabolic process Effects 0.000 claims description 11
- 238000006731 degradation reaction Methods 0.000 claims description 11
- 239000012141 concentrate Substances 0.000 claims description 9
- 239000002537 cosmetic Substances 0.000 claims description 8
- 239000005909 Kieselgur Substances 0.000 claims description 7
- 239000007921 spray Substances 0.000 claims description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 6
- 239000002158 endotoxin Substances 0.000 claims description 6
- 239000002253 acid Substances 0.000 claims description 5
- 239000006185 dispersion Substances 0.000 claims description 5
- 230000008439 repair process Effects 0.000 claims description 5
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 3
- 239000003814 drug Substances 0.000 claims description 3
- 238000009281 ultraviolet germicidal irradiation Methods 0.000 claims description 3
- 239000004909 Moisturizer Substances 0.000 claims description 2
- 229940121363 anti-inflammatory agent Drugs 0.000 claims description 2
- 239000002260 anti-inflammatory agent Substances 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims description 2
- QWPPOHNGKGFGJK-UHFFFAOYSA-N hypochlorous acid Chemical compound ClO QWPPOHNGKGFGJK-UHFFFAOYSA-N 0.000 claims description 2
- 239000000314 lubricant Substances 0.000 claims description 2
- 230000001333 moisturizer Effects 0.000 claims description 2
- 238000002604 ultrasonography Methods 0.000 claims description 2
- WCDDVEOXEIYWFB-VXORFPGASA-N (2s,3s,4r,5r,6r)-3-[(2s,3r,5s,6r)-3-acetamido-5-hydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-4,5,6-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@@H]1C[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](C(O)=O)O[C@@H](O)[C@H](O)[C@H]1O WCDDVEOXEIYWFB-VXORFPGASA-N 0.000 claims 1
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical class [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims 1
- 229940014041 hyaluronate Drugs 0.000 claims 1
- 235000017557 sodium bicarbonate Nutrition 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 12
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (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-carboxylic acid Chemical compound 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 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 abstract description 11
- 229920002674 hyaluronan Polymers 0.000 abstract description 11
- 229960003160 hyaluronic acid Drugs 0.000 abstract description 11
- 230000003020 moisturizing effect Effects 0.000 abstract description 7
- 230000003110 anti-inflammatory effect Effects 0.000 abstract description 6
- 238000002156 mixing Methods 0.000 abstract description 2
- 230000000052 comparative effect Effects 0.000 description 37
- 210000004027 cell Anatomy 0.000 description 19
- 239000000706 filtrate Substances 0.000 description 8
- 239000000047 product Substances 0.000 description 8
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- 239000000203 mixture Substances 0.000 description 6
- 210000001519 tissue Anatomy 0.000 description 6
- 102000015696 Interleukins Human genes 0.000 description 3
- 108010063738 Interleukins Proteins 0.000 description 3
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 3
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 230000005855 radiation Effects 0.000 description 3
- 210000001179 synovial fluid Anatomy 0.000 description 3
- 210000004127 vitreous body Anatomy 0.000 description 3
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 2
- 231100000949 EpiSkin SIT Toxicity 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 210000001339 epidermal cell Anatomy 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 230000037394 skin elasticity Effects 0.000 description 2
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 description 1
- 241000239218 Limulus Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 description 1
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 238000005904 alkaline hydrolysis reaction Methods 0.000 description 1
- AEMOLEFTQBMNLQ-WAXACMCWSA-N alpha-D-glucuronic acid Chemical compound O[C@H]1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-WAXACMCWSA-N 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 210000001188 articular cartilage Anatomy 0.000 description 1
- 230000003796 beauty Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- CDQSJQSWAWPGKG-UHFFFAOYSA-N butane-1,1-diol Chemical compound CCCC(O)O CDQSJQSWAWPGKG-UHFFFAOYSA-N 0.000 description 1
- 208000037887 cell injury Diseases 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- -1 compound sodium hyaluronate Chemical class 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 239000010200 folin Substances 0.000 description 1
- 210000000245 forearm Anatomy 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 229950006780 n-acetylglucosamine Drugs 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 210000001525 retina Anatomy 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000013334 tissue model Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 210000003954 umbilical cord Anatomy 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- 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
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/03—Organic compounds
- A23L29/035—Organic compounds containing oxygen as heteroatom
- A23L29/04—Fatty acids or derivatives
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/03—Organic compounds
- A23L29/045—Organic compounds containing nitrogen as heteroatom
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
-
- 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
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
- A61K31/728—Hyaluronic acid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
- A61K8/73—Polysaccharides
- A61K8/735—Mucopolysaccharides, e.g. hyaluronic acid; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
- B01D61/02—Reverse osmosis; Hyperfiltration ; Nanofiltration
- B01D61/027—Nanofiltration
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
- B01D61/02—Reverse osmosis; Hyperfiltration ; Nanofiltration
- B01D61/04—Feed pretreatment
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D69/00—Semi-permeable membranes for separation processes or apparatus characterised by their form, structure or properties; Manufacturing processes specially adapted therefor
- B01D69/02—Semi-permeable membranes for separation processes or apparatus characterised by their form, structure or properties; Manufacturing processes specially adapted therefor characterised by their properties
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D2311/00—Details relating to membrane separation process operations and control
- B01D2311/04—Specific process operations in the feed stream; Feed pretreatment
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D2311/00—Details relating to membrane separation process operations and control
- B01D2311/26—Further operations combined with membrane separation processes
- B01D2311/2626—Absorption or adsorption
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D2311/00—Details relating to membrane separation process operations and control
- B01D2311/26—Further operations combined with membrane separation processes
- B01D2311/2699—Drying
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D2325/00—Details relating to properties of membranes
- B01D2325/34—Molecular weight or degree of polymerisation
Definitions
- the present application relates to the technical field of biomaterials, in particular to a sodium hyaluronate with a full molecular weight distribution and its preparation method and application.
- Hyaluronic acid is a linear polysaccharide linked by D-glucuronic acid and N-acetyl-D-glucosamine, with a molecular weight of hundreds of thousands to millions of daltons, widely distributed in In animal tissues, especially in human skin, umbilical cord, eye vitreous body and joint synovial fluid, the content is the highest, and it is also widely distributed in plants and microorganisms. In nature, HA has the same structure, no racial differences, and no immunogenicity.
- HA When HA is in a high concentration (1%), the intermolecular network exists in the form of a high viscoelasticity.
- the aqueous solution of HA is a viscoelastic fluid, which fills the space between cells and collagen fibers and covers some epidermal tissues. Its main function is to protect and lubricate cells, and regulate the movement of cells on this elastic matrix.
- HA In the skin, HA can lock moisture, make the skin smooth and tender, and maintain skin elasticity.
- HA in the synovial fluid of human joints can lubricate joints, isolate the stimulation of harmful signal factors, and protect articular cartilage.
- HA in the vitreous body supports the eye chamber, protects the retina, and brightens the eyes.
- HA is an internationally recognized ideal natural moisturizing factor, which can be applied to various cosmetics to achieve the effect of beauty and moisturizing. After oral absorption, HA can participate in the synthesis of HA in the body and distribute in the human skin, joint cavity and vitreous body of the eye. It can replenish hyaluronic acid in the synovial fluid of the joints and increase skin elasticity.
- HA mostly exists in the form of sodium hyaluronate during production and application, and the efficacy of different types of sodium hyaluronate is quite different.
- Low molecular weight sodium hyaluronate (molecular weight 10,000-500,000) and oligomeric sodium hyaluronate (molecular weight ⁇ 10,000) have better advantages in skin penetration and oral absorption.
- hyaluronic acid products mainly focus on hyaluronic acid with a single molecular weight distribution, which has a narrow molecular weight distribution width and uneven quality.
- the existing method wants to obtain sodium hyaluronate with a wider molecular weight distribution, it is necessary to mix and compound sodium hyaluronate of each molecular weight segment.
- the viscosity and other properties of sodium hyaluronate products with different molecular weights The difference is large, and the homogeneity of the product obtained by direct mixing is difficult to guarantee.
- Some physical, chemical or biological sodium hyaluronate degradation methods are also provided in the prior art.
- the molecular weight distribution of sodium hyaluronate treated by the existing degradation methods is not uniform, and the stability is poor, so it is difficult to further improve The quality of sodium hyaluronate products.
- the present invention aims to provide a high-quality sodium hyaluronate with good uniformity, high stability and full molecular weight distribution, as well as its preparation method and application.
- the application provides a method for preparing sodium hyaluronate with full molecular weight distribution, the method comprising:
- Step 1) Spraying hydrogen peroxide and ultraviolet irradiation on the sodium hyaluronate solid raw material to obtain the sodium hyaluronate degradation material;
- Step 2) dissolving the sodium hyaluronate degradation material in water, and adjusting the pH to alkaline with NaOH solution to obtain a sodium hyaluronate alkaline solution;
- Step 3 Ultrasonic treatment of the sodium hyaluronate alkaline solution
- Step 4) The sodium hyaluronate solid raw material is prepared into a sodium hyaluronate solution with a concentration of 0.1% to 1% (w/v), and the addition ratio of 20% to 60% (v/v) is compared with the step 3) The sodium hyaluronate alkaline solution after the obtained ultrasound is mixed evenly;
- Step 5) adopt diatomite and activated carbon to carry out adsorption treatment respectively, and adopt nanofiltration membrane to filter and concentrate, obtain the sodium hyaluronate of described total molecular weight distribution after drying.
- step 1) 50-100 ml of hydrogen peroxide with a mass concentration of 1%-5% is sprayed per kilogram of the sodium hyaluronate solid raw material, the ultraviolet irradiation dose is 300-1500 ⁇ W/cm 2 , and the irradiation time is 50 ⁇ 70min.
- the ultraviolet radiation dose mentioned in this application refers to the luminous flux of ultraviolet light received per unit area.
- the step of ultraviolet irradiation is to irradiate under an ultraviolet lamp, wherein the irradiation wavelength of the ultraviolet lamp is 200-400nm, preferably 220-320nm.
- the molecular weight of the sodium hyaluronate solid raw material is 1 million to 2 million.
- the molecular weight of sodium hyaluronate in this application refers to its weight average molecular weight.
- the used sodium hyaluronate solid raw material is selected from one or more of cosmetic-grade sodium hyaluronate, food-grade sodium hyaluronate, and pharmaceutical-grade sodium hyaluronate.
- the volume concentration of the sodium hyaluronate degradation material dissolved in water is 1%-10% (w/v); and/or,
- the frequency of ultrasonic treatment is 10-100 kHz, and the time is 15-180 min.
- the step of adopting diatomite adsorption treatment includes: adding diatomite based on the mass of the sodium hyaluronate alkaline solution of 0.1% to 1% (w/v), stirring and adsorbing at 45-80°C for 30-60min, and filtering ;and / or,
- the steps of activated carbon adsorption treatment include: adjusting the pH to 6-7 with dilute acid solution, adding activated carbon based on the mass of sodium hyaluronate solution at 0.1%-1% (w/v), stirring and adsorbing at 45-80°C for 30- 60min, filter; preferably, the dilute acid solution is selected from one or more of dilute hydrochloric acid solution, dilute sulfuric acid solution, dilute acetic acid solution, and dilute hypochlorous acid solution.
- the molecular weight cut-off of the nanofiltration membrane is 200-300Da
- the operating pressure is controlled at 15-30 bar
- the temperature is controlled at 30-50°C.
- the present application also provides sodium hyaluronate with full molecular weight distribution prepared by the above method, the weight average molecular weight range of the full molecular weight distribution sodium hyaluronate is 0.2 million to 1.5 million, and the molecular weight dispersion coefficient Mw /Mn is above 5;
- the endotoxin content in the sodium hyaluronate is ⁇ 0.01EU, and the protein content is ⁇ 0.01%;
- the OD value of the sodium hyaluronate at 280nm is ⁇ 0.01, and the OD value at 260nm is ⁇ 0.01.
- the present application also provides sodium hyaluronate prepared by the above preparation method in the preparation of moisturizers, lubricants, anti-inflammatory agents and/or cell repair agents for medicine, food and/or cosmetics Applications.
- the preparation method provided by the present invention can quickly and efficiently prepare sodium hyaluronate with full molecular weight distribution, and the prepared sodium hyaluronate has good uniformity and high stability, and can make full use of the sodium hyaluronate of various molecular weights. Efficacy advantages, the molecular weight dispersion coefficient Mw/Mn is above 5.
- the preparation method of full molecular weight sodium hyaluronate provided by the present invention combines ultraviolet irradiation degradation, ultrasonic-alkaline hydrolysis treatment, diatomite filtration, activated carbon adsorption and nano-membrane filtration at the same time, and the endotoxin of sodium hyaluronate obtained is Content ⁇ 0.01EU, protein content ⁇ 0.01%, OD value at 280nm ⁇ 0.01, OD value at 260nm ⁇ 0.01, has higher biocompatibility, can reach a higher quality level, and is beneficial to improve the sodium hyaluronate in Application effects in skin moisturizing, cell protection, oral administration, etc.
- UV irradiation degradation and ultrasonic degradation are placed before the filtration of high-viscosity hyaluronic acid solution, which greatly reduces the difficulty of filtration of high-viscosity solution and improves the preparation efficiency and yield. rate, improving the impurity removal efficiency.
- the preparation method of full molecular weight sodium hyaluronate provided by the present invention the obtained high-quality sodium hyaluronate with full molecular weight distribution has superior activities such as intelligent moisturizing, anti-inflammatory and cell protection.
- This embodiment provides a method for preparing sodium hyaluronate with full molecular weight distribution, which specifically includes the following steps:
- Step 1) Take 1 kg of food-grade sodium hyaluronate raw material with a molecular weight of 1 million, evenly spray 50 mL of 1% hydrogen peroxide on the surface of the raw material sodium hyaluronate, and irradiate it with ultraviolet light at a dose of 300 ⁇ W/cm 2 for 60 min;
- Step 2) Dissolving the sodium hyaluronate raw material after ultraviolet irradiation in water at a concentration of 1% (w/v), and stirring at room temperature to completely dissolve it;
- Step 3) Use 2% NaOH solution to adjust the pH to 10, and then place it under 10 kHz ultrasonic treatment for 15 minutes;
- Step 4) Prepare a 1 million sodium hyaluronate solution with a concentration of 1% (w/v), and mix it with the above-mentioned treated sodium hyaluronate alkaline solution at an addition ratio of 60% (v/v);
- Step 5 using 0.1% diatomaceous earth, stirring at 45°C, absorbing for 30 minutes, and filtering;
- Step 6) After adjusting the pH to 5.0 with 2% dilute hydrochloric acid solution, use 0.1% activated carbon at 45°C, stir, absorb for 30 minutes, and filter;
- Step 7) Concentrate the filtrate with a nanofiltration membrane system, wherein the molecular weight cut-off of the nanofiltration membrane is 300 Da, the operating pressure is 15 bar, and the temperature is controlled at 40°C to obtain a nanofiltration concentrated solution, which is spray-dried to obtain a high-quality total molecular weight distribution Sodium hyaluronate sample.
- This embodiment provides a method for preparing sodium hyaluronate with full molecular weight distribution, which specifically includes the following steps:
- Step 1) Take 1 kg of food-grade sodium hyaluronate raw material with a molecular weight of 1.2 million, evenly spray 80 mL of 3% hydrogen peroxide onto the surface of the sodium hyaluronate material, and irradiate it with ultraviolet rays for 60 minutes at a dose of 600 ⁇ W/cm 2 ;
- Step 2) Dissolving the sodium hyaluronate raw material after ultraviolet irradiation in water at a concentration of 3% (w/v), stirring at room temperature to completely dissolve it;
- Step 3) Use 5% NaOH solution to adjust the pH to 12, and then place it under 30 kHz ultrasonic treatment for 30 minutes;
- Step 4) Prepare a 1.2 million sodium hyaluronate raw material solution with a concentration of 0.8% (v/v), and mix it with the above-mentioned treated sodium hyaluronate alkaline solution at an addition ratio of 40%;
- Step 5) use 0.2% diatomaceous earth, stir at 50°C, absorb for 40 minutes, and filter;
- Step 6) After adjusting the pH to 5.0 with 2% dilute sulfuric acid solution, use 0.3% activated carbon at 50°C, stir, absorb for 50 minutes, and filter;
- Step 7) Concentrate the filtrate with a nanofiltration membrane system, wherein the molecular weight cut-off of the nanofiltration membrane is 250Da, the operating pressure is 20bar, and the temperature is controlled at 50°C to obtain a nanofiltration concentrated solution, which is spray-dried to obtain a high-quality total molecular weight distribution Sodium hyaluronate sample.
- This embodiment provides a method for preparing sodium hyaluronate with full molecular weight distribution, which specifically includes the following steps:
- Step 1) Take 1 kg of cosmetic-grade sodium hyaluronate raw material with a molecular weight of 1.2 million, evenly spray 80 mL of 5% hydrogen peroxide onto the surface of the sodium hyaluronate material, and irradiate with ultraviolet rays for 60 minutes at a dose of 900 ⁇ W/cm 2 ;
- Step 2) Dissolving the sodium hyaluronate material after ultraviolet irradiation in water at a concentration of 5% (w/v), stirring at room temperature to completely dissolve it;
- Step 3) Use 5% (w/v) NaOH solution to adjust the pH to 12, and then place it under 60kHz ultrasonic treatment, and the ultrasonic treatment time is 60min;
- Step 4) Prepare a 1.2 million high molecular weight sodium hyaluronate solution with a concentration of 0.6% (w/v), and mix it with the above-mentioned treated sodium hyaluronate alkaline solution at an addition ratio of 40% (v/v);
- Step 5 use 0.4% diatomaceous earth, stir at 55°C, absorb for 40 minutes, and filter;
- Step 6) After adjusting the pH to 5.0 with 2% dilute sulfuric acid solution, use 0.3% activated carbon at 55°C, stir, absorb for 50 minutes, and filter;
- Step 7) Concentrate the filtrate with a nanofiltration membrane system, wherein the molecular weight cut-off of the nanofiltration membrane is 200Da, the operating pressure is 30bar, and the temperature is controlled at 50°C to obtain a nanofiltration concentrated solution, which is spray-dried to obtain a high-quality total molecular weight distribution Sodium hyaluronate sample.
- This embodiment provides a method for preparing sodium hyaluronate with full molecular weight distribution, which specifically includes the following steps:
- Step 1) Take 1 kg of cosmetic-grade sodium hyaluronate raw material with a molecular weight of 1.4 million, evenly spray 100 mL of 5% hydrogen peroxide onto the surface of the sodium hyaluronate material, and irradiate with ultraviolet radiation for 60 minutes at a dose of 1200 ⁇ W/cm 2 ;
- Step 2) Dissolving the sodium hyaluronate raw material after ultraviolet irradiation in water at a concentration of 5% (w/v), stirring at room temperature to completely dissolve it;
- Step 3) Use 6% NaOH solution to adjust the pH to 12, then place it under 90kHz ultrasonic treatment, and the ultrasonic treatment time is 60min;
- Step 4) Prepare a 1.4 million high molecular weight sodium hyaluronate solution with a concentration of 0.3% (w/v), and mix it with the above-mentioned treated sodium hyaluronate alkaline solution at an addition ratio of 30% (v/v);
- Step 5 use 0.6% diatomaceous earth, stir at 55°C, absorb for 60 minutes, and filter;
- Step 6) After adjusting the pH to 5.0 with 2% dilute sulfuric acid solution, use 0.4% activated carbon at 55°C, stir, absorb for 60 minutes, and filter;
- Step 7) Concentrate the filtrate with a nanofiltration membrane system, wherein the molecular weight cut-off of the nanofiltration membrane is 200Da, the operating pressure is 30bar, and the temperature is controlled at 50°C to obtain a nanofiltration concentrated solution, which is spray-dried to obtain a high-quality total molecular weight distribution Sodium hyaluronate sample.
- This embodiment provides a method for preparing sodium hyaluronate with full molecular weight distribution, which specifically includes the following steps:
- Step 1) Take 1 kg of cosmetic-grade sodium hyaluronate raw material with a molecular weight of 1.5 million, evenly spray 100 mL of 5% hydrogen peroxide onto the surface of the sodium hyaluronate material, and irradiate with ultraviolet rays for 60 minutes at a dose of 1500 ⁇ W/cm 2 ;
- Step 2) Dissolving the sodium hyaluronate raw material after ultraviolet irradiation in water at a concentration of 3% (w/v), stirring at room temperature to completely dissolve it;
- Step 3) Use 5% NaOH solution to adjust the pH to 12, and then place it under 90kHz ultrasonic treatment, and the ultrasonic treatment time is 60min;
- Step 4) Prepare a 1.5 million high molecular weight sodium hyaluronate solution with a concentration of 0.3% (w/v), and mix it with the above-mentioned treated sodium hyaluronate alkaline solution at an addition ratio of 20% (v/v);
- Step 5 using 0.8% diatomaceous earth, stirring at 55°C, absorbing for 30 minutes, and filtering;
- Step 6) After adjusting the pH to 5.0 with 2% dilute sulfuric acid solution, use 0.6% activated carbon at 55°C, stir, absorb for 30 minutes, and filter;
- Step 7) Concentrate the filtrate with a nanofiltration membrane system, wherein the molecular weight cut-off of the nanofiltration membrane is 300Da, the operating pressure is 30bar, and the temperature is controlled at 50°C to obtain a nanofiltration concentrated solution, which is spray-dried to obtain a high-quality total molecular weight distribution Sodium hyaluronate sample.
- Embodiment 6 is a diagrammatic representation of Embodiment 6
- This embodiment provides a method for preparing sodium hyaluronate with full molecular weight distribution, which specifically includes the following steps:
- Step 1) Take 1 kg of cosmetic-grade sodium hyaluronate raw material with a molecular weight of 2 million, evenly spray 100 mL of 5% hydrogen peroxide onto the surface of the sodium hyaluronate material, and irradiate with ultraviolet radiation for 60 minutes at a dose of 1500 ⁇ W/cm 2 ;
- Step 2) Dissolving the sodium hyaluronate raw material after ultraviolet irradiation in water at a concentration of 1% (w/v), and stirring at room temperature to completely dissolve it;
- Step 3) Use 5% NaOH solution to adjust the pH to 12, then place it under 100kHz ultrasonic treatment, and the ultrasonic treatment time is 60min;
- Step 4) Prepare a 2 million high molecular weight sodium hyaluronate solution with a concentration of 0.1% (w/v), and mix it with the above-mentioned treated sodium hyaluronate alkaline solution at an addition ratio of 30% (v/v);
- Step 5 using 1% diatomaceous earth, stirring at 55°C, absorbing for 50 minutes, and filtering;
- Step 6) After adjusting the pH to 5.0 with 2% dilute sulfuric acid solution, use 1% activated carbon at 55°C, stir, absorb for 50 minutes, and filter;
- Step 7) Concentrate the filtrate with a nanofiltration membrane system, wherein the molecular weight cut-off of the nanofiltration membrane is 300Da, the operating pressure is 30bar, and the temperature is controlled at 50°C to obtain a nanofiltration concentrated solution, which is spray-dried to obtain a high-quality total molecular weight distribution Sodium hyaluronate sample.
- step 1) only 50 mL of 1% hydrogen peroxide is uniformly sprayed without ultraviolet irradiation, and the nanofiltration membrane system is no longer used after adsorption by activated carbon The filtrate was concentrated by filtration.
- step 1) only a dose of 300 ⁇ W/cm 2 is used for ultraviolet irradiation for 60 minutes without hydrogen peroxide treatment, and it is not used after adsorption by activated carbon
- step 2) only a dose of 300 ⁇ W/cm 2 is used for ultraviolet irradiation for 60 minutes without hydrogen peroxide treatment, and it is not used after adsorption by activated carbon
- the nanofiltration membrane system filters and concentrates the filtrate.
- Embodiment 7 is a diagrammatic representation of Embodiment 7:
- the sodium hyaluronate obtained in Examples 1 to 6 has a uniform appearance and good stability; while the sodium hyaluronate obtained in Comparative Example 3 and Comparative Example 4 has a relatively uniform appearance, but the stability is slightly poor, and The viscosity is relatively high, the filtration efficiency is low, and it is difficult to use a nanofiltration membrane system to filter.
- the sodium hyaluronate obtained in Comparative Example 5 has poor uniformity and stability.
- Embodiment 8 is a diagrammatic representation of Embodiment 8
- the molecular weight distribution of the sodium hyaluronate samples obtained in the above examples and comparative examples was determined, wherein the weight average molecular weight Mw was between 0.2 million to 10,000, 10,000 to 300,000, 300,000 to 1 million, and >1 million.
- the proportion of sodium hyaluronate molecules is shown in Table 2.
- the molecular weight distribution range of the sodium hyaluronate raw materials obtained in Examples 1-6 is wider, and the molecular weight dispersion coefficient Mw/Mn is above 5.
- the molecular weight distribution ranges of comparative examples 3 and 4 are relatively narrow, and the molecular weight dispersion coefficient is below 2.5. It can be seen that the effect of UV irradiation or hydrogen peroxide treatment alone is not ideal.
- Embodiment 9 is a diagrammatic representation of Embodiment 9:
- HaCaT human immortalized epidermal cells induced by interleukin IL-1 ⁇ was used as a model to verify that the sodium hyaluronate with full molecular weight distribution prepared by the present invention has anti-inflammatory effect.
- HaCaT Human immortalized epidermal cells
- 96-well plates at 5 ⁇ 10 3 cells/mL, 100 ⁇ L per well, with 6 replicate wells in each group, and replaced with 10 ng/mL interleukin IL-1 ⁇ after 24 hours of culture.
- the culture medium of 20 mg/mL full molecular weight sodium hyaluronate of the present invention (Examples 1-6) and control example sodium hyaluronate (Comparison examples 1-5)
- cells without adding IL-1 ⁇ and sodium hyaluronate As a normal control group, the cells that only added IL-1 ⁇ without adding sodium hyaluronate were used as the model control group.
- the expression levels of interleukin IL-1 ⁇ and tumor necrosis factor TNF- ⁇ were detected with ELISA kits .
- the hyaluronic acid in different examples and comparative examples can reduce the expression levels of IL-1 ⁇ and TNF- ⁇ in cells, and all have shown a certain anti-inflammatory effect.
- the prepared sodium hyaluronate with full molecular weight distribution has a better anti-inflammatory effect than the ordinary sodium hyaluronate of the comparative example.
- Sodium dodecyl sulfate (SDS)-induced skin model damage was used as a model to verify the anti-cell damage and cell repair effects of the sodium hyaluronate with full molecular weight distribution prepared by the present invention.
- Tissue cell activity (%) A p (t) / A n ⁇ 100%
- a p (t) is the absorbance value of the positive control group or the experimental group;
- a n is the absorbance value of the negative control group, the results are as follows:
- the sodium hyaluronate with full molecular weight distribution prepared by the present invention has a strong inhibitory effect on the damage of the EpiSkin skin model induced by SDS, and can effectively promote the repair of damaged cells.
- the sodium hyaluronate with full molecular weight distribution prepared by the process of the present invention has a better effect of promoting cell repair.
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Abstract
本发明提供了一种全分子量分布的透明质酸钠的制备方法，所述方法包括：步骤1)：对透明质酸钠固体原料进行喷淋双氧水和紫外照射处理，获得透明质酸钠降解物料；步骤2)：将透明质酸钠降解物料溶解于水中，用NaOH溶液调pH至碱性，获得透明质酸钠碱性溶液；步骤3)：对透明质酸钠碱性溶液进行超声处理；步骤4)：将透明质酸固体原料配制成浓度为0.1％～1％(w/v)的透明质酸钠溶液，以20％～60％(v/v)的添加比例与超声后的透明质酸钠碱性溶液混合均匀；步骤5)：分别采用硅藻土和活性炭进行吸附处理，并采用纳米滤膜过滤浓缩，干燥后即得。本发明的制备方法，能够获得较好补水保湿、抗炎及细胞保护等活性的透明质酸钠。
Description
相关申请的交叉引用
本申请要求享有于2021年7月30日提交的名称为“一种全分子量分布的透明质酸钠及其制备方法和应用”的中国专利申请CN 202110874694.1的优先权，上述申请的全部内容通过引用并入本文中。
本申请涉及生物材料技术领域，具体涉及一种全分子量分布的透明质酸钠及其制备方法和应用。
透明质酸(Haluronic acid，HA)是由D-葡糖醛酸和N-乙酰-D-葡糖胺链接而成的线性多糖，分子量为数十万至数百万道尔顿，广泛分布于动物组织中，特别是在人皮肤、脐带、眼玻璃体及关节滑液中含量最高，也广泛分布于植物和微生物中。在自然界中，HA结构一致，没有种族差异性，无免疫原性。
HA在高浓度(1％)时，分子间以网状形式存在，有很高的黏弹性。HA的水溶液为黏弹性流体，填充在细胞与胶原纤维空间之中且覆盖在某些表皮组织上，主要功能为保护及润滑细胞，调节细胞在此弹性基质上的移动。在皮肤中，HA可锁住水分，使皮肤光滑细嫩，维持皮肤弹性。人体关节滑液中的HA，可起到润滑关节，隔离有害信号因子的刺激，保护关节软骨的功能。眼玻璃体中的HA，起到支撑眼房、保护视网膜、使眼睛明亮的功能。HA是国际公认的理想的天然保湿因子，可以应用到各种化妆品中，起到美容保湿的功效。HA口服吸收后，可参与体内HA的合成，分布在人体皮肤、关节腔及眼玻璃体中，可补充关节滑液内玻尿酸，增加皮肤弹性。
HA在生产及应用时多以透明质酸钠的形式存在，不同种类的透明质酸钠功效差别较大，例如，高分子量透明质酸钠(分子量>50万)具有较好的黏膜保护作用，低分子量透明质酸钠(分子量1万～50万)和寡聚透明质酸钠(分子量<1万)在皮肤渗透能力及口服吸收能力方面具有较好的优势。目前，透明质酸产品主要集中在单一分子量分布的透明质酸，分子量分布宽度较窄，且质量参差不齐。因此，现有的方法想要获得较宽分子量分布的透明质酸钠，需将各分子量段的透明质酸钠进行混合复配，然而，不同分子量的透明质酸钠产品之间的黏度等性能差异较大，直接混合获得的产物均匀性很难保证。现有技术中还提供了一些物理、化学或生物类的透明质酸钠降解方法，然而现有的降解方法处理后的透明质酸钠分子量分布并不均匀，稳定性也较差，难以进一步提升透明质酸钠产品的质量。
发明内容
为了解决上述问题，本发明旨在提供一种均匀性好、稳定性高且为全分子量分布的高质量透明质酸钠，以及其制备方法和应用。
一方面，本申请提供了一种全分子量分布的透明质酸钠的制备方法，所述方法包括：
步骤1)：对透明质酸钠固体原料进行喷淋双氧水和紫外照射处理，获得透明质酸钠降解物料；
步骤2)：将所述透明质酸钠降解物料溶解于水中，并用NaOH溶液调pH至碱性，获得透明质酸钠碱性溶液；
步骤3)：对所述透明质酸钠碱性溶液进行超声处理；
步骤4)：将所述透明质酸钠固体原料配制成浓度为0.1％～1％(w/v)的透明质酸钠溶液，以20％～60％(v/v)的添加比例与步骤3)所得超声后的透明质酸钠碱性溶液混合均匀；
步骤5)：分别采用硅藻土和活性炭进行吸附处理，并采用纳米滤膜过滤 浓缩，干燥后即得所述全分子量分布的透明质酸钠。
进一步地，所述步骤1)中，每千克所述透明质酸钠固体原料喷淋质量浓度1％～5％的双氧水50～100毫升，紫外照射剂量为300～1500μW/cm
2，照射时长50～70min。
可以理解的是，本申请中所述紫外照射剂量指单位面积上所接受紫外光的光通量。可选的，所述紫外照射的步骤为置于紫外灯下进行照射，其中紫外灯的照射波长为200-400nm，优选220-320nm。
进一步地，所述透明质酸钠固体原料的分子量为100～200万。
可以理解的是，本申请中所述透明质酸钠的分子量均指其重均分子量。
进一步地，所用透明质酸钠固体原料选自化妆品级透明质酸钠、食品级透明质酸钠、医药级透明质酸钠中的一种或多种。
进一步地，所述步骤2)中，所述透明质酸钠降解物料溶解于水后的体积浓度为1％～10％(w/v)；和/或，
用NaOH溶液调pH至10～12。
进一步地，所述步骤3)中，超声处理的频率为10～100kHz，时间为15～180min。
进一步地，所述步骤5)中，
采用硅藻土吸附处理的步骤包括：加入基于所述透明质酸钠碱性溶液质量0.1％～1％(w/v)的硅藻土，于45-80℃下搅拌吸附30～60min，过滤；和/或，
采用活性炭吸附处理的步骤包括：用稀酸溶液调pH至6～7，加入基于透明质酸钠溶液质量0.1％～1％(w/v)的活性炭，于45-80℃下搅拌吸附30～60min，过滤；优选的，所述稀酸溶液选自稀盐酸溶液、稀硫酸溶液、稀醋酸溶液、稀次氯酸溶液中的一种或多种。
进一步地，所述步骤5)中，所述纳米滤膜的截留分子量为200-300Da，操作压力控制在15-30bar，温度控制在30-50℃。
另一方面，本申请还提供了采用上述方法所制备获得的全分子量分布的透 明质酸钠，所述全分子量分布透明质酸钠的重均分子量范围为0.2万～150万，分子量分散系数Mw/Mn在5以上；
优选的，所述透明质酸钠中内毒素含量<0.01EU，蛋白质含量<0.01％；
优选的，所述透明质酸钠在280nm处OD值<0.01，在260nm处OD值<0.01。
另一方面，本申请还提供了采用上述制备方法所制备获得的透明质酸钠在制备医药用、食品用和/或化妆品用的保湿剂、润滑剂、抗炎剂和/或细胞修复剂中的应用。
与现有技术相比，本发明的有益效果为：
1、本发明提供的制备方法能够快速、高效地制备获得全分子量分布的透明质酸钠，并且制得的透明质酸钠均匀性好、稳定性高，可充分利用各分子量透明质酸钠的功效优势，分子量分散系数Mw/Mn在5以上。
2、本发明提供的全分子量透明质酸钠的制备方法，同时融合了紫外照射降解、超声-碱解处理、硅藻土过滤、活性炭吸附和纳米膜过滤，获得的透明质酸钠的内毒素含量<0.01EU，蛋白质含量<0.01％，280nm处OD值<0.01，260nm处OD值<0.01，具有更高的生物相容性，可达到较高的质量水平，有利于提高透明质酸钠在皮肤保湿、细胞保护、口服等方面的应用效果。
3、本发明提供的全分子量透明质酸钠的制备方法，将紫外照射降解、超声降解置于高黏度透明质酸溶液过滤之前，大大降低了高黏度溶液过滤的难度，提高了制备效率及收率，提高了杂质去除效率。
4、本发明提供的全分子量透明质酸钠的制备方法，获得的高品质全分子量分布透明质酸钠，具备优越的智能补水保湿、抗炎及细胞保护等活性。
为了更清楚的阐释本申请的整体构思，下面以实施例的方式进行详细说 明。本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限制本发明的范围。
实施例1：
该实施例提供了一种全分子量分布透明质酸钠的制备方法，具体包括如下步骤：
步骤1)：取分子量100万的食品级透明质酸钠原料1kg，均匀喷洒1％的双氧水50mL至透明质酸钠原料表面，采用300μW/cm
2的剂量进行紫外照射60min；
步骤2)：将紫外照射后的透明质酸钠原料溶于水中，浓度为1％(w/v)，室温下搅拌，使其完全溶解；
步骤3)：采用2％的NaOH溶液调节pH为10，然后置于10kHz的超声下处理，超声处理时间15min；
步骤4)：配置浓度为1％(w/v)的100万透明质酸钠溶液，按添加比例60％(v/v)与上述处理后的透明质酸钠碱性溶液混合；
步骤5)：采用0.1％的硅藻土，45℃，搅拌，吸附处理30min，过滤；
步骤6)：采用2％稀盐酸溶液调pH 5.0后，用0.1％活性炭，45℃，搅拌，吸附处理30min，过滤；
步骤7)：采用纳滤膜系统对滤液进行浓缩，其中纳滤膜截留分子量300Da，操作压力15bar，温度控制为40℃，得纳滤浓缩液，对其进行喷雾干燥，获得高品质全分子量分布透明质酸钠样品。
实施例2：
该实施例提供了一种全分子量分布透明质酸钠的制备方法，具体包括如下步骤：
步骤1)：取分子量120万的食品级透明质酸钠原料1kg，均匀喷洒3％的双氧水80mL至透明质酸钠物料表面，采用600μW/cm
2的剂量进行紫外照射60min；
步骤2)：将紫外照射后的透明质酸钠原料溶于水中，浓度为3％(w/v)，室温下搅拌，使其完全溶解；
步骤3)：采用5％的NaOH溶液调节pH为12，然后置于30kHz的超声下处理，超声处理时间30min；
步骤4)：配置浓度为0.8％(v/v)的120万的透明质酸钠原料溶液，按添加比例40％与上述处理后的透明质酸钠碱性溶液混合；
步骤5)：采用0.2％的硅藻土，50℃，搅拌，吸附处理40min，过滤；
步骤6)：采用2％稀硫酸溶液调pH 5.0后，用0.3％活性炭，50℃，搅拌，吸附处理50min，过滤；
步骤7)：采用纳滤膜系统对滤液进行浓缩，其中纳滤膜截留分子量250Da，操作压力20bar，温度控制为50℃，得纳滤浓缩液，对其进行喷雾干燥，获得高品质全分子量分布透明质酸钠样品。
实施例3：
该实施例提供了一种全分子量分布透明质酸钠的制备方法，具体包括如下步骤：
步骤1)：取分子量120万的化妆品级透明质酸钠原料1kg，均匀喷洒5％的双氧水80mL至透明质酸钠物料表面，采用900μW/cm
2的剂量进行紫外照射60min；
步骤2)：将紫外照射后的透明质酸钠物料溶于水中，浓度为5％(w/v)，室温下搅拌，使其完全溶解；
步骤3)：采用5％(w/v)的NaOH溶液调节pH为12，然后置于60kHz的超声下处理，超声处理时间60min；
步骤4)：配置浓度为0.6％(w/v)的120万的高分子量透明质酸钠溶液，按添加比例40％(v/v)与上述处理后的透明质酸钠碱性溶液混合；
步骤5)：采用0.4％的硅藻土，55℃，搅拌，吸附处理40min，过滤；
步骤6)：采用2％稀硫酸溶液调pH 5.0后，用0.3％活性炭，55℃，搅拌，吸附处理50min，过滤；
步骤7)：采用纳滤膜系统对滤液进行浓缩，其中纳滤膜截留分子量200Da，操作压力30bar，温度控制为50℃，得纳滤浓缩液，对其进行喷雾干燥，获得高品质全分子量分布透明质酸钠样品。
实施例4：
该实施例提供了一种全分子量分布透明质酸钠的制备方法，具体包括如下步骤：
步骤1)：取分子量140万的化妆品级透明质酸钠原料1kg，均匀喷洒5％的双氧水100mL至透明质酸钠物料表面，采用1200μW/cm
2的剂量进行紫外照射60min；
步骤2)：将紫外照射后的透明质酸钠原料溶于水中，浓度为5％(w/v)，室温下搅拌，使其完全溶解；
步骤3)：采用6％的NaOH溶液调节pH为12，然后置于90kHz的超声下处理，超声处理时间60min；
步骤4)：配置浓度为0.3％(w/v)的140万高分子量透明质酸钠溶液，按添加比例30％(v/v)与上述处理后的透明质酸钠碱性溶液混合；
步骤5)：采用0.6％的硅藻土，55℃，搅拌，吸附处理60min，过滤；
步骤6)：采用2％稀硫酸溶液调pH 5.0后，用0.4％活性炭，55℃，搅拌，吸附处理60min，过滤；
步骤7)：采用纳滤膜系统对滤液进行浓缩，其中纳滤膜截留分子量200Da，操作压力30bar，温度控制为50℃，得纳滤浓缩液，对其进行喷雾干燥，获得高品质全分子量分布透明质酸钠样品。
实施例5：
该实施例提供了一种全分子量分布透明质酸钠的制备方法，具体包括如下步骤：
步骤1)：取分子量150万的化妆品级透明质酸钠原料1kg，均匀喷洒5％的双氧水100mL至透明质酸钠物料表面，采用1500μW/cm
2的剂量进行紫外照射60min；
步骤2)：将紫外照射后的透明质酸钠原料溶于水中，浓度为3％(w/v)，室温下搅拌，使其完全溶解；
步骤3)：采用5％的NaOH溶液调节pH为12，然后置于90kHz的超声下处理，超声处理时间60min；
步骤4)：配置浓度为0.3％(w/v)的150万高分子量透明质酸钠溶液，按添加比例20％(v/v)与上述处理后的透明质酸钠碱性溶液混合；
步骤5)：采用0.8％的硅藻土，55℃，搅拌，吸附处理30min，过滤；
步骤6)：采用2％稀硫酸溶液调pH 5.0后，用0.6％活性炭，55℃，搅拌，吸附处理30min，过滤；
步骤7)：采用纳滤膜系统对滤液进行浓缩，其中纳滤膜截留分子量300Da，操作压力30bar，温度控制为50℃，得纳滤浓缩液，对其进行喷雾干燥，获得高品质全分子量分布透明质酸钠样品。
实施例6：
该实施例提供了一种全分子量分布透明质酸钠的制备方法，具体包括如下步骤：
步骤1)：取分子量200万的化妆品级透明质酸钠原料1kg，均匀喷洒5％的双氧水100mL至透明质酸钠物料表面，采用1500μW/cm
2的剂量进行紫外照射60min；
步骤2)：将紫外照射后的透明质酸钠原料溶于水中，浓度为1％(w/v)，室温下搅拌，使其完全溶解；
步骤3)：采用5％的NaOH溶液调节pH为12，然后置于100kHz的超声下处理，超声处理时间60min；
步骤4)：配置浓度为0.1％(w/v)的200万高分子量透明质酸钠溶液，按添加比例30％(v/v)与上述处理后的透明质酸钠碱性溶液混合；
步骤5)：采用1％的硅藻土，55℃，搅拌，吸附处理50min，过滤；
步骤6)：采用2％稀硫酸溶液调pH 5.0后，用1％活性炭，55℃，搅拌，吸附处理50min，过滤；
步骤7)：采用纳滤膜系统对滤液进行浓缩，其中纳滤膜截留分子量300Da，操作压力30bar，温度控制为50℃，得纳滤浓缩液，对其进行喷雾干燥，获得高品质全分子量分布透明质酸钠样品。
对比例1：
该对比例选取某商品化的市售透明质酸钠1号(化妆品级，重均分子量125万)。
对比例2：
该对比例选取某商品化的市售透明质酸钠2号(化妆品级，重均分子量10万)。
对比例3：
该对比例与实施例1的制备方法大致相同，区别仅在于，在步骤1)中只均匀喷洒1％的双氧水50mL，而不进行紫外照射，并且在采用活性炭吸附后不再使用纳滤膜系统对滤液进行过滤浓缩。
对比例4：
该对比例与实施例1的制备方法大致相同，区别仅在于，在步骤1)中只采用300μW/cm
2的剂量进行紫外照射60min，而不使用双氧水处理，并且在采用活性炭吸附后不再使用纳滤膜系统对滤液进行过滤浓缩。
对比例5：
该对比例分别将单独的重均分子量200万的高分子量透明质酸钠、重均分子量100万的透明质酸钠、重均分子量30万的低分子量透明质酸钠和重均分 子量小于1万的寡聚透明质酸钠按照1:1:1:1的质量比进行混合搅拌60min，获得透明质酸钠样品。
实施例7：
对上述实施例1-6以及对比例3-5获得的不同透明质酸钠进行不同位置取样，测定均匀性和稳定性，在25℃下采用NDJ-1型旋转黏度计3号转子，60r/min分别对1％的不同透明质酸钠溶液进行黏度测定。各样品分别于制备后4℃保存，并于6个月后取样检测黏度。结果如表1所示，实施例1～6获得的透明质酸钠外观均匀，稳定性好；而对比例3和对比例4获得的透明质酸钠外观较为均匀，但稳定性稍差，并且黏度较大，过滤效率低，且难以使用纳滤膜系统进行过滤，对比例5获得的透明质酸钠均匀性和稳定性均较差。
表1透明质酸钠均匀度和稳定性检测结果
实施例8：
对上述实施例和对比例所得透明质酸钠样品进行分子量分布测定，其中，重均分子量Mw分别在0.2万～1万、1万～30万、30万～100万、＞100万之间的透明质酸钠分子占比如表2所示。
表2透明质酸钠分子量分布检测结果
由表2中的数据可知，与市售不同分子量规格透明质酸钠比较，实施例1～6所得透明质酸钠原料分子量分布范围较宽，分子量分散系数Mw/Mn在5以上。对比例3和对比例4的分子量分布范围较窄，分子量分散系数在2.5以下，由此可见，单独使用紫外照射或双氧水处理的效果并不理想。
实施例9：
分别采用鲎试剂检测法、Folin酚法对上述实施例1～6所制备的透明质酸钠的内毒素和杂蛋白进行检测，所得结果如表3所示：
表3透明质酸质量检查结果
| 示例 | 内毒素(EU) | 杂蛋白 |
| 实施例1 | 0.045 | <0.01％ |
| 实施例2 | 0.031 | <0.01％ |
| 实施例3 | 0.043 | <0.01％ |
| 实施例4 | 0.032 | <0.01％ |
| 实施例5 | 0.026 | <0.01％ |
| 实施例6 | 0.028 | <0.01％ |
| 对比例1 | 0.132 | 0.09 |
| 对比例2 | 0.126 | 0.08 |
| 对比例3 | 0.125 | 0.08 |
| 对比例4 | 0.113 | 0.07 |
| 对比例5 | 0.142 | 0.11 |
由表3中的结果可知，采用实施例1～6的制备方法获得的透明质酸的内毒素含量<0.01EU，蛋白质含量<0.01％，并测得280nm处OD值<0.01，260nm处OD值<0.01，具有更高的生物相容性和较高的质量水平。
实施例10：
选择有效受试志愿者45名，年龄在20～55岁，受试部位前2～3d不能使用任何产品，包括化妆品和外用药品等。试验前，受试者统一清洁双手前臂内侧，对4×4cm测试区域做好标记，配方1～8样品(分别为实施例1～6全分子量透明质酸钠1％，配方7为市售120万透明质酸钠1％，配方8为对比例5透明质酸钠1％，配方中其余成分为甘油2％，丁二醇2％)，均匀涂布随机分布在左右侧手臂上，用量3mg/cm
2，用皮肤水分测试仪测试产品涂抹前及涂抹后30min、1h、3h、6h时受试皮肤区域的含水量，每个区域平行测定5次，以平均值作为测试者，同一受试者的测试由同一测定人员完成，所有测试者测试前后处于温湿度恒定的室内空间。皮肤水分增加量(％)＝(涂抹后含水量-涂抹前含水量)/涂抹前含水量×100％。
表4透明质酸对皮肤水分的影响
| 30min | 1h | 3h | 6h | |
| 配方1 | 32.2±1.98 | 31.6±1.45 | 25.0±1.67 | 19.6±1.86 |
| 配方2 | 33.3±2.12 | 32.3±2.12 | 27.8±2.01 | 20.1±2.02 |
| 配方3 | 32.3±1.24 | 31.3±21.3 | 28.3±2.08 | 21.3±1.39 |
| 配方4 | 33.2±1.53 | 32.6±1.85 | 29.6±1.75 | 21.8±0.98 |
| 配方5 | 32.3±2.12 | 31.4±1.86 | 28.7±1.78 | 21.4±1.57 |
| 配方6 | 32.2±2.32 | 31.4±2.31 | 29.3±1.82 | 22.6±1.68 |
| 配方7 | 27.1±1.35 | 25.2±1.62 | 21.4±1.25 | 16.7±1.72 |
| 配方8 | 30.1±1.30 | 28.1±1.41 | 22.5±1.28 | 17.2±1.56 |
由表3中的结果可知，本发明制备的全分子量透明质酸钠的保湿性能优越，且优于市售透明质酸钠产品，用于化妆品可发挥较好的保湿效果。
实施例11
以白细胞介素IL-1β诱导人永生化表皮细胞(HaCaT)的炎症反应为模型验证本发明制备的全分子量分布的透明质酸钠具有抗炎效果。
将人永生化表皮细胞(HaCaT)以5×10
3cells/mL，每孔100μL接种于96孔板中，每组设6个复孔，培养24h后更换含10ng/mL白细胞介素IL-1β与20mg/mL本发明全分子量透明质酸钠(实施例1～6)及对照例透明质酸钠(对照例1～5)的培养基，以不加IL-1β和透明质酸钠的细胞作为正常对照组，以 只加入IL-1β不加入透明质酸钠的细胞作为模型对照组，继续培养24h后，用ELISA试剂盒检测白细胞介素IL-1α以及肿瘤坏死因子TNF-α的表达水平。
表5透明质酸钠的抗炎效果
| 组别 | IL-1α(ng/ml) | TNF-α(ng/ml) |
| 正常对照 | 43.36 | 1.81 |
| 模型对照 | 314.23 | 4.35 |
| 实施例1 | 102.82 | 2.14 |
| 实施例2 | 95.19 | 2.00 |
| 实施例3 | 92.17 | 2.07 |
| 实施例4 | 97.32 | 1.98 |
| 实施例5 | 82.83 | 2.05 |
| 实施例6 | 88.67 | 1.92 |
| 对比例1 | 134.73 | 2.93 |
| 对比例2 | 154.20 | 3.17 |
| 对比例3 | 123.89 | 2.38 |
| 对比例4 | 116.28 | 2.29 |
| 对比例5 | 117.02 | 2.41 |
由表5数据可以看出，不同实施例与对比例的透明质酸均能降低细胞IL-1α及TNF-α的表达水平，都体现出了有一定的抗炎效果，而本发明的工艺所制备的全分子量分布透明质酸钠与对比例普通透明质酸钠相比，有更好的抗炎效果。
实施例12
以十二烷基硫酸钠(SDS)诱导皮肤模型损伤作为模型验证本发明制备的全分子量分布透明质酸钠的抗细胞损伤及细胞修复效果。
将EpiSkin皮肤模型转移至12孔板中培养，培养24h后更换含0.5％(v/v)SDS的培养基，孵育2h后，每孔直接加入等体积含1％(w/v)本发明全分子量透明质酸钠(实施例1～6)及对照例透明质酸钠(对照例1～5)的培养基，得到培养基含透明质酸钠的终浓度为0.5％(w/v)，以不加SDS和透明质酸钠的细胞作为正常对照组，以只加入SDS不加入透明质酸钠的细胞作为模型对照组，继续培养24h后，取出皮肤组织模型，以MTT法用酶标仪在570nm测吸光度值，组织细胞活性按如下公式计算：
组织细胞活性(％)＝A
p(t)/A
n×100％
其中，A
p(t)是阳性对照组或实验组的吸光度值；A
n是阴性对照组的吸光度值，结果如下表：
表6 SDS与透明质酸作用于皮肤模型后的组织细胞活性
| 组别 | 组织细胞活性(％) |
| 正常对照 | 95.11 |
| 模型对照 | 54.58 |
| 实施例1 | 86.34 |
| 实施例2 | 88.16 |
| 实施例3 | 88.12 |
| 实施例4 | 89.57 |
| 实施例5 | 89.28 |
| 实施例6 | 92.78 |
| 对比例1 | 74.37 |
| 对比例2 | 6819 |
| 对比例3 | 76.34 |
| 对比例4 | 72.28 |
| 对比例5 | 77.56 |
由表6中数据可以看出，本发明所制备的全分子量分布透明质酸钠对SDS诱导的EpiSkin皮肤模型损伤有很强的抑制作用，可有效促进损伤细胞的修复。本发明的工艺所制备的全分子量分布透明质酸钠与对比例普通透明质酸钠相比，有更好的促进细胞修复效果。
以上所述仅为本申请的实施例而已，并不用于限制本申请。对于本领域技术人员来说，本申请可以有各种更改和变化。凡在本申请的精神和原理之内所作的任何修改、等同替换、改进等，均应包含在本申请的权利要求范围之内。
Claims (10)
- 一种全分子量分布的透明质酸钠的制备方法，其特征在于，所述方法包括：步骤1)：对透明质酸钠固体原料进行喷淋双氧水和紫外照射处理，获得透明质酸钠降解物料；步骤2)：将所述透明质酸钠降解物料溶解于水中，用NaOH溶液调pH至碱性，获得透明质酸钠碱性溶液；步骤3)：对所述透明质酸钠碱性溶液进行超声处理；步骤4)：将所述透明质酸钠固体原料配制成浓度为0.1％～1％(w/v)的透明质酸钠溶液，以20％～60％(v/v)的添加比例与步骤3)所得超声后的透明质酸钠碱性溶液混合均匀；步骤5)：分别采用硅藻土和活性炭进行吸附处理，并采用纳米滤膜过滤浓缩，干燥后即得所述全分子量分布的透明质酸钠。
- 根据权利要求1所述的全分子量分布的透明质酸钠的制备方法，其特征在于，所述步骤1)中，每千克所述透明质酸钠固体原料喷淋质量浓度1％～5％的双氧水50～100mL，紫外照射剂量为300～1500μW/cm 2，照射时长50～70min。
- 根据权利要求1所述的全分子量分布的透明质酸钠的制备方法，其特征在于，所述透明质酸钠固体原料的分子量为100～200万。
- 根据权利要求3所述的全分子量分布的透明质酸钠的制备方法，其特征在于，所用透明质酸钠固体原料选自化妆品级透明质酸钠、食品级透明质酸钠、医药级透明质酸钠中的一种或多种。
- 根据权利要求1所述的全分子量分布的透明质酸钠的制备方法，其特征在于，所述步骤2)中，所述透明质酸钠降解物料溶解于水后的体积浓度为1％～10％(w/v)；和/或，用NaOH溶液调pH至10～12。
- 根据权利要求1所述的全分子量分布的透明质酸钠的制备方法，其特征在于，所述步骤3)中，超声处理的频率为10～100kHz，时间为15～180min。
- 根据权利要求1所述的全分子量分布的透明质酸钠的制备方法，其特征在于，所述步骤5)中，采用硅藻土吸附处理的步骤包括：加入基于所述透明质酸钠碱性溶液质量0.1％～1％(w/v)的硅藻土，于45-80℃下搅拌吸附30～60min，过滤；和/或，采用活性炭吸附处理的步骤包括：用稀酸溶液调pH至6～7，加入基于透明质酸钠溶液质量0.1％～1％(w/v)的活性炭，于45-80℃下搅拌吸附30～60min，过滤；优选的，所述稀酸溶液选自稀盐酸溶液、稀硫酸溶液、稀醋酸溶液、稀次氯酸溶液中的一种或多种。
- 根据权利要求1所述的全分子量分布的透明质酸钠的制备方法，其特征在于，所述步骤5)中，所述纳米滤膜的截留分子量为200-300Da，操作压力控制在15-30bar，温度控制在30-50℃。
- 如权利要求1～8任一所述的制备方法所制备获得的全分子量分布的透明质酸钠，其特征在于，所述全分子量分布的透明质酸钠的重均分子量范围为0.2万～150万，分子量分散系数Mw/Mn在5以上；优选的，所述透明质酸钠中内毒素含量<0.01EU，蛋白质含量<0.01％；优选的，所述透明质酸钠在280nm处OD值<0.01，在260nm处OD值<0.01。
- 如权利要求1～8任一所述的制备方法所制备获得的全分子量透明质酸钠在制备医药用、食品用和/或化妆品用的保湿剂、润滑剂、抗炎剂和/或细胞修复剂中的应用。
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