CN116850086A - Epicatechin hydrogel and preparation method and application thereof - Google Patents
Epicatechin hydrogel and preparation method and application thereof Download PDFInfo
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- CN116850086A CN116850086A CN202310793343.7A CN202310793343A CN116850086A CN 116850086 A CN116850086 A CN 116850086A CN 202310793343 A CN202310793343 A CN 202310793343A CN 116850086 A CN116850086 A CN 116850086A
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- hydrogel
- epicatechin
- hyaluronic acid
- metal ions
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- 239000000017 hydrogel Substances 0.000 title claims abstract description 168
- PFTAWBLQPZVEMU-ZFWWWQNUSA-N (+)-epicatechin Natural products C1([C@@H]2OC3=CC(O)=CC(O)=C3C[C@@H]2O)=CC=C(O)C(O)=C1 PFTAWBLQPZVEMU-ZFWWWQNUSA-N 0.000 title claims abstract description 115
- LPTRNLNOHUVQMS-UHFFFAOYSA-N epicatechin Natural products Cc1cc(O)cc2OC(C(O)Cc12)c1ccc(O)c(O)c1 LPTRNLNOHUVQMS-UHFFFAOYSA-N 0.000 title claims abstract description 115
- 235000012734 epicatechin Nutrition 0.000 title claims abstract description 115
- PFTAWBLQPZVEMU-UKRRQHHQSA-N (-)-epicatechin Chemical compound C1([C@H]2OC3=CC(O)=CC(O)=C3C[C@H]2O)=CC=C(O)C(O)=C1 PFTAWBLQPZVEMU-UKRRQHHQSA-N 0.000 title claims abstract description 114
- 238000002360 preparation method Methods 0.000 title claims abstract description 20
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- USHAGKDGDHPEEY-UHFFFAOYSA-L potassium persulfate Chemical compound [K+].[K+].[O-]S(=O)(=O)OOS([O-])(=O)=O USHAGKDGDHPEEY-UHFFFAOYSA-L 0.000 description 1
- ZDWVWKDAWBGPDN-UHFFFAOYSA-O propidium Chemical compound C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 ZDWVWKDAWBGPDN-UHFFFAOYSA-O 0.000 description 1
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Classifications
-
- 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
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
- A61K8/04—Dispersions; Emulsions
- A61K8/042—Gels
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
- A61K8/4973—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with oxygen as the only hetero atom
- A61K8/498—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with oxygen as the only hetero atom having 6-membered rings or their condensed derivatives, e.g. coumarin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
- A61Q17/005—Antimicrobial preparations
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
- A61Q17/04—Topical preparations for affording protection against sunlight or other radiation; Topical sun tanning preparations
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
- A61K2800/52—Stabilizers
- A61K2800/522—Antioxidants; Radical scavengers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
- A61K2800/59—Mixtures
- A61K2800/592—Mixtures of compounds complementing their respective functions
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Epidemiology (AREA)
- Birds (AREA)
- Dermatology (AREA)
- Chemical & Material Sciences (AREA)
- Dispersion Chemistry (AREA)
- Gerontology & Geriatric Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses epicatechin hydrogel and a preparation method and application thereof. According to the invention, epicatechin is introduced into hyaluronic acid, a part of hydrogen bonds are formed into a weak hydrogen bond network by adopting a hyaluronic acid chain with a certain carboxylic acid/carboxylate ratio under a proper pH value, metal ions are introduced to form a metal-carboxylate coordination strong bond, and simultaneously, phenolic hydroxyl groups on the molecular structure of epicatechin and hydroxyl groups on the hyaluronic acid form a hydrogen bond effect, and a complexation effect is formed between the phenolic hydroxyl groups and the metal ions, so that a multiple cross-linked network structure is formed by assembly, the mechanical property of the hyaluronic acid hydrogel is improved, and the hyaluronic acid hydrogel with excellent photodamage resistance, tissue repair property and good tissue adhesion capability is obtained; meanwhile, the synergistic effect of the metal ions and epicatechin acid also improves the antibacterial capability of the hydrogel, has long-term tissue adhesion stability, and ensures the timeliness of the hydrogel in tissue action.
Description
Technical Field
The invention belongs to the technical field of hydrogel preparation, and particularly relates to epicatechin hydrogel, and a preparation method and application thereof.
Technical Field
Hydrogels are networks of hydrophilic polymer chains in which water is the dispersing medium. The hydrogel has special swelling property and water-retaining function by virtue of a hydrophilic three-dimensional network structure, and is widely applied to the fields of crude oil dehydration, medical drug carriers, sensing and the like. Wherein, the hyaluronic acid hydrogel belongs to natural hydrogel. Hyaluronic Acid (HA) is a linear natural glycosaminoglycan, which is widely found in animal skin, connective tissue nerve tissue. The repeating units of HA consist of D-glucuronic acid and N-acetylglucosamine. The negative charge in HA is combined with different cations (such as Na + 、Ca 2+ 、K + ) Balanced with each other, and the HA HAs high hydrophilicity. And a large number of hydroxyl and carboxyl functional groups in the HA molecular chain have strong binding capacity to water molecules under the action of intermolecular hydrogen bonds, so that the HA obtains excellent water-retaining property. The existence of carboxyl, hydroxyl and acetamido also enables HA to have good chemical modification potential in aqueous solution, thus laying foundation for application in the field of hydrogel. However, since biological materials based on hyaluronic acid generally exhibit poor mechanical properties, hyaluronic acid is chemically modified in the main chain and used in combination with a crosslinking agent to improve the mechanical properties of the gel in many cases.
Polyphenols become a biomaterial construction material with various advantages due to their functional diversity, biocompatibility and biodegradability and the ability to be assembled by different mechanisms. Biological materials with different physical and chemical characteristics, stimulus response and different scales can be prepared by simply mixing polyphenol and polymer. The polyphenols have multiple hydrophobic aromatic rings and hydrophilic phenolic hydroxyl groups, which provide rich reaction sites for the polyphenols, and can also interact with other groups or substances through non-covalent interactions, such as hydrogen bonds, hydrophobic interactions and van der Waals forces, which provides the possibility for the polyphenols to be applied in the hydrogel field.
Epicatechin is a class of polyphenol hydroxy organic compounds extracted from green tea and has various pharmacological effects. However, epicatechin is often used as a functional modifier (used as an auxiliary agent in a low dosage) in the prior art, and is added and applied to hyaluronic acid or hydrogel to modify the viscosity thereof; besides excellent antioxidant and anti-aging properties, epicatechin has many effects which are not developed and applied, and the epicatechin is not used as a main active substance for preparing hydrogel materials for resisting photodamage and repairing tissues.
Disclosure of Invention
The invention aims to overcome the defects and shortcomings of the problems and provide a preparation method and application of epicatechin hydrogel.
It is a first object of the present invention to provide an epicatechin hydrogel.
A second object of the present invention is to provide the use of the epicatechin hydrogel.
A third object of the present invention is to provide a method for producing epicatechin hydrogel.
The above object of the present invention is achieved by the following technical scheme:
the invention provides epicatechin hydrogel which is formed by mixing hyaluronic acid and epicatechin and introducing metal ions for crosslinking.
Epicatechin is also known as Epicatechin (EC) of formula C 15 H 14 O 6 Molecular weight: 290.27. the epicatechin flavonoids have effects of resisting oxidation and scavenging free radicals, and can promote heart health. Studies on epicatechin have found that increasing epicatechin levels enhances the ability of plasma to scavenge free radicals and inhibit lipid peroxidation and destruction.
Hyaluronic Acid (HA) is a linear natural glycosaminoglycanIs widely present in animal skin and connective tissue nerve tissue. The repeating units of HA consist of D-glucuronic acid and N-acetylglucosamine. The negative charge in HA is combined with different cations (such as Na + 、Ca 2+ 、K + ) Balanced with each other, and the HA HAs high hydrophilicity. And a large number of hydroxyl and carboxyl functional groups in the HA molecular chain have strong binding capacity to water molecules under the action of intermolecular hydrogen bonds, so that the HA obtains excellent water-retaining property. The existence of carboxyl, hydroxyl and acetamido also enables HA to have good chemical modification potential in aqueous solution, thus laying foundation for application in the field of hydrogel.
The invention uses epicatechin with excellent photodamage resistance and tissue repair performance as a main active ingredient, and introduces the epicatechin into hyaluronic acid to prepare the functional hydrogel with metal ions, wherein the epicatechin not only serves as a functional modifier to change the mechanical properties of the hyaluronic acid, but also serves as a cross-linking agent to increase the cross-linking degree of the hyaluronic acid through hydrogen bonding, so that the mechanical properties are further improved. The epicatechin is made to act on the hyaluronic acid by the hydrogen bonding action of the phenolic hydroxyl group on the epicatechin molecular structure and the hydroxyl group on the hyaluronic acid; under proper pH value, metal ions are simultaneously introduced, strong bonds are formed through metal-carboxylate coordination, and complexation is formed between phenolic hydroxyl groups and the metal ions, so that the epicatechin hydrogel obtains strong mechanical properties, the action strength and the toughness adhesion capability of the epicatechin hydrogel and skin tissues are improved, and the epicatechin hydrogel has better stability in the subsequent application process. And the synergistic effect of the metal ions and epicatechin acid improves the antibacterial capability of the hydrogel, and simultaneously has long-term tissue adhesion stability, so that the timeliness of the hydrogel in tissue action is ensured.
Further, the hyaluronic acid is a 10.0wt% aqueous solution.
Further, the epicatechin is 10.0 to 25.0 weight percent aqueous solution.
Further, the metal ion is Fe 3+ ，Cu 2+ ，Co 2+ ，Ni 2+ ，Zn 2+ ，Cd 2+ And Ag + Is a kind of the above-mentioned materials.
Preferably, the concentration of the metal ions is 0.3 to 0.7mol/mL.
More preferably, the metal ion is Fe 3+ . The iron ions can effectively absorb ultraviolet radiation, and the iron ions are used as a good ultraviolet resistant agent, and cooperate with epicatechin to further improve the photodamage resistance and tissue repair capability of the hyaluronic acid hydrogel, so that the hyaluronic acid hydrogel has obvious photodamage resistance and tissue repair effects.
The method introduces Fe after mixing hyaluronic acid and epicatechin 3+ Crosslinking to achieve the purpose of functional modification, epicatechin and Fe 3+ Has synergistic effect, and can improve tissue repair ability, antibacterial performance, DPPH and ABTS of hyaluronic acid hydrogel + The free radical scavenging capability ensures that the hyaluronic acid hydrogel has better photodamage resistance and tissue repair performance, can resist ultraviolet light and repair tissue damage generated by the ultraviolet light in time.
The invention provides a preparation method of hydrogel, which comprises the steps of mixing hyaluronic acid with epicatechin, adjusting pH to be acidic, introducing metal ions for crosslinking, and dialyzing to obtain the hydrogel.
Preferably, the volume ratio of the hyaluronic acid to epicatechin is 1-2: 1.
further preferably, the volume ratio of hyaluronic acid to epicatechin is 1:1.
Preferably, the pH is adjusted to be acidic to be 2.5-2.8, specifically acetate is adopted for adjustment, and the volume ratio of acetate to hyaluronic acid is 1: 120-130.
As the most preferred embodiment, the invention provides a preparation method of epicatechin hydrogel, which comprises the following steps:
hyaluronic acid powder was dissolved in deionized water at room temperature, and was gently stirred to prepare an HA (10.0 wt%) stock solution, which was placed in a centrifuge tube and centrifuged at 10000rpm/min at 37℃for 10min to remove air bubbles. The epicatechin powder is prepared by mixing epicatechin powder withDissolving in deionized water at room temperature to obtain epicatechin (10.0-25.0 wt%) aqueous solution, placing the epicatechin aqueous solution in a centrifuge tube, and centrifuging at 37deg.C and 10000rpm/min for 10min to remove air bubbles. Taking the 10mL of HA stock solution, 10mL of epicatechin solution and 0.6mL of HNO 3 (1.0M) solution was mixed uniformly, and 4mL of Fe (NO) was added thereto 3 ) 3 (0.5M) solution. Immediately and uniformly mixing to form elastic hydrogel, sealing and preserving for 24 hours at 25 ℃, then placing the elastic hydrogel in a dialysis bag, placing the dialysis bag in a 1000mL large beaker, and dialyzing for 24 hours at room temperature by using deionized water to obtain the gel.
The invention also provides application of the hydrogel in photodamage resistance, antibacterial property, tissue repair and/or antioxidation.
The invention also provides application of the hydrogel in preparation of photodamage-resistant, antibacterial, tissue repair and/or antioxidant products.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides epicatechin hydrogel which is formed by mixing hyaluronic acid and epicatechin and introducing metal ions for crosslinking. The multi-crosslinked network formed by hydrogen bond crosslinking between epicatechin and hyaluronic acid and metal-carboxylate coordination strong crosslinking between metal ions and hyaluronic acid obviously improves the mechanical property of the hyaluronic acid hydrogel, so that the hyaluronic acid hydrogel has long-term tissue adhesion stability, and the service life and the service stability of the hydrogel in the subsequent application process are ensured. Meanwhile, the introduced metal ions and epicatechin have a synergistic effect, so that the hyaluronic acid hydrogel has good antibacterial performance, can effectively absorb ultraviolet radiation through the metal ions, is used as a good ultraviolet inhibitor, and can further improve the photodamage resistance and tissue repair capability of the hyaluronic acid hydrogel in cooperation with epicatechin, and can timely repair tissue damage generated by ultraviolet light while resisting ultraviolet light, so that the hyaluronic acid hydrogel has remarkable photodamage resistance and tissue repair effects.
The epicatechin hydrogel provided by the invention has the advantages of simple preparation method and short preparation time, and can achieve obvious action and effect, thereby ensuring the effective action time and long service life of epicatechin and other functional components and tissues.
Detailed Description
The invention is further illustrated below in connection with specific examples which are intended to be illustrative of the invention and are not to be construed as limiting the invention, the specific techniques or conditions not identified in the examples being according to the techniques or conditions described in the literature in this field or according to the product specifications.
The hyaluronic acid powder used in the following examples was purchased from Guangzhou Huayun biotechnology Co., ltd and epicatechin powder was purchased from Shanghai Ji biotechnology Co., ltd.
EXAMPLE 1 preparation of epicatechin hydrogel
Dissolving hyaluronic acid powder in deionized water at room temperature, slightly stirring to obtain 10.0wt% HA stock solution, placing the HA stock solution in a centrifuge tube, and centrifuging at 37deg.C and 10000rpm/min for 10min to remove air bubbles for use.
Dissolving epicatechin powder in deionized water at room temperature, slightly stirring to obtain 10.0wt% epicatechin aqueous solution, placing epicatechin aqueous solution in a centrifuge tube, and centrifuging at 37deg.C and 10000rpm/min for 10min to remove bubbles for use.
Taking 10mL of HA stock solution, 10mL of epicatechin solution and 0.6mL of HNO 3 (1.0M) solution was mixed uniformly, and 4mL of Fe (NO) was added thereto 3 ) 3 (0.5M) solution. Immediately and uniformly mixing to form elastic hydrogel, sealing and preserving for 24 hours at 25 ℃, then placing the hydrogel into a dialysis bag, placing the dialysis bag into a 1000mL large beaker, and dialyzing the dialysis bag for 24 hours at room temperature by using deionized water to obtain the hydrogel HA-1.
EXAMPLE 2 preparation of epicatechin hydrogel
Dissolving hyaluronic acid powder in deionized water at room temperature, slightly stirring to obtain 10.0wt% HA stock solution, placing the HA stock solution in a centrifuge tube, and centrifuging at 37deg.C and 10000rpm/min for 10min to remove air bubbles for use.
Dissolving epicatechin powder in deionized water at room temperature, slightly stirring to obtain epicatechin aqueous solution of 15.0wt%, placing epicatechin aqueous solution in a centrifuge tube, and centrifuging at 37deg.C and 10000rpm/min for 10min to remove air bubbles for use.
Taking the 10mL of HA stock solution, 10mL of epicatechin solution and 0.6mL of HNO 3 (1.0M) solution was mixed uniformly, and 4mL of Fe (NO) was added thereto 3 ) 3 (0.5M) solution. Immediately and uniformly mixing to form elastic hydrogel, sealing and preserving for 24 hours at 25 ℃, then placing the hydrogel into a dialysis bag, placing the dialysis bag into a 1000mL large beaker, and dialyzing the dialysis bag for 24 hours at room temperature by using deionized water to obtain the hydrogel HA-2.
EXAMPLE 3 preparation of epicatechin hydrogel
Dissolving hyaluronic acid powder in deionized water at room temperature, slightly stirring to obtain 10.0wt% HA stock solution, placing the HA stock solution in a centrifuge tube, and centrifuging at 37deg.C and 10000rpm/min for 10min to remove air bubbles for use.
Dissolving epicatechin powder in deionized water at room temperature, slightly stirring to obtain 20.0wt% epicatechin aqueous solution, placing epicatechin aqueous solution in a centrifuge tube, and centrifuging at 37deg.C and 10000rpm/min for 10min to remove bubbles for use.
Taking the 10mL of HA stock solution, 10mL of epicatechin solution and 0.6mL of HNO 3 (1.0M) solution was mixed uniformly, and 4mL of Fe (NO) was added thereto 3 ) 3 (0.5M) solution. Immediately and uniformly mixing to form elastic hydrogel, sealing and preserving for 24 hours at 25 ℃, then placing the hydrogel into a dialysis bag, placing the dialysis bag into a 1000mL large beaker, and dialyzing the dialysis bag for 24 hours at room temperature by using deionized water to obtain the hydrogel HA-3.
EXAMPLE 4 preparation of epicatechin hydrogel
Dissolving hyaluronic acid powder in deionized water at room temperature, slightly stirring to obtain 10.0wt% HA stock solution, placing the HA stock solution in a centrifuge tube, and centrifuging at 37deg.C and 10000rpm/min for 10min to remove air bubbles for use.
Dissolving epicatechin powder in deionized water at room temperature, slightly stirring to obtain 25.0wt% epicatechin aqueous solution, placing epicatechin aqueous solution in a centrifuge tube, and centrifuging at 37deg.C and 10000rpm/min for 10min to remove bubbles for use.
Taking the 10mL of HA stock solution, 10mL of epicatechin solution and 0.6mL of HNO 3 (1.0M) solution was mixed uniformly, and 4mL of Fe (NO) was added thereto 3 ) 3 (0.5M) solution. Immediately and uniformly mixing to form elastic hydrogel, sealing and preserving for 24 hours at 25 ℃, then placing the hydrogel into a dialysis bag, placing the dialysis bag into a 1000mL large beaker, and dialyzing the dialysis bag for 24 hours at room temperature by using deionized water to obtain the hydrogel HA-4.
EXAMPLE 5 preparation of epicatechin hydrogel
Dissolving hyaluronic acid powder in deionized water at room temperature, slightly stirring to obtain 10.0wt% HA stock solution, placing the HA stock solution in a centrifuge tube, centrifuging at 37deg.C and 10000rpm/min for 10min to remove bubbles, and sealing at 25deg.C for 24 hr.
Dissolving epicatechin powder in deionized water at room temperature, slightly stirring to obtain 25.0wt% epicatechin aqueous solution, placing epicatechin aqueous solution in a centrifuge tube, and centrifuging at 37deg.C and 10000rpm/min for 10min to remove bubbles for use.
Mixing the 10mL of HA stock solution and 10mL of epicatechin solution uniformly to form hydrogel, sealing and preserving for 24 hours at 25 ℃, then placing the hydrogel in a dialysis bag, placing the dialysis bag in a 1000mL large beaker, and dialyzing for 24 hours at room temperature by using deionized water to obtain hydrogel HA-EC.
Comparative example 1 preparation of hyaluronic acid hydrogel
Dissolving hyaluronic acid powder in deionized water at room temperature, slightly stirring to obtain 10.0wt% of HA stock solution, placing the HA stock solution in a centrifuge tube, centrifuging at 37 ℃ and 10000rpm/min for 10min to remove bubbles, sealing and preserving at 25 ℃ for 24h, placing in a dialysis bag, placing in a 1000ml large beaker, and dialyzing with deionized water at room temperature for 24h to obtain hydrogel HA-0.
Comparative example 2 preparation of Metal ion hyaluronic acid hydrogel
Dissolving hyaluronic acid powder in deionized water at room temperature, slightly stirring to obtain 10.0wt% HA stock solution, placing the HA stock solution in a centrifuge tube, and centrifuging at 37deg.C and 10000rpm/min for 10min to remove air bubbles for use.
Taking 10mL of HA stock solution and 0.3mL HNO 3 (1.0M) solution was mixed uniformly, and 2mL of Fe (NO) was added thereto 3 ) 3 (0.5M) solution. Immediately and uniformly mixing to form elastic hydrogel, sealing and preserving for 24 hours at 25 ℃, then placing the hydrogel into a dialysis bag, placing the dialysis bag into a 1000ml large beaker, and dialyzing the dialysis bag for 24 hours at room temperature by deionized water to obtain the hydrogel HA-Fe.
Test example 1 mechanical Property test
The hydrogels prepared in examples 1-5 and comparative examples 1-2 were made into standard dumbbell-shaped bars, the bars were mounted on clamps of a universal tensile machine with a distance of (20) mm between the clamps and a tensile rate of (50) mm/min, tensile testing was performed, all bars were measured three times or more, and the average was calculated.
As a result, as shown in table 1, from the results of table 1, it can be seen that the tensile strength of the hydrogel in examples 1 to 5 was significantly improved as compared with the hydrogel in comparative examples 1 to 2 to which epicatechin had not been added, and that the mechanical properties of the epicatechin-added hydrogel in example 5 were also improved as compared with comparative example 1 to which epicatechin had not been added as the epicatechin content was increased. This is because the phenolic hydroxyl groups of the added epicatechin as cross-linking points cross-link the hyaluronic acid molecules through hydrogen bonds to increase the cross-linking density of the hydrogel and thus the tensile strength of the hydrogel. Adding Fe into 3+ The hydrogel of comparative example 2 of (2) compared with the one without Fe added 3+ The hydrogel in comparative example 1 of which the tensile strength is improved by approximately 3 to 10 times shows that, under a proper pH value, a part of hydrogen bonds are formed into a weak hydrogen bond network by adopting a hyaluronic acid chain with a certain carboxylic acid/carboxylate ratio, metal ions are introduced, and a strong cross-linking bond coordinated by metal-carboxylate is formed between the metal ions and the hyaluronic acid, so that the mechanical property of the hyaluronic acid hydrogel is remarkably improved. The multi-crosslinked network formed by hydrogen bond crosslinking between epicatechin and hyaluronic acid and metal-carboxylate coordination strong crosslinking between metal ions and hyaluronic acid ensures that the hydrogel has strong mechanical properties, and the higher mechanical properties ensure the service life and the service stability of the hydrogel in the subsequent application process.
Table 1: tensile Strength
Test example 2 adhesion Performance test
1. Tensile adhesion Strength test
The hydrogels prepared in examples 1-5 and comparative examples 1-2 above were tested for tensile bond strength to pigskin based on the ASTM F2258 standard. After the skin layer was left by removing the fat layer from the fresh pigskin, it was cut into a shape of 25mm long and 25mm wide and fixed to a tray with 502 adhesive, and then the hydrogels prepared in examples 1 to 5 and comparative examples 1 to 2 described above were made into a sheet-like hydrogel of 25mm long, 25mm wide and 0.5mm thick, which was placed between two pigskins, and the pigskins were held for 30s with a pressing pressure of 8kPa to make them adhesive. Then at room temperature using a universal tester at 5mm min -1 The bond strength was measured at the tensile speed of (c). Each group was set with 3 replicates and averaged.
As shown in Table 2, it can be seen from the results of Table 2 that the adhesive strength of the hydrogels prepared in examples 1 to 5 of the present invention to wet pig skin was 31.2 to 91.2kPa, which is significantly improved as compared with the comparative examples 1 to 2 in which epicatechin was not added, and the increase in epicatechin content was significantly improved. This is because hydroxyl groups and amino groups on the surface of the skin of pigs can form hydrogen bonds with polysaccharide hydroxyl groups and polyphenol hydroxyl groups in the hydrogel of the embodiment of the invention, and the quinone structure generated by partial oxidation of epicatechin can form Schiff base bonds with amino groups on the surface of the skin, so that the action strength of the hydrogel and skin tissues is improved. Since epicatechin itself can be used for hydrogel modification, the hydrogel containing epicatechin in example 5 also has a good tensile adhesion strength but is formed by Fe 3+ Can further increase its tensile adhesion strength. The higher adhesive strength is beneficial to the stability of the hydrogel in use in the subsequent application process.
Table 2: tensile adhesion Strength
2. Adhesion toughness test
The hydrogels prepared in examples 1-5 and comparative examples 1-2 above were tested for adhesion toughness to pigskin based on the ASTM F2256 standard. After the skin layer was left by removing the fat layer from the fresh pigskin, it was cut into a shape of 50mm long and 25mm wide, and then the hydrogels prepared in examples 1 to 5 and comparative examples 1 to 2 above were made into a sheet-like hydrogel of 25mm long, 10mm wide and 0.5mm thick, which was then adhered to the surface of the pigskin, and pressed and held for 30 seconds with a pressing pressure of 8kPa to adhere. Then at room temperature using a universal tester at 5mm min -1 Is tested for adhesion toughness at the tensile speed of (c). Each group was set with 3 replicates and averaged.
As shown in Table 3, it can be seen from the results of Table 3 that the adhesive toughness of the hydrogels prepared in examples 1 to 5 of the present invention can reach 97.6 to 251.1Jm -2 And the adhesive toughness of the hydrogel is improved by 5-14 times compared with the hydrogel prepared in the comparative examples 1-2 without epicatechin, which proves that the hydrogel prepared by the invention has excellent tissue adhesive toughness. When the hydrogel is broken, the breaking of the hydrogel body by hydrogen bond cross-linking consumes a lot of energy, while the bonding and topological entanglement of the hydrogel with skin tissue at the adhesion interface also consumes a lot of energy due to the breaking, so the high adhesion energy derives from the synergy of the high bulk toughness and strong interface interactions of the hydrogel.
Since epicatechin itself can be used for hydrogel modification, the hydrogel containing epicatechin in example 5 also has good adhesion toughness but is modified by Fe 3+ Can further increase its adhesion toughness. The book is provided withThe strong adhesion capability of the hydrogel is derived from interfacial adhesion mainly based on interactions such as hydrogen bonds formed between epicatechin and skin tissues, and is also a synergistic effect of bulk toughness network and interfacial adhesion formed by hydrogen bonds between epicatechin and hyaluronic acid.
Table 3: adhesion toughness
3. Adhesion time stability test
The hydrogels prepared in examples 1-5 and comparative examples 1-2 above were tested for tensile tissue adhesive strength to pigskin based on ASTM F2255 standard. The hydrogels prepared in examples 1-5 and comparative examples 1-2 above were stored at a temperature of 4℃for 0, 1, 3, 7, 14 days and removed for testing. After the skin layer was left by removing the fat layer from the fresh pigskin, it was cut into a shape of 50mm long and 25mm wide, and then the hydrogels prepared in examples 1 to 5 and comparative examples 1 to 2 above were made into a sheet-like hydrogel of 25mm long, 10mm wide and 0.5mm thick, which was placed between two pigskins, and the pigskins were held for 30s with a pressing pressure of 8kPa to adhere them. Then at room temperature using a universal tester at 5 mm.min- 1 The bond strength was measured at the tensile speed of (c). Each group was set with 3 replicates and averaged.
The results are shown in Table 4, and from the results of Table 4, we can see that the adhesion of the hydrogels in examples 1 to 5 of the present invention did not significantly decrease with the storage time during the period of 0 to 14 days, indicating that the hydrogels prepared in the examples of the present invention had good stability over time. Since epicatechin itself can be used for hydrogel modification, the hydrogel containing epicatechin in example 5 also has good adhesion toughness but is modified by Fe 3+ The addition of (3) can further increase the stability of the adhesive time.
For practical application of the hydrogel, the hydrogel has tough tissue adhesion performance and also requires the product to have high time stability, and the tissue adhesion performance of the hydrogel in the embodiment of the invention shows good time stability, which indicates that the hydrogel has high practical production application potential.
Table 4: time stability of adhesion
Test example 3 photodamage resistance test
1. Photodamage apoptosis test of ultraviolet light induced human epidermal cell HaCaT
The photodamage-resistant effect of the hydrogels was evaluated by uv-light induced photodamage apoptosis of human epidermal cells HaCaT (purchased from marpranopsis life technologies limited). The specific method comprises the following steps: haCaT was used at 5X 10 per well 5 The individual cells were inoculated evenly in well plates and placed in a cell incubator for overnight incubation. Setting 10 groups of treatment: blank, model, control (treated with epicatechin alone at the same concentration as in example 4), comparative examples 1-2, inventive examples 1-5, 3 replicates per group; then the groups except the blank group were irradiated with UVB 300mJ/cm 2 Performing ultraviolet induced damage treatment, and establishing an ultraviolet damage model; then, except for the blank group and the model group, 10 μl of sample (the final concentration of each group of samples is uniformly controlled) was added to each well of the other groups, and the culture was continued for 48 hours. After the medium was aspirated, the cells were collected by washing 3 times with PBS, digestion with EDTA-free pancreatin, centrifugation at 1000prm for 5min, and washing 3 times with PBS. 5 Xbinding Buffer was treated with deionized water 1:4 dilutions were added to 100. Mu.L of 1 Xbinding Buffer. Adding 2 mu L of Annexin V-FITC (AV) and 1 mu L Propidium lodide (PI), mixing uniformly, keeping away from light for 15min, adding 200 mu L of 1 Xbinding Buffer into each sample, mixing uniformly, filtering, and detecting. The effect of each group on the apoptosis of ultraviolet damaged skin cells HaCaT was examined using flow cytometry.
The test results are shown in Table 5, and the apoptosis of the model group is obviously increased after ultraviolet irradiation compared with that of the blank group, while the apoptosis inhibition effect of the invention in examples 1-5 is obviously enhanced compared with that of other groups, which indicates that the hydrogel of the invention has better photodamage resistance.
Table 5: influence of UV damaged skin on cell HaCaT apoptosis
| Group of | Apoptotic cells (%) |
| Example 1 | 11.2 |
| Example 2 | 9.5 |
| Example 3 | 7.9 |
| Example 4 | 6.7 |
| Example 5 | 9.6 |
| Comparative example 1 | 17.5 |
| Comparative example 2 | 14.4 |
| Control group | 8.42 |
| Model group | 18.1 |
| Blank group | 3.2 |
2. Mouse skin photodamage resistance test
Healthy female mice 90 (purchased from medical laboratory animal center in Guangdong province) were selected, and the weight was 20-25g, and the animals were divided into 9 groups (blank group, model group, comparative examples 1-2, inventive examples 1-5) of 10 animals each. The backs Mao Ti of the mice were cleaned and the area was 2cm. Times.2 cm. Except for the blank groups, the remaining 8 groups of mice were irradiated under UV lamp for a distance of 50cm,1h/d, 5 times per week for 8 consecutive weeks. Before ultraviolet irradiation for 20min, the mice in examples 1-4 and comparative examples 1-3 were uniformly coated with hydrogels prepared in the corresponding groups (the final concentration of each group of samples was uniformly controlled) on the dehairing area, and the model groups were directly irradiated. After the test, 1g of the whole skin layer of the ultraviolet irradiation part of each animal was taken, and skin tissue homogenates were prepared, and the levels of MDA, SOD, GSH to Px, CAT, hyp were measured respectively.
The results of the test are shown in Table 6, and the skin tissues of examples 1-5 showed significantly decreased SOD, GSH-Px, CAT and Hyp levels (< 0.05 or P < 0.01) and significantly increased MDA levels (< 0.01) compared to the model group. Comparative examples 1-3 showed reduced levels of SOD, GSH-Px, CAT and Hyp in skin tissue and increased levels of MDA compared to the model group, but the effect was not significant (P > 0.1). The hydrogel provided by the invention has obvious photodamage resistance effect.
Table 6: skin tissue SOD, GSH-Px, CAT and Hyp levels
Test example 4 tissue repair Capacity test
1. Test for repairing photodamage of zebra fish tail fin
Healthy AB-line zebra fish embryos developed to 3dpf (days post fertilization) (purchased from Shanghai Fei Xi Biotechnology Co., ltd.) were selected for experimental study and placed in 6-well cell culture plates, 1 tail/well, 9 total groups (blank, model, inventive examples 1-5, comparative examples 1-2) with 3 replicates per group. 5.0mL of the experimental solution is added into each hole (the measured final concentration of each group of samples is uniformly controlled to be the same), after the samples are hatched for 2 hours at 25 ℃ in a dark place, the rest groups are irradiated under 305nm ultraviolet light except for a blank group, and an ultraviolet damage model (shown as tail storage shrinkage) is built. And radiating for 30s every 0.5h within 2.5h, absorbing out the sample liquid after radiating, changing the sample liquid into culture water, and placing the culture water in an incubator for incubation. Before and 24 hours and 48 hours after the experiment, the damage repair condition of the zebra fish embryo fish fin and the change of the growth area of the tail fin are observed under a microscope, and the record is photographed. The data were collected using advanced image processing software, fish tail data were analyzed, and the change in zebra fish tail area was determined, under the present experimental conditions, based on 100 units of ultraviolet radiation front fin area.
The experimental results are shown in table 7, under the experimental conditions, the fish fins of the blank control group normally develop after 24h and 48h, the area of the tail fin is increased, and the tail fin of the model group is damaged, which indicates that the model structure is effective. After the sample of the embodiment of the invention is acted, the area of the zebra fish tail fin irradiated by ultraviolet rays is increased with the passage of time, and the hydrogel of the invention has effective repairing effect.
Table 7: zebra fish tail fin photodamage repair
2. Cell migration test
Uniformly marking the bottom center of the 6-hole cell culture plate with a cross marker with a spacing of 0.5-1cm. Inoculating 5X 10 per well 5 The individual/mL HaCaT cells are plated on a culture plate; after conventional culture to cell monolayer fusion and overnight with 1640 medium containing 1% new born calf serum, vertical scratches were made on the bottom of each well in the culture plate with 200 μl of sterile pipette tips, and 9 sets of treatments were set: blank, model, inventive examples 1-5, comparative example 1-2 groups (uniform control of the final concentrations measured for each group of samples) with 3 replicates each. An in vitro cell damage model is established, cells are washed for 3 times by PBS, the scraped cells are eluted, the cells are added into a serum-free culture medium only containing RHC to serve as a blank group, each group of holes are treated according to grouping and sample adding, and the adding amount is the same. At 37 ℃,5% CO 2 Culturing in an incubator, and sampling time is 24 hours. The overall migration distance of cells between different time periods was calculated according to the following formula.
S i ＝S 0 -S t
Wherein: s is S i Distance of overall migration of cells per length of time
S 0 Distance length at time 0
S t Distance length per time point
The results are shown in Table 8, and it can be seen from the results of Table 8 that the hydrogels prepared in examples 1 to 5 of the present invention have a cell mobility improved by nearly 2 times as compared with the hydrogels prepared in comparative examples 1 to 2, showing that the hydrogels of the present invention have a remarkable cell migration effect. In comparison with examples 4 and 5, which added the same amount of epicatechin, fe was added 3+ Example 4 compared to no Fe addition 3+ Also, the mobility of cells was improved, and it was also demonstrated that epicatechin and Fe in the hydrogel prepared in the example of the present invention 3+ Has synergistic effect, and can improve tissue repair ability of hyaluronic acid hydrogel after adding. The results prove that the hydrogel provided by the invention has good tissue repair capability.
Table 8: cell migration results
| Group of | Cell migration distance (mm) |
| Example 1 | 3.8±0.2 |
| Example 2 | 3.9+0.3 |
| Example 3 | 4.1±0.1 |
| Example 4 | 4.3±0.1 |
| Example 5 | 4.0±0.1 |
| Comparative example 1 | 2.0±0.2 |
| Comparative example 2 | 2.2±0.4 |
| Blank group | 0.5±0.1 |
Test example 5 antibacterial Property test
The hydrogels prepared in example 4 were tested for qualitative antibacterial activity by the agar plate method, using hydrogels without iron ions and epicatechin in comparative example 1, hydrogels without epicatechin in comparative example 2, and hydrogels without iron ions in example 5 as controls (the final concentrations measured for each group of samples were uniformly controlled). Using Escherichia coli and Staphylococcus aureus as bacterial models, mixing 100 μl,1×10 5 CFU/mL of bacterial suspension containing Staphylococcus aureus and E.coli at the exponential growth phase was spread evenly on LB agar plates. The obtained hydrogel was then cut into round pieces with a diameter of about 8mm, placed on LB agar plates, and subjected to a temperature of 37 ℃Culturing for 24 hours. Finally, the antibacterial activity is determined by measuring the diameter of the inhibition zone formed around the sample.
As shown in Table 9, it can be seen from the results of Table 9 that the hydrogels prepared in example 4 of the present invention have significantly improved antibacterial properties as compared to the hydrogels prepared in comparative examples 1-2, whereas epicatechin and Fe in the hydrogels prepared in the examples of the present invention 3+ Has synergistic effect, and Fe is added 3+ The antibacterial performance of the hyaluronic acid hydrogel is improved. The hydrogel of the invention has a certain antibacterial property.
Table 9: results of zone of inhibition
| Group of | Staphylococcus aureus (mm) | Coli (mm) |
| Example 4 | 17.1 | 16.8 |
| Example 5 | 15.3 | 14.8 |
| Comparative example 1 | 11.3 | 10.6 |
| Comparative example 2 | 13.6 | 13.1 |
Test example 6 antioxidant Capacity test
1. DPPH free radical scavenging ability
Accurately weighing DPPH 0.0050g, dissolving with absolute ethyl alcohol, carrying out ultrasonic light-shielding ultrasound for 30min, fully dissolving, and then preparing a DPPH solution with the volume of 50 mug/mL by using the absolute ethyl alcohol to reach 100mL to serve as a DPPH measuring solution. Accurately weighing 0.0100g of the sample, fixing the volume to 1.0mL by deionized water, and fully mixing to prepare 10.0mg/mL mother liquor. After adding 3.0mL of DPPH solution and 1.0mL of sample solution to be detected and fully mixing uniformly, carrying out light-proof reaction at room temperature for 30min, measuring the absorbance value (sample solvent zeroing calibration) by using an ultraviolet spectrophotometer under the condition of 517nm wavelength, evaluating the effect of the hydrogels prepared in examples 1-5 and comparative examples 1-2 on the clearance of DPPH free radicals (uniformly controlling the measurement final concentration of each group of samples to be the same), and taking the hydrogel without being added as a blank group, and testing each sample for 3 times. The DPPH radical scavenging rate was calculated as follows.
Wherein: a is that x Absorbance value of mixed solution of to-be-detected liquid and DPPH solution
A c Absorbance value of mixed solution of liquid to be detected and absolute ethyl alcohol solution
A b Absorbance of mixture of DPPH solution and sample solvent solution
As shown in Table 10, it can be seen from the results of Table 6 that the hydrogels of examples 1 to 5 of the present invention have good DPPH radical scavenging ability and DPPH radical scavenging ability is significantly improved with the increase of epicatechin content, and in example 4, DPPH radical scavenging rate reaches 98%, which indicates that epicatechin is introduced to provide good oxidation resistance to hyaluronic acid hydrogel and that Fe is added 3+ The hydrogel of comparative example 2 of (1) was compared with comparative example 1 without Fe added 3+ The DPPH free radical scavenging ability of the hydrogel is also improvedIn comparison with example 4 and example 5, which added the same epicatechin amount, fe was added 3+ Example 4 compared to no Fe addition 3+ Example 5 of the present invention, which shows that the DPPH radical scavenging ability is also improved, epicatechin and Fe in the hydrogel prepared in the present invention 3+ Has synergistic effect, and can improve DPPH free radical scavenging ability of hyaluronic acid hydrogel after adding. The hydrogel in the embodiment of the invention has good DPPH free radical scavenging capability and better antioxidant activity.
Table 10: DPPH free radical scavenging ability
2、ABTS + Free radical scavenging ability
Accurately weighing 0.2000g of ABTS, 0.0344g of potassium persulfate, dissolving in 52mL of deionized water, fully dissolving, and standing at room temperature in a dark place for 24 hours to obtain an ABTS mother solution. And taking a proper amount of ABTS mother solution, and diluting the ABTS mother solution with 95% ethanol until the absorbance value is within 0.7+/-0.02, so as to obtain an ABTS assay solution. Accurately weighing 0.0100g of sample, and fixing the volume to 1.0mL with deionized water, and fully and uniformly mixing to prepare 10.0mg/mL mother liquor. Adding 3.6mL of ABTS solution and 0.4mL of sample solution to be detected, fully mixing, reacting at room temperature in a dark place for 5min, measuring absorbance value (sample solvent zero setting calibration) by using a spectrophotometer under the condition of wavelength 734nm, and evaluating the pair of ABTS of the hydrogels prepared in examples 1-5 and comparative examples 1-2 + The scavenging effect of free radicals (uniform control of the final concentration measured for each group of samples) was repeated 3 times for each sample with no hydrogel added as a blank. ABTS + The radical scavenging rate was calculated as follows.
Wherein: a is that x Absorbance value of mixed solution of to-be-detected liquid and ABTS solution
A b ABTS solutionAbsorbance of sample solvent solution mixture
The results are shown in Table 11, and from the results of Table 11, we can see that the hydrogels in examples 1 to 5 of the present invention have good ABTS + Free radical scavenging ability, and ABTS + The radical scavenging ability was significantly increased with increasing epicatechin content, ABTS in example 4 + The free radical clearance reaches 95%, which indicates that the introduction of epicatechin provides good antioxidant capacity for hyaluronic acid hydrogel, and Fe is added 3+ The hydrogel of comparative example 2 of (1) was compared with comparative example 1 without Fe added 3+ Is a hydrogel of ABTS + The free radical scavenging ability was also improved, and Fe was added as compared with example 4 and example 5 in which the same amount of epicatechin was added 3+ Example 4 compared to no Fe addition 3+ Example 5, ABTS thereof + The free radical scavenging ability was also improved, demonstrating that epicatechin and Fe in the hydrogels prepared in the examples of the present invention 3+ Has synergistic effect, and can improve ABTS of hyaluronic acid hydrogel after adding + Free radical scavenging ability. The hydrogel in the embodiment of the invention has good ABTS + Free radical scavenging ability and good antioxidant activity.
Table 11: ABTS + Free radical scavenging ability
The invention provides epicatechin hydrogel, a preparation method and a process thereof, and a method and a way for realizing the technical scheme are numerous, the above is only a feasible implementation mode of the invention, and it should be pointed out that a plurality of improvements and modifications can be made to a person skilled in the art without departing from the principle of the invention, and the improvements and modifications are considered as the protection scope of the invention. The components not explicitly described in this embodiment can be implemented by using the prior art.
Claims (10)
1. A epicatechin hydrogel is characterized by being prepared by mixing hyaluronic acid and epicatechin and introducing metal ions for crosslinking.
2. The hydrogel of claim 1, wherein the hyaluronic acid is a 10.0wt% aqueous solution.
3. The hydrogel of claim 1, wherein the epicatechin is in the form of a 10.0 to 25.0 weight percent aqueous solution.
4. The hydrogel of claim 1, wherein the metal ion is Fe 3+ ，Cu 2+ ，Co 2+ ，Ni 2+ ，Zn 2+ ，Cd 2+ Or Ag + The concentration of the metal ions is 0.3 to 0.7mol/mL.
5. The method for preparing the hydrogel according to any one of claims 1 to 4, wherein hyaluronic acid and epicatechin are mixed, the pH is adjusted to be acidic, metal ions are introduced for crosslinking, and the hydrogel is obtained by dialysis.
6. The method according to claim 5, wherein the volume ratio of hyaluronic acid to epicatechin is 1-2: 1.
7. the method of claim 5, wherein the adjusting the pH to acidity is adjusting the pH to 2.5 to 2.8.
8. The method according to claim 5, wherein the metal ion is Fe 3+ 。
9. Use of the hydrogel according to any one of claims 1 to 4 for the protection against photodamage, for the protection against bacteria, for tissue repair and/or for the protection against oxidation.
10. Use of a hydrogel according to any one of claims 1 to 4 for the preparation of a photodamage, antibacterial, tissue repair and/or antioxidant product.
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