CN116948057A - Degradation method of hyaluronic acid - Google Patents
Degradation method of hyaluronic acid Download PDFInfo
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- CN116948057A CN116948057A CN202311015923.XA CN202311015923A CN116948057A CN 116948057 A CN116948057 A CN 116948057A CN 202311015923 A CN202311015923 A CN 202311015923A CN 116948057 A CN116948057 A CN 116948057A
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- 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 title claims abstract description 128
- 229920002674 hyaluronan Polymers 0.000 title claims abstract description 128
- 229960003160 hyaluronic acid Drugs 0.000 title claims abstract description 128
- 238000006731 degradation reaction Methods 0.000 title claims abstract description 50
- 230000015556 catabolic process Effects 0.000 title claims abstract description 49
- 238000000034 method Methods 0.000 title claims abstract description 49
- 239000007857 degradation product Substances 0.000 claims abstract description 47
- 238000006243 chemical reaction Methods 0.000 claims abstract description 44
- 230000003197 catalytic effect Effects 0.000 claims abstract description 16
- ZOMNIUBKTOKEHS-UHFFFAOYSA-L dimercury dichloride Chemical class Cl[Hg][Hg]Cl ZOMNIUBKTOKEHS-UHFFFAOYSA-L 0.000 claims abstract description 6
- 238000005286 illumination Methods 0.000 claims abstract description 6
- 229910010413 TiO 2 Inorganic materials 0.000 claims abstract description 3
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims description 75
- 230000000593 degrading effect Effects 0.000 claims description 19
- -1 iron ions Chemical class 0.000 claims description 16
- 229910052742 iron Inorganic materials 0.000 claims description 14
- 239000011521 glass Substances 0.000 claims description 13
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical group [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 11
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 11
- 238000003756 stirring Methods 0.000 claims description 8
- 239000010453 quartz Substances 0.000 claims description 5
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 5
- 230000001105 regulatory effect Effects 0.000 claims description 3
- 239000013049 sediment Substances 0.000 claims description 2
- 230000008961 swelling Effects 0.000 claims description 2
- 230000007613 environmental effect Effects 0.000 abstract description 2
- 150000004676 glycans Chemical class 0.000 abstract description 2
- 229920001282 polysaccharide Polymers 0.000 abstract description 2
- 239000005017 polysaccharide Substances 0.000 abstract description 2
- 230000035484 reaction time Effects 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 61
- GWEVSGVZZGPLCZ-UHFFFAOYSA-N Titan oxide Chemical compound O=[Ti]=O GWEVSGVZZGPLCZ-UHFFFAOYSA-N 0.000 description 14
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 10
- 239000004408 titanium dioxide Substances 0.000 description 7
- 229910052724 xenon Inorganic materials 0.000 description 7
- FHNFHKCVQCLJFQ-UHFFFAOYSA-N xenon atom Chemical compound [Xe] FHNFHKCVQCLJFQ-UHFFFAOYSA-N 0.000 description 7
- 239000002244 precipitate Substances 0.000 description 6
- 230000000052 comparative effect Effects 0.000 description 5
- 239000012528 membrane Substances 0.000 description 5
- 230000001590 oxidative effect Effects 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 239000008367 deionised water Substances 0.000 description 4
- 229910021641 deionized water Inorganic materials 0.000 description 4
- 238000001035 drying Methods 0.000 description 4
- 230000014759 maintenance of location Effects 0.000 description 4
- ZNNZYHKDIALBAK-UHFFFAOYSA-M potassium thiocyanate Chemical compound [K+].[S-]C#N ZNNZYHKDIALBAK-UHFFFAOYSA-M 0.000 description 4
- 238000001556 precipitation Methods 0.000 description 4
- 238000007639 printing Methods 0.000 description 4
- 239000002994 raw material Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical group N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 3
- 239000005695 Ammonium acetate Substances 0.000 description 3
- 229920002307 Dextran Polymers 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 3
- 229940043376 ammonium acetate Drugs 0.000 description 3
- 235000019257 ammonium acetate Nutrition 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 239000003054 catalyst Substances 0.000 description 3
- 238000002144 chemical decomposition reaction Methods 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 230000007515 enzymatic degradation Effects 0.000 description 3
- 238000004192 high performance gel permeation chromatography Methods 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 239000001856 Ethyl cellulose Substances 0.000 description 2
- ZZSNKZQZMQGXPY-UHFFFAOYSA-N Ethyl cellulose Chemical compound CCOCC1OC(OC)C(OCC)C(OCC)C1OC1C(O)C(O)C(OC)C(CO)O1 ZZSNKZQZMQGXPY-UHFFFAOYSA-N 0.000 description 2
- 241000230491 Gulo Species 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- WUOACPNHFRMFPN-UHFFFAOYSA-N alpha-terpineol Chemical compound CC1=CCC(C(C)(C)O)CC1 WUOACPNHFRMFPN-UHFFFAOYSA-N 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 238000001354 calcination Methods 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- SQIFACVGCPWBQZ-UHFFFAOYSA-N delta-terpineol Natural products CC(C)(O)C1CCC(=C)CC1 SQIFACVGCPWBQZ-UHFFFAOYSA-N 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000003792 electrolyte Substances 0.000 description 2
- 229920001249 ethyl cellulose Polymers 0.000 description 2
- 235000019325 ethyl cellulose Nutrition 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 230000003020 moisturizing effect Effects 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 230000005622 photoelectricity Effects 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 150000003254 radicals Chemical class 0.000 description 2
- 238000006479 redox reaction Methods 0.000 description 2
- 238000002390 rotary evaporation Methods 0.000 description 2
- 238000007650 screen-printing Methods 0.000 description 2
- 239000004065 semiconductor Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 230000002195 synergetic effect Effects 0.000 description 2
- 229940116411 terpineol Drugs 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 229920002683 Glycosaminoglycan Polymers 0.000 description 1
- 108010003272 Hyaluronate lyase Proteins 0.000 description 1
- 102000001974 Hyaluronidases Human genes 0.000 description 1
- 206010062028 Keratitis bacterial Diseases 0.000 description 1
- NIPNSKYNPDTRPC-UHFFFAOYSA-N N-[2-oxo-2-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 NIPNSKYNPDTRPC-UHFFFAOYSA-N 0.000 description 1
- AFCARXCZXQIEQB-UHFFFAOYSA-N N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CCNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 AFCARXCZXQIEQB-UHFFFAOYSA-N 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 230000033115 angiogenesis Effects 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 230000007969 cellular immunity Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 238000006356 dehydrogenation reaction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 125000000600 disaccharide group Chemical group 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000005684 electric field Effects 0.000 description 1
- 238000005562 fading Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 229940097043 glucuronic acid Drugs 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 229960002773 hyaluronidase Drugs 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 238000005805 hydroxylation reaction Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- RUTXIHLAWFEWGM-UHFFFAOYSA-H iron(3+) sulfate Chemical group [Fe+3].[Fe+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O RUTXIHLAWFEWGM-UHFFFAOYSA-H 0.000 description 1
- 229910000360 iron(III) sulfate Inorganic materials 0.000 description 1
- 230000011164 ossification Effects 0.000 description 1
- 239000007800 oxidant agent Substances 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 238000010525 oxidative degradation reaction Methods 0.000 description 1
- 239000003002 pH adjusting agent Substances 0.000 description 1
- 238000007146 photocatalysis Methods 0.000 description 1
- 230000001699 photocatalysis Effects 0.000 description 1
- 239000000276 potassium ferrocyanide Substances 0.000 description 1
- 229940116357 potassium thiocyanate Drugs 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000027756 respiratory electron transport chain Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 230000037394 skin elasticity Effects 0.000 description 1
- 238000001694 spray drying Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- XOGGUFAVLNCTRS-UHFFFAOYSA-N tetrapotassium;iron(2+);hexacyanide Chemical compound [K+].[K+].[K+].[K+].[Fe+2].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-] XOGGUFAVLNCTRS-UHFFFAOYSA-N 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W10/00—Technologies for wastewater treatment
- Y02W10/30—Wastewater or sewage treatment systems using renewable energies
- Y02W10/37—Wastewater or sewage treatment systems using renewable energies using solar energy
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Materials Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The invention relates to the technical field of polysaccharide degradation, and particularly discloses a degradation method of hyaluronic acid. The degradation method of the hyaluronic acid comprises the following steps: a. adding the swelled hyaluronic acid solution and H into a photochemical reactor 2 O 2 Solution and Fe (CH) 3 COO) 3 Obtaining a mixed reaction solution; b. with TiO 2 The modified ITO electrode is a working electrode, the Pt electrode is a counter electrode, and the saturated calomel electrode is a reference electrode, so that a three-electrode photoelectrocatalysis reaction system is formed; and (3) under the conditions of externally applied bias and illumination, carrying out catalytic degradation on the mixed reaction solution to obtain a hyaluronic acid degradation product. The degradation method of the hyaluronic acid has the advantages of simple operation, high degradation efficiency, environmental protection, no pollution, short reaction time, low running cost and low molecular weight of degradation products.
Description
Technical Field
The invention relates to the technical field of polysaccharide degradation, in particular to a degradation method of hyaluronic acid.
Background
Hyaluronic Acid (HA) is a linear macromolecular acid mucopolysaccharide, mainly composed of β -1, 4-glucuronic acid and β -1, 3-N-acetylglucose as repeating disaccharide units, commonly known as hyaluronic acid. Hyaluronic acid is a high-molecular water-soluble substance, is widely distributed in human skin tissues and accounts for 2% -4% of the total weight of the human body, has the effects of improving rough skin, fading color spots, increasing skin elasticity and the like, and is known as a moisturizing agent of the skin and an ideal natural moisturizing factor. The application range of the hyaluronic acid is wide, and the hyaluronic acid is mainly used in various fields of cosmetics, medicines, health products, foods and the like. Research shows that the hyaluronic acid with low molecular weight (molecular weight range is less than 500 kDa) has higher bioactivity, has the effects of activating cell immunity, treating bacterial corneal ulcer, promoting angiogenesis, promoting osteogenesis and the like, is easier to be absorbed by human body compared with the hyaluronic acid with high molecular weight, and has potential application prospect.
Currently, degradation of hyaluronic acid mainly includes three major categories, physical degradation, chemical degradation and enzymatic degradation. The physical degradation method does not need to add chemical reagents, is simple to operate and mainly comprises an ultrasonic method, a microwave method and a high-pressure homogenizing degradation method, but has lower efficiency and is easy to cause side reactions. The chemical degradation method mainly comprises hydrolysis method, oxidative degradation and the like, and the hyaluronic acid is degraded by adding acid, alkali or oxidant, but the chemical degradation is easy to cause the difficulty in separating and purifying the product. The enzymatic degradation method is that the hyaluronic acid breaks the glycosidic bond under the action of hyaluronidase, and the enzymatic degradation condition is mild, but the cost is high.
Therefore, the development of the hyaluronic acid degradation method has the advantages of green pollution-free, high degradation efficiency, low product molecular weight, easy separation and purification and low degradation cost.
Disclosure of Invention
In order to overcome the problems of the prior degradation of hyaluronic acid, the invention provides a degradation method of hyaluronic acid, which introduces a photoelectric-Fenton combination system into the degradation process of hyaluronic acid to combine Fe (CH) 3 COO) 3 Can realize the full degradation of the hyaluronic acid,has the advantages of simple operation, high degradation efficiency, environmental protection, no pollution, short reaction time, low running cost and low molecular weight of degradation products.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a method for degrading hyaluronic acid, comprising the steps of:
a. adding the swelled hyaluronic acid solution and H into a photochemical reactor 2 O 2 Solution and Fe (CH) 3 COO) 3 Obtaining a mixed reaction solution;
b. with TiO 2 The modified ITO electrode is a working electrode, the Pt electrode is a counter electrode, and the saturated calomel electrode is a reference electrode, so that a three-electrode photoelectrocatalysis reaction system is formed; and (3) under the conditions of externally applied bias and illumination, carrying out catalytic degradation on the mixed reaction solution to obtain a hyaluronic acid degradation product.
Preferably, the working electrode is prepared as follows: cutting ITO conductive glass into 2.5cm×1.5cm, respectively ultrasonic treating in deionized water, acetone, and absolute ethanol for 30min, washing with deionized water, and drying. Titanium dioxide with the particle size of 25nm, ethyl cellulose and terpineol are taken as raw materials, ethanol is taken as a solvent, the raw materials are fully mixed and uniformly dispersed according to the mass ratio of 2:1:7, and the ethanol is removed by rotary evaporation, so that the uniformly dispersed membrane paste is prepared. And (3) printing the prepared paste film on the surface of ITO conductive glass through a screen printing method by 140 meshes, putting the conductive glass into an oven after printing, and drying for 30min at 150 ℃. And (3) placing the dried electrode slice in a muffle furnace, and carrying out annealing treatment for 2 hours at 500 ℃ with a heating rate of 5 ℃/min. And taking out after calcining, and naturally cooling to room temperature to obtain the titanium dioxide modified ITO electrode, namely the working electrode.
ITO conductive glass for making the working electrode is purchased from Luoyang gulo glass Co., ltd., tiO 2 Purchased from Yingchang De Guest company; the counter electrode is purchased from Sangzhi, inc. of Tianjin, model number chi115; the reference electrode was purchased from Tianjin Aida Heng Cheng, model R0232.
Preferably, in step a, the photochemical reactor is a quartz photochemical glass reaction vessel.
Preferably, in the step a, the swelling method of the hyaluronic acid solution is as follows: and (3) placing the hyaluronic acid solution at 18-26 ℃ and stirring for 4-6 hours to obtain the hyaluronic acid.
Preferably, in the step a, the concentration of the hyaluronic acid in the hyaluronic acid solution is 0.5-10mg/mL.
Preferably, in step a, H in the mixed reaction solution 2 O 2 Is 0.1% -0.5% by volume.
Preferably, in step a, H in the mixed reaction solution 2 O 2 Is 0.5% by volume.
Preferably, in step a, fe (CH 3 COO) 3 Is 1-10mM.
Preferably, in step a, fe (CH 3 COO) 3 Is 8mM.
Preferably, in step b, the externally applied bias voltage is 2-2.5V.
Preferably, in the step b, the light source of illumination is sunlight or simulated sunlight (100W-500W xenon lamp), and the distance between the light source and the mixed reaction solution is 5cm.
Preferably, in the step b, the time of the catalytic degradation is more than 0 and less than or equal to 30min.
Preferably, the degradation method of hyaluronic acid further comprises: and regulating the pH value of the hyaluronic acid degradation product to 9-11, centrifuging to remove sediment, and obtaining the hyaluronic acid degradation product with iron ions removed.
In the invention, iron ions and Fe are introduced into a photoelectric Fenton system 3+ The existence of the catalyst can influence the subsequent determination of the molecular weight and structure of the hyaluronic acid and the degradation products thereof, and after the pH value of the degradation products reaches a specific value by adding ammonia water, the iron ions can be precipitated in the system, so that the iron ions in the degradation products are sufficiently removed, and the subsequent detection and application of the low molecular weight hyaluronic acid are not influenced.
Preferably, the pH adjuster used to adjust the pH of the hyaluronic acid degradation product is aqueous ammonia.
Preferably, the pH of the hyaluronic acid degradation product is adjusted to 10.
Compared with the prior art, the invention adds Fe (CH) into the hyaluronic acid degradation system 3 COO) 3 And a photoelectric-Fenton combination system is introduced, and Fe is realized under the simultaneous action of illumination and current 3+ /Fe 2+ High rate of reaction recycle with H 2 O 2 In the system, the efficiency of generating OH by decomposition is extremely high, so that the capability of oxidizing and decomposing organic matters into small molecular substances is extremely high. The photoelectric-Fenton system is combined with the addition of specific iron ions to perform catalytic degradation, so that the photoelectric-Fenton system is a novel means for degrading hyaluronic acid, and H is decomposed fully and efficiently through the synergistic effect of photoelectricity and iron ions 2 O 2 The method can produce more active substances, greatly shortens the time for degrading the hyaluronic acid by using the photocatalysis alone (within 30 min), improves the degradation efficiency of the hyaluronic acid, and obtains the hyaluronic acid with low molecular weight (3.7 kDa), and has the advantages of simple operation and low running cost. In particular Fe (CH) 3 COO) 3 At a concentration of 8mM, the degradation efficiency is significantly better.
In the specific process of the invention for the photo-Fenton catalytic degradation of hyaluronic acid, photo-generated electron-hole pairs generated by the irradiation of light on the surface of a semiconductor contacted with electrolyte are separated by the electric field of the semiconductor or the electrolyte junction and then undergo oxidation-reduction reaction with ions in the solution, so that super-oxyanion (O) with extremely strong oxidizing property is generated in a reaction system 2 - ) Hydroxyl free radical (OH) and the like, and then the hyaluronic acid is degraded through the processes of addition, substitution and electron transfer between the free radical and the hyaluronic acid. The generated free radicals and holes have strong oxidizing property, meanwhile, fenton system is introduced, and through the synergistic effect of photoelectricity and iron ions, H is firstly 2 O 2 Is activated with Fe 2+ The reaction produces more OH, fe 2+ Quilt H 2 O 2 Oxidation to Fe 3+ The method comprises the steps of carrying out a first treatment on the surface of the Fe formed in the reaction 3+ Re-associated with H 2 O 2 React and be reduced to Fe 2+ ，H 2 O 2 Oxidized to HO 2 The method comprises the steps of carrying out a first treatment on the surface of the Generated byFe 2+ Re-associated with H 2 O 2 Reaction to produce OH, fe 2+ And Fe (Fe) 3+ The cyclic reaction between them completes the catalysis of H 2 O 2 Process of generating OH. Since OH has strong oxidizing power and high electronegativity or electrophilicity, it attacks hyaluronic acid through oxidation-reduction reaction, dehydrogenation reaction and hydroxylation reaction, so that it is degraded into small molecular substances.
Drawings
FIG. 1 shows the Fe (CH) values of example 1 3 COO) 3 High-efficiency gel permeation chromatogram of degradation products of hyaluronic acid at concentration;
FIG. 2 shows the different H in example 1 2 O 2 High-efficiency gel permeation chromatogram of degradation products of hyaluronic acid at concentration;
FIG. 3 is a high performance gel permeation chromatogram of the degradation products of hyaluronic acid at various catalytic degradation times in example 3;
FIG. 4 is a high performance gel permeation chromatogram of the degradation product of hyaluronic acid in comparative example 2;
FIG. 5 is a high performance gel permeation chromatogram of the degradation product of hyaluronic acid in comparative example 3.
Detailed Description
The following describes in further detail the embodiments of the present invention with reference to the drawings and examples. The following examples are illustrative of the invention and are not intended to limit the scope of the invention.
Example 1
A method for degrading hyaluronic acid, comprising the steps of:
a. preparing 2mg/mL hyaluronic acid solution (molecular weight of 1526kDa of hyaluronic acid), magnetically stirring at 20deg.C for 5 hr to make it fully swell to obtain swelled hyaluronic acid solution; 70mL of the swelled hyaluronic acid solution, H 2 O 2 Solution and Fe (CH) 3 COO) 3 Adding the solution into a quartz photochemical glass reactor (50 mm. Times.50 mm), stirring uniformly to obtain H 2 O 2 The final volume fraction reaches 0.5%, and a mixed reaction solution is obtained;
wherein Fe (CH) 3 COO) 3 The addition amount of the solution was 0, 1mM, 2mM, 5mM, 8mM, 10mM.
b. Simulating solar light irradiation with 500W xenon lamp, wherein the distance between the xenon lamp and the mixed reaction solution is 5cm, and under the condition of 2.5V external bias voltage, using titanium dioxide modified ITO electrode as working electrode (effective area of electrode is 2×1.5 cm) 2 ) The Pt electrode is a counter electrode, the Saturated Calomel Electrode (SCE) is a reference electrode, and a three-electrode photoelectrocatalytic reaction system is formed, and the above-mentioned different Fe (CH) are respectively 3 COO) 3 Catalyzing the mixed reaction solution obtained in the step a for 30min under the condition of adding amount to obtain a hyaluronic acid degradation product;
the working electrode was prepared as follows: cutting ITO conductive glass into 2.5cm×1.5cm, respectively ultrasonic treating in deionized water, acetone, and absolute ethanol for 30min, washing with deionized water, and drying. Titanium dioxide with the particle size of 25nm, ethyl cellulose and terpineol are taken as raw materials, ethanol is taken as a solvent, the raw materials are fully mixed and uniformly dispersed according to the mass ratio of 2:1:7, and the ethanol is removed by rotary evaporation, so that the uniformly dispersed membrane paste is prepared. And (3) printing the prepared paste film on the surface of ITO conductive glass through a screen printing method by 140 meshes, putting the conductive glass into an oven after printing, and drying for 30min at 150 ℃. And (3) placing the dried electrode slice in a muffle furnace, and carrying out annealing treatment for 2 hours at 500 ℃ with a heating rate of 5 ℃/min. And taking out after calcining, and naturally cooling to room temperature to obtain the titanium dioxide modified ITO electrode, namely the working electrode.
ITO conductive glass for making the working electrode is purchased from Luoyang gulo glass Co., ltd., tiO 2 Purchased from Yingchang De Guest company; the counter electrode is purchased from Sangzhi, inc. of Tianjin, model number chi115; the reference electrode was purchased from Tianjin Aida Heng Cheng, model R0232.
c. And adding ammonia water into the hyaluronic acid degradation product after the reaction is finished, wherein the pH value of the hyaluronic acid degradation product reaches 10, generating precipitate, centrifuging to remove the precipitate, and obtaining the hyaluronic acid degradation product with iron ions removed.
To identify Fe in solution 2+ And Fe (Fe) 3+ Whether or not to removeThen, 5mL of the reaction solution was taken in a test tube, and potassium thiocyanate (KSCN) and potassium ferrocyanide (K) were added to the solutions, respectively 3 Fe(CN) 6 ) Qualitative identification of Fe contained in solution according to phenomenon change and color reaction 2+ And Fe (Fe) 3+ . The solution became red after the KSCN was added dropwise, indicating free Fe 3+ Dripping K 3 Fe(CN) 6 Post blue precipitation indicates free Fe 2+ 。
When ammonia water is not added, KSCN and K are added dropwise 3 Fe(CN) 6 The color of the solution changes obviously, which proves that a large amount of Fe exists in the degraded solution 2+ And Fe (Fe) 3+ . When the pH value of the ammonia water solution is increased, the color of the solution becomes light, fe in the solution 2+ And Fe (Fe) 3+ The decrease starts. When ammonia water is added dropwise until the pH value of the solution is 10, the solution basically does not change color, fe 2+ And Fe (Fe) 3+ The precipitation was complete. Therefore, when the pH is adjusted to 10, free Fe in the solution after the reaction can be completely removed 2+ And Fe (Fe) 3+ 。
1mL of the hyaluronic acid degradation product was passed through a 0.22 μm aqueous membrane, and the relative molecular mass (Mw) was measured by high performance gel permeation chromatography using a TSK-gel G5000PWxl (7.8 mm. Times.30 cm) column with a differential refractive index detector at a column box temperature of 30 ℃. The mobile phase is ammonium acetate buffer (0.1 mol/L, pH 6.0) with a flow rate of 0.4mL/min; dextran (relative molecular masses 5, 12, 25, 50, 150, 410 and 670kDa, respectively) was used as standard. Taking the retention time tR of a chromatographic peak as an abscissa and Mw of lg as an ordinate as a standard curve, comparing the molecular weight of the hyaluronic acid after catalytic degradation under different conditions, wherein the retention time is inversely proportional to the molecular weight of the hyaluronic acid or degradation products thereof;
as shown in FIG. 1, under the same conditions, fe (CH) 3 COO) 3 The addition of the Fe (CH) can obviously improve the degradation efficiency, and the Fe (CH) with a certain concentration 3 COO) 3 The smaller the molecular weight of the catalytic degradation hyaluronic acid is, the more the addition amount is increased, but Fe (CH) 3 COO) 3 The product at 8mM has a smaller molecular weight than the product at 10mM, indicating that Fe (CH) 3 COO) 3 The highest degradation efficiency at a concentration of 8mM。
Example 2
A method for degrading hyaluronic acid, comprising the steps of:
a. preparing 0.5mg/mL hyaluronic acid solution (molecular weight of 1526kDa of hyaluronic acid), magnetically stirring at 18deg.C for 4 hr to make it fully swell to obtain swelled hyaluronic acid solution; 70mL of the swelled hyaluronic acid solution, H 2 O 2 Solution and Fe (CH) 3 COO) 3 Adding the solution into a quartz photochemical glass reactor (50 mm. Times.50 mm), stirring uniformly to obtain Fe (CH) 3 COO) 3 The final concentration reaches 8mM, and a mixed reaction solution is obtained;
wherein H is added 2 O 2 The amount of solution is such that H in the final mixed reaction solution 2 O 2 The volume fractions of (a) respectively reach 0%,0.1%,0.2%,0.3%,0.4% and 0.5%;
b. simulating solar light irradiation with 500W xenon lamp, wherein the distance between the xenon lamp and the mixed reaction solution is 5cm, and under the condition of 2.2V external bias voltage, using titanium dioxide modified ITO electrode as working electrode (effective area of electrode is 2×1.5 cm) 2 The preparation method is the same as in example 1), the Pt electrode is a counter electrode, the Saturated Calomel Electrode (SCE) is a reference electrode, and a three-electrode photoelectrocatalytic reaction system is formed, and the different Fe are respectively 2+ Catalyzing the mixed reaction solution obtained in the step a for 30min under the condition of adding amount to obtain a hyaluronic acid degradation product;
c. and adding ammonia water into the hyaluronic acid degradation product after the reaction is finished, so that the pH of the hyaluronic acid degradation product reaches 9, generating precipitate, centrifuging to remove the precipitate, and obtaining the degradation product of the hyaluronic acid with iron ions removed.
The degradation products were tested for free iron ions by the method of example 1, and when aqueous ammonia was added dropwise to a pH of the solution of 9, the solution showed substantially no color change, fe 2+ And Fe (Fe) 3+ The precipitation was complete. Therefore, when the pH is adjusted to 9, free Fe in the solution after the reaction can be completely removed 2+ And Fe (Fe) 3+ 。
1mL of the hyaluronic acid degradation product was passed through a 0.22 μm aqueous membrane, and the relative molecular mass (Mw) was measured by high performance gel permeation chromatography using a TSK-gel G5000PWxl (7.8 mm. Times.30 cm) column with a differential refractive index detector at a column box temperature of 30 ℃. The mobile phase is ammonium acetate buffer (0.1 mol/L, pH 6.0) with a flow rate of 0.4mL/min; dextran (relative molecular masses 5, 12, 25, 50, 150, 410 and 670kDa, respectively) was used as standard. Comparing the molecular weight of the hyaluronic acid after catalytic degradation under different conditions by taking the retention time tR of the chromatographic peak as an abscissa and the Mw of lg as an ordinate as a standard curve, wherein HA represents the undegraded hyaluronic acid;
under the same conditions, H as shown in FIG. 2 2 O 2 The addition of the catalyst can obviously improve the degradation efficiency, and the catalyst is along with H within a certain range 2 O 2 The smaller the molecular weight of the catalytic degradation hyaluronic acid, the higher the degradation efficiency.
Example 3
A method for degrading hyaluronic acid, comprising the steps of:
a. preparing 10mg/mL hyaluronic acid solution (molecular weight of 1526kDa of hyaluronic acid), magnetically stirring at 26 ℃ for 6 hours to fully swell the hyaluronic acid solution, and obtaining a swelled hyaluronic acid solution; 70mL of the swelled hyaluronic acid solution, H 2 O 2 Solution and Fe (CH) 3 COO) 3 Adding the solution into a quartz photochemical glass reactor (50 mm. Times.50 mm), stirring uniformly to obtain Fe (CH) 3 COO) 3 Final concentration reached 8mM, H 2 O 2 The final volume fraction reached 0.5%, yielding a mixed reaction solution.
b. Simulating solar light irradiation with 500W xenon lamp, wherein the distance between the xenon lamp and the mixed reaction solution is 5cm, and under the condition of 2V external bias voltage, using titanium dioxide modified ITO electrode as working electrode (effective area of electrode is 2×1.5 cm) 2 The preparation method is the same as in example 1), the Pt electrode is a counter electrode, the Saturated Calomel Electrode (SCE) is a reference electrode, a three-electrode photoelectrocatalysis reaction system is formed, and the mixed reaction solution obtained in the step a is catalyzed for different time to obtain a hyaluronic acid degradation product;
the catalytic time was 0min,5min,10min,15min,20min,25min,30min in this order.
c. And adding ammonia water into the hyaluronic acid degradation product after the reaction is finished, so that the pH of the hyaluronic acid degradation product reaches 11, generating precipitate, centrifuging and removing the precipitate to obtain the degradation product of hyaluronic acid.
The degradation products were tested for free iron ions by the method of example 1, and when aqueous ammonia was added dropwise to a pH of the solution of 11, the solution showed substantially no color change, fe 2+ And Fe (Fe) 3+ The precipitation was complete. Therefore, when the pH is adjusted to 11, free Fe in the solution after the reaction can be completely removed 2+ And Fe (Fe) 3+ 。
1mL of the hyaluronic acid degradation product was respectively taken and passed through a 0.22 μm aqueous membrane, and the relative molecular mass (Mw) was measured by high performance gel permeation chromatography using a TSK-gel G5000PWxl (7.8 mm. Times.30 cm) column with a differential refractive index detector at a column box temperature of 30 ℃. The mobile phase is ammonium acetate buffer (0.1 mol/L, pH 6.0) with a flow rate of 0.4mL/min; dextran (relative molecular masses 5, 12, 25, 50, 150, 410 and 670kDa, respectively) was used as standard. Taking the retention time tR of a chromatographic peak as an abscissa and Mw of lg as an ordinate as a standard curve, and comparing the molecular weight of hyaluronic acid after catalytic degradation under different conditions;
as shown in FIG. 3, the hyaluronic acid used in this example has an original molecular weight of 1526kDa, a relative molecular weight of 16kDa after 5min degradation, a molecular weight of 6.4kDa after 10min degradation, a molecular weight of 3.8kDa after 15min degradation, a relative molecular weight of 3.8kDa after 20min degradation, a relative molecular weight of 3.7kDa after 25min degradation, and a relative molecular weight of 3.7kDa after 30min degradation.
Comparative example 1
A method for degrading hyaluronic acid, wherein Fe (CH) in example 3 is replaced with ferric sulfate 3 COO) 3 The concentration of iron ions in the mixed reaction solution was 8mM, and the amounts of other reagents and the method were the same as in example 3, to obtain a hyaluronic acid degradation product.
In the dry powder obtained after spray drying of the hyaluronic acid degradation product, the inorganic salt content is 18%, which indicates that iron ions in the dry powder cannot be sufficiently removed, so that the impurity content of the degradation product of the hyaluronic acid is high, and the normal use of the degradation product is affected.
Comparative example 2
A method for degrading hyaluronic acid, wherein Fe (CH) is added in step a 3 COO) 3 The amount of the solution added was 8mM, and the setting of the working electrode, counter electrode and reference electrode was omitted in step b, and the other reagent amounts and methods were the same as in example 3, to obtain a hyaluronic acid degradation product.
The molecular weight of hyaluronic acid in the hyaluronic acid degradation product obtained at the time of catalytic degradation for 30min was measured, and as shown in fig. 4, the molecular weight of hyaluronic acid was reduced to 115kDa.
Comparative example 3
A method for degrading hyaluronic acid, wherein Fe (CH) is omitted in step a 3 COO) 3 And H 2 O 2 The solution was added in the same amount and method as in example 3 to obtain a hyaluronic acid degradation product.
The molecular weight of hyaluronic acid in the hyaluronic acid degradation product obtained at the time of catalytic degradation for 30min was measured, and as shown in fig. 5, the molecular weight of hyaluronic acid was reduced to 68kDa.
Claims (10)
1. A method for degrading hyaluronic acid, characterized in that: the method comprises the following steps:
a. adding the swelled hyaluronic acid solution and H into a photochemical reactor 2 O 2 Solution and Fe (CH) 3 COO) 3 Obtaining a mixed reaction solution;
b. with TiO 2 The modified ITO electrode is a working electrode, the Pt electrode is a counter electrode, and the saturated calomel electrode is a reference electrode, so that a three-electrode photoelectrocatalysis reaction system is formed; and (3) under the conditions of externally applied bias and illumination, carrying out catalytic degradation on the mixed reaction solution to obtain a hyaluronic acid degradation product.
2. The method for degrading hyaluronic acid according to claim 1, wherein: in the step a, the photochemical reactor is a quartz photochemical glass reaction vessel.
3. The method for degrading hyaluronic acid according to claim 1, wherein: in the step a, the swelling method of the hyaluronic acid solution comprises the following steps: and (3) placing the hyaluronic acid solution at 18-26 ℃ and stirring for 4-6 hours to obtain the hyaluronic acid.
4. A method for degrading hyaluronic acid according to claim 1 or 3, characterized in that: in the step a, the concentration of hyaluronic acid in the hyaluronic acid solution is 0.5-10mg/mL.
5. The method for degrading hyaluronic acid according to claim 1, wherein: in step a, H in the mixed reaction solution 2 O 2 Is 0.1-0.5% by volume;
and/or in step a, fe (CH) 3 COO) 3 Is 1-10mM.
6. The method for degrading hyaluronic acid according to claim 1, wherein: in the step b, the externally applied bias voltage is 2-2.5V.
7. The method for degrading hyaluronic acid according to claim 1, wherein: in the step b, the light source of illumination is sunlight or simulated sunlight, and the distance between the light source and the mixed reaction solution is 5cm.
8. The method for degrading hyaluronic acid according to claim 1, wherein: in the step b, the time of catalytic degradation is more than 0 and less than or equal to 30min.
9. The method for degrading hyaluronic acid according to claim 1, wherein: further comprises: and regulating the pH value of the hyaluronic acid degradation product to 9-11, centrifuging to remove sediment, and obtaining the hyaluronic acid degradation product with iron ions removed.
10. The method for degrading hyaluronic acid according to claim 9, wherein: the pH regulator used for regulating the pH of the hyaluronic acid degradation product is ammonia water; and/or the number of the groups of groups,
the pH of the hyaluronic acid degradation product was adjusted to 10.
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