CN113893385A - Composition for preparing hydrogel, hydrogel and preparation method and application thereof - Google Patents
Composition for preparing hydrogel, hydrogel and preparation method and application thereof Download PDFInfo
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- CN113893385A CN113893385A CN202111176594.8A CN202111176594A CN113893385A CN 113893385 A CN113893385 A CN 113893385A CN 202111176594 A CN202111176594 A CN 202111176594A CN 113893385 A CN113893385 A CN 113893385A
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- cordyceps
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- hydrogel
- carboxymethyl chitosan
- cordyceps sinensis
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- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0009—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing macromolecular materials
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0057—Ingredients of undetermined constitution or reaction products thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0061—Use of materials characterised by their function or physical properties
- A61L26/0066—Medicaments; Biocides
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- A—HUMAN NECESSITIES
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- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0061—Use of materials characterised by their function or physical properties
- A61L26/008—Hydrogels or hydrocolloids
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- A—HUMAN NECESSITIES
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
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- A—HUMAN NECESSITIES
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
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- A—HUMAN NECESSITIES
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/404—Biocides, antimicrobial agents, antiseptic agents
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- A—HUMAN NECESSITIES
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
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Abstract
The invention relates to the technical field of biological medicines, and discloses a composition for preparing hydrogel, the hydrogel, a preparation method and application of the hydrogel. The composition contains cordyceps polysaccharide, carboxymethyl chitosan and cordyceps fermentation alcohol extract, wherein the mass ratio of the cordyceps polysaccharide to the carboxymethyl chitosan to the cordyceps fermentation alcohol extract is 1:0.8-4.5: 1-5. The hydrogel has good compatibility of each component, can ensure the permeability of the hydrogel, and has good antibacterial effect and wound healing promoting effect.
Description
Technical Field
The invention relates to the technical field of biomedicine, and particularly relates to a composition for preparing hydrogel, the hydrogel and a preparation method and application thereof.
Background
The skin is used as the first immunological defense line of human body, and has the functions of protecting human body tissue and organ from external mechanical and chemical stimulation and virus and bacteria invasion. After the skin is wounded, the healing process of the wound surface is long and complicated, and the risk of bacterial infection and suppuration exists. The dressing is used as a wound surface covering material, can prevent infection to a certain extent, provides a beneficial microenvironment for wound surface healing, and has the function of promoting wound healing. Gauze self that traditional processing used lacks etc. effectual anti-infection function, is in the same place with wound adhesion easily when changing, to the secondary damage that causes the patient, because opaque, also can't in time see through the dressing and observe the particular case of wound.
Hydrogels are high molecular weight polymeric materials that are hydrophilic and insoluble in water, and that have a cross-linked structure that can absorb a large amount of water (typically greater than 50% of the total mass). The hydrogel dressing can maintain the moisture of the wound; adhesive coverage of sensitive underlying tissue; pain relief also promotes epithelialization and cell migration to reduce the risk of scarring, which has a positive impact on the healing process of the wound. Therefore, hydrogel dressings are known as the hot spot of the present research. Although the hydrogel also has the function of a drug release carrier, drug molecules with an anti-inflammatory effect or active factors for promoting wound healing can be embedded in the hydrogel according to needs, so that the purposes of diminishing inflammation, inhibiting bacteria and promoting wound healing are achieved, in the prior art, the porous structure of the hydrogel needs to be formed by adding a cross-linking agent, and the cross-linking agent has certain side effect on a body, is not easy to degrade and is not beneficial to the ecological environment. In addition, in order to improve various properties (such as antibacterial property and wound healing promotion function) of the hydrogel in the use process in the prior art, different drug molecules can be added according to different functions, and the compatibility between the drug molecules and the hydrogel cannot be guaranteed, so that the permeability of the hydrogel is even affected.
Disclosure of Invention
The invention aims to solve the technical problem of providing a composition for preparing hydrogel, the hydrogel as well as a preparation method and application thereof.
In order to achieve the above object, the invention provides a composition for preparing hydrogel, which comprises cordyceps polysaccharide, carboxymethyl chitosan and cordyceps fermentation ethanol extract, wherein the mass ratio of the cordyceps polysaccharide to the carboxymethyl chitosan to the cordyceps fermentation ethanol extract is 1:0.8-4.5: 1-5.
Preferably, the mass ratio of the cordyceps polysaccharide to the carboxymethyl chitosan to the cordyceps fermentation alcohol extract is 1:1.5-2.5: 1.5-3.
Preferably, the preparation method of the cordyceps sinensis fermentation alcohol extract comprises the following steps: carrying out alcohol precipitation treatment on the cordyceps sinensis fermentation liquor I, then carrying out solid-liquid separation I, collecting supernatant I and concentrating.
Further preferably, the preparation method of the cordyceps sinensis fermentation liquid comprises the following steps: inoculating the fine cordyceps sinensis into a fermentation culture medium for aerobic fermentation, then carrying out solid-liquid separation II on the fermentation product, and collecting supernatant II, namely the cordyceps sinensis fermentation liquor.
Preferably, the inoculation amount of the fine cordyceps sinensis is 3-8%.
Preferably, the aerobic fermentation conditions include: the temperature is 18-25 deg.C, and the time is 14-24 days.
Preferably, the alcohol precipitation treatment I comprises: mixing the cordyceps sinensis fermentation liquor and an alcohol precipitation solvent I according to the volume ratio of 1:3-5, and standing, wherein the alcohol precipitation solvent I is 90-98 mass% of ethanol water solution, and the alcohol precipitation temperature is 2-6 ℃.
Preferably, the concentrating comprises: concentrating the supernatant I to 0.025-0.1 of the volume of the supernatant I.
In a second aspect, the present invention provides a hydrogel, comprising the composition for preparing a hydrogel according to the first aspect and water, wherein the mass ratio of the composition to the water is 1: 7-15.
In a third aspect, the present invention provides a method for preparing a hydrogel, comprising the steps of:
(1) mixing cordyceps polysaccharide, carboxymethyl chitosan and water to obtain a mixed solution I;
(2) mixing the mixed solution I with the cordyceps sinensis fermentation alcohol extract to obtain a mixed solution II;
(3) heating the mixed solution II, adjusting the pH value to 6-7, and standing to obtain the hydrogel;
wherein the mass ratio of the cordyceps polysaccharide to the carboxymethyl chitosan to the cordyceps fermentation alcohol extract is 1:0.8-4.5:1-5, and the ratio of the total mass of the cordyceps polysaccharide to the carboxymethyl chitosan to the cordyceps fermentation alcohol extract to the mass of water is 1: 7-15.
Preferably, the mass ratio of the cordyceps polysaccharide to the carboxymethyl chitosan to the cordyceps fermentation alcohol extract is 1:1.5-2.5: 1.5-3.
Preferably, the preparation method of the cordyceps sinensis fermentation alcohol extract comprises the following steps: carrying out alcohol precipitation treatment on the cordyceps sinensis fermentation liquor I, then carrying out solid-liquid separation I, collecting supernatant I and concentrating.
Further preferably, the preparation method of the cordyceps sinensis fermentation liquid comprises the following steps: inoculating the fine cordyceps sinensis into a fermentation culture medium for aerobic fermentation, then carrying out solid-liquid separation II on the fermentation product, and collecting supernatant II, namely the cordyceps sinensis fermentation liquor.
Preferably, the inoculation amount of the fine cordyceps sinensis is 3-8%.
Preferably, the aerobic fermentation conditions include: the temperature is 18-25 deg.C, and the time is 14-24 days.
Preferably, the alcohol precipitation treatment I comprises: mixing the cordyceps sinensis fermentation liquor and an alcohol precipitation solvent I according to the volume ratio of 1:3-5, and standing, wherein the alcohol precipitation solvent is 90-98 mass% of ethanol water solution, and the alcohol precipitation temperature is 2-6 ℃.
Preferably, the concentrating comprises: concentrating the supernatant I to 0.025-0.1 of the volume of the supernatant I.
Preferably, in the step (1), carboxymethyl chitosan and water are mixed to obtain a carboxymethyl chitosan solution, and then the carboxymethyl chitosan solution and the cordyceps polysaccharide are mixed.
Preferably, in the step (3), the conditions of the heat treatment include: the temperature is 45-55 deg.C, and the time is 15-30 min.
Through the technical scheme, the invention has the beneficial effects that:
the composition for preparing the hydrogel provided by the invention adopts cordyceps polysaccharide, carboxymethyl chitosan and cordyceps fermentation alcohol extract with good biocompatibility, and various active molecules contained in the cordyceps fermentation alcohol extract can achieve the purposes of improving immunity, resisting inflammation, resisting bacteria and the like, can improve the antibacterial and anti-inflammatory effects of the hydrogel, and can promote the healing of wounds. The addition of a cross-linking agent is not required in the process of forming the hydrogel, so that the adverse effect brought by the cross-linking agent is prevented. After the hydrogel is formed, a plurality of active components in the cordyceps sinensis fermentation alcohol extract can be dispersed and dissolved in the hydrogel, so that the permeability of the hydrogel can be ensured, and the medicine can smoothly reach an affected part.
Drawings
FIG. 1 is an SEM photograph of the hydrogel prepared in example 2;
FIG. 2 is a graph showing measured permeability values of hydrogels prepared in examples 1 to 7 and comparative examples 1 to 3.
Detailed Description
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.
As described above, the first aspect of the present invention provides a composition for preparing hydrogel, the composition comprises cordyceps polysaccharide, carboxymethyl chitosan and cordyceps fermentation alcoholic extract, and the mass ratio of the cordyceps polysaccharide to the carboxymethyl chitosan to the cordyceps fermentation alcoholic extract is 1:0.8-4.5: 1-5.
The inventor finds that when the cordyceps polysaccharide, the carboxymethyl chitosan and the cordyceps fermentation alcohol extract are used for preparing the hydrogel, the cordyceps polysaccharide, the carboxymethyl chitosan and the cordyceps fermentation alcohol extract are matched with each other, so that active components in the cordyceps fermentation alcohol extract can be well dispersed in the hydrogel, the permeability of the hydrogel can be ensured, and the medicine can smoothly reach an affected part. And the antibacterial and anti-inflammatory effects of the hydrogel can be improved through active components in the cordyceps sinensis fermentation alcohol extract, and the wound healing is promoted.
Preferably, the mass ratio of the cordyceps polysaccharide to the carboxymethyl chitosan to the cordyceps fermentation alcohol extract is 1:1.5-2.5: 1.5-3. Researches show that under the condition of the proportioning, the matching effect among the cordyceps polysaccharide, the carboxymethyl chitosan and the cordyceps fermentation alcohol extract can be improved, and further, the permeability and the antibacterial and anti-inflammatory effects of the hydrogel are improved.
Preferably, the preparation method of the cordyceps sinensis fermentation alcohol extract comprises the following steps: carrying out alcohol precipitation treatment on the cordyceps sinensis fermentation liquor I, then carrying out solid-liquid separation I, collecting supernatant I and concentrating.
The alcohol precipitation treatment is an alcohol precipitation treatment method disclosed in the prior art, and specifically, in the invention, 50 mass% or more of ethanol aqueous solution is slowly added into cordyceps sinensis fermentation liquor, and the ethanol aqueous solution and the cordyceps sinensis fermentation liquor are fully mixed by continuously stirring in the process of adding the ethanol aqueous solution, and then the mixture is subjected to standing precipitation treatment.
Preferably, the alcohol precipitation treatment I comprises: mixing the cordyceps sinensis fermentation liquor and an alcohol precipitation solvent I according to the volume ratio of 1:3-5, and standing, wherein the alcohol precipitation solvent I is 90-98 mass% of ethanol water solution, and the alcohol precipitation temperature is 2-6 ℃. And (3) carrying out alcohol precipitation treatment I under the conditions, so that macromolecular impurities in the fermentation liquor can be removed to the maximum extent, and the permeability of the hydrogel can be effectively ensured.
The solid-liquid separation I can adopt solid-liquid separation modes disclosed in the prior art, such as filtration and centrifugation. Preferably by centrifugation.
Specifically, the concentrating comprises: concentrating the supernatant I to 0.025-0.1 of the volume of the supernatant I. Can reduce the alcohol extract of the cordyceps while ensuring the sterilization and anti-inflammation functions of the hydrogel. Wherein, the concentration mode can be a conventional evaporation method, and preferably a rotary evaporator is adopted to remove the ethanol. In the present invention, the conditions of rotary evaporation include: the rotating speed is 80-120rpm, and the temperature is 30-50 ℃.
The cordyceps sinensis fermentation liquor can be obtained by fermentation by adopting a method disclosed in the prior art. Preferably, the preparation method of the cordyceps sinensis fermentation liquid comprises the following steps: inoculating the fine cordyceps sinensis into a fermentation culture medium for aerobic fermentation, then carrying out solid-liquid separation II on the fermentation product, and collecting supernatant II, namely the cordyceps sinensis fermentation liquor.
Wherein, the fermentation culture medium can be a culture medium which can be used for fermenting the fine cordyceps sinensis and is disclosed in the prior art. According to the invention, the formula of the fermentation medium is as follows: 10g/L of peptone, 20g/L of glucose, 2g/L of magnesium sulfate heptahydrate, 2g/L of monopotassium phosphate and 0.5g/L of ammonium sulfate. Of course, the above formula is only one preferable formula in the embodiment of the present invention, and it is also feasible to replace some components in the above fermentation medium or adjust the content of each component in the fermentation medium during the actual use process, as long as the nutrition of the fine cordyceps sinensis fermentation can be supplemented. The solid-liquid separation II can be realized by adopting a centrifugal or filtering mode, preferably a filtering mode, and the aperture of a filter screen is 100 meshes.
Aerobic fermentation may be by aerobic fermentation means as disclosed in the prior art. Preferably, the aerobic fermentation is carried out on a shaker. Not only can the fermentation culture medium be uniformly distributed, but also the aerobic fermentation effect of the fine cordyceps can be improved, thereby effectively improving the content of active components in the fermentation liquor.
Preferably, the inoculation amount of the fine cordyceps is 3-8%, and under the condition of the inoculation amount, the fine cordyceps has a good fermentation effect and can effectively improve the content of active components in fermentation liquor.
Preferably, the aerobic fermentation conditions include: the temperature is 18-25 deg.C, and the time is 14-24 days. Researches show that the fermentation liquor has a good fermentation effect under the condition, and the content of active components in the fermentation liquor can be effectively improved.
The cordyceps polysaccharide can be obtained commercially or prepared by self. Preferably, the preparation method of the cordyceps polysaccharide comprises the following steps: collecting mycelium in the preparation process of the cordyceps sinensis fermentation liquor, and removing impurities after freeze drying I to obtain cordyceps sinensis polysaccharide. The method can effectively utilize mycelium and fermentation liquor in the fermentation of the fine cordyceps sinensis, effectively reduce the cost and reduce the waste.
In order to increase the purity of cordyceps polysaccharide and thus the permeability of the resulting hydrogel, preferably, the removing comprises: and (3) grinding the product subjected to freeze drying I, dissolving the product in water, then carrying out alcohol precipitation treatment II, and freeze drying the collected precipitate II.
Preferably, the conditions of freeze-drying II include: the temperature is-30-50 ℃ and the time is 36-60 h.
In order to further improve the purity of the cordyceps polysaccharide and thus the permeability of the obtained hydrogel, the mass ratio of the product after freeze drying I to water is more preferably 1:5-15 in the impurity removal process.
Preferably, the conditions for freeze-drying I include: the temperature is-30-50 ℃ and the time is 36-60 h.
Preferably, in the impurity removing process, the alcohol precipitation treatment II comprises: mixing the cordyceps sinensis fermentation liquor and an alcohol precipitation solvent II according to the volume ratio of 1:3-5, and standing, wherein the alcohol precipitation solvent II is 90-98 mass% of ethanol water solution, and the alcohol precipitation temperature is 2-6 ℃. The optimization method can effectively improve the purity of the prepared cordyceps polysaccharide.
As described above, according to a second aspect of the present invention, there is provided a hydrogel comprising the composition for producing a hydrogel according to the first aspect and water in a mass ratio of 1:7 to 15. The hydrogel has good permeability and bactericidal performance, and can promote wound healing when used as a dressing.
As described above, the third aspect of the present invention provides a method for producing a hydrogel, comprising the steps of:
(1) mixing cordyceps polysaccharide, carboxymethyl chitosan and water to obtain a mixed solution I;
(2) mixing the mixed solution I with the cordyceps sinensis fermentation alcohol extract to obtain a mixed solution II;
(3) heating the mixed solution II, adjusting the pH value to 6-7, and standing to obtain the hydrogel;
wherein the mass ratio of the cordyceps polysaccharide to the carboxymethyl chitosan to the cordyceps fermentation alcohol extract is 1:0.8-4.5:1-5, and the ratio of the total mass of the cordyceps polysaccharide to the carboxymethyl chitosan to the cordyceps fermentation alcohol extract to the mass of water is 1: 7-15.
According to the present invention, the mixing in step (1) and step (2) may be stirring mixing, ultrasonic mixing, or other mixing methods. The pH can be adjusted by adding an acid solution or an alkali solution dropwise to the mixed solution II. Preferably, the pH is adjusted by dropwise addition of a hydrogen chloride solution and/or a sodium hydroxide solution. The concentrations of the hydrogen chloride solution and the sodium hydroxide solution can be determined by those skilled in the art according to actual conditions, and as one embodiment of the present invention, the concentration of the hydrogen chloride solution is 1M and the concentration of the sodium hydroxide is 1M.
Preferably, the mass ratio of the cordyceps polysaccharide to the carboxymethyl chitosan to the cordyceps fermentation alcohol extract is 1:1.5-2.5: 1.5-3. Researches show that under the condition of the proportioning, the matching effect among the cordyceps polysaccharide, the carboxymethyl chitosan and the cordyceps fermentation alcohol extract can be improved, and further, the permeability and the antibacterial and anti-inflammatory effects of the hydrogel are improved.
Preferably, the preparation method of the cordyceps sinensis fermentation alcohol extract comprises the following steps: carrying out alcohol precipitation treatment on the cordyceps sinensis fermentation liquor I, then carrying out solid-liquid separation I, collecting supernatant I and concentrating.
The alcohol precipitation treatment is an alcohol precipitation treatment method disclosed in the prior art, and specifically, in the invention, 50 mass% or more of ethanol aqueous solution is slowly added into cordyceps sinensis fermentation liquor, and the ethanol aqueous solution and the cordyceps sinensis fermentation liquor are fully mixed by continuously stirring in the process of adding the ethanol aqueous solution, and then the mixture is subjected to standing precipitation treatment.
Preferably, the alcohol precipitation treatment I comprises: mixing the cordyceps sinensis fermentation liquor and an alcohol precipitation solvent I according to the volume ratio of 1:3-5, and standing, wherein the alcohol precipitation solvent I is 90-98 mass% of ethanol water solution, and the alcohol precipitation temperature is 2-6 ℃. And (3) carrying out alcohol precipitation treatment I under the conditions, so that macromolecular impurities in the fermentation liquor can be removed to the maximum extent, and the permeability of the hydrogel can be effectively ensured.
The solid-liquid separation I can adopt solid-liquid separation modes disclosed in the prior art, such as filtration and centrifugation. Preferably by centrifugation.
Specifically, the concentrating comprises: concentrating the supernatant I to 0.025-0.1 of the volume of the supernatant I. Can reduce the alcohol extract of the cordyceps while ensuring the sterilization and anti-inflammation functions of the hydrogel. Wherein, the concentration mode can be a conventional evaporation method, and preferably a rotary evaporator is adopted to remove the ethanol. In the present invention, the conditions of rotary evaporation include: the rotating speed is 80-120rpm, and the temperature is 30-50 ℃.
The cordyceps sinensis fermentation liquor can be obtained by fermentation by adopting a method disclosed in the prior art. Preferably, the preparation method of the cordyceps sinensis fermentation liquid comprises the following steps: inoculating the fine cordyceps sinensis into a fermentation culture medium for aerobic fermentation, then carrying out solid-liquid separation II on the fermentation product, and collecting supernatant II, namely the cordyceps sinensis fermentation liquor.
Wherein, the fermentation culture medium can be a culture medium which can be used for fermenting the fine cordyceps sinensis and is disclosed in the prior art. According to the invention, the formula of the fermentation medium is as follows: 10g/L of peptone, 20g/L of glucose, 2g/L of magnesium sulfate heptahydrate, 2g/L of monopotassium phosphate and 0.5g/L of ammonium sulfate. Of course, the above formula is only one preferable formula in the embodiment of the present invention, and it is also possible to replace some components in the fermentation medium or adjust the content of each component in the fermentation medium during the actual use. The solid-liquid separation II can be realized by adopting a centrifugal or filtering mode, preferably a filtering mode, and the aperture of a filter screen is 100 meshes.
Aerobic fermentation may be by aerobic fermentation means as disclosed in the prior art. Preferably, the aerobic fermentation is carried out on a shaker. Not only can the fermentation culture medium be uniformly distributed, but also the aerobic fermentation effect of the fine cordyceps can be improved, thereby effectively improving the content of active components in the fermentation liquor.
Preferably, the inoculation amount of the fine cordyceps is 3-8%, and under the condition of the inoculation amount, the fine cordyceps has a good fermentation effect and can effectively improve the content of active components in fermentation liquor.
Preferably, the aerobic fermentation conditions include: the temperature is 18-25 deg.C, and the time is 14-24 days. Researches show that the fermentation liquor has a good fermentation effect under the condition, and the content of active components in the fermentation liquor can be effectively improved.
The cordyceps polysaccharide can be obtained commercially or prepared by self. Preferably, the preparation method of the cordyceps polysaccharide comprises the following steps: collecting mycelium in the preparation process of the cordyceps sinensis fermentation liquor, and removing impurities after freeze drying I to obtain cordyceps sinensis polysaccharide. The method can effectively utilize mycelium and components in the fermentation of the fine cordyceps sinensis, effectively reduce the cost and reduce the waste.
In order to increase the purity of the cordyceps polysaccharide and thus the permeability of the resulting hydrogel, preferably, the removing comprises: and (3) grinding the product subjected to freeze drying I, dissolving the product in water, then carrying out alcohol precipitation treatment II, and freeze drying the collected precipitate II.
Preferably, the conditions of freeze-drying II include: the temperature is-30-50 ℃ and the time is 36-60 h. As a specific embodiment of the invention, the temperature of the freeze-drying II is-40 ℃ and the time is 48 h.
In order to further improve the purity of the cordyceps polysaccharide and thus the permeability of the obtained hydrogel, the mass ratio of the product after freeze drying I to water is more preferably 1:5-15 in the impurity removal process.
Preferably, the conditions for freeze-drying I include: the temperature is-30-50 ℃ and the time is 36-60 h. As a specific embodiment of the invention, the temperature of the freeze-drying II is-40 ℃ and the time is 48 h.
Preferably, in the impurity removing process, the alcohol precipitation treatment II comprises: mixing the cordyceps sinensis fermentation liquor and an alcohol precipitation solvent II according to the volume ratio of 1:3-5, and standing, wherein the alcohol precipitation solvent II is 90-98 mass% of ethanol water solution, and the alcohol precipitation temperature is 2-6 ℃. The optimization method can effectively improve the purity of the prepared cordyceps polysaccharide.
Preferably, in the step (1), carboxymethyl chitosan and water are mixed to form a carboxymethyl chitosan solution, and then the carboxymethyl chitosan solution and the cordyceps polysaccharide are mixed. The reaction of the cordyceps polysaccharide and the carboxymethyl chitosan can be more sufficient, so that the permeability of the hydrogel is ensured.
Preferably, the carboxymethyl chitosan and water are mixed in a manner that: dissolving carboxymethyl chitosan in water at 50-60 deg.C.
Preferably, in the step (3), the conditions of the heat treatment include: the temperature is 45-55 deg.C, and the time is 15-30 min. So that the carboxymethyl chitosan, the cordyceps polysaccharide and the active components in the cordyceps alcohol extract can be fully reacted, and the slow release effect of the hydrogel is further improved.
As described above, the fourth aspect of the present invention provides the use of the hydrogel of the second aspect or the hydrogel produced by the production method of the second aspect as a dressing. The hydrogel provided by the invention is applied to dressings, has good permeability and good antibacterial and anti-inflammatory properties, and can promote the healing of wounds.
As a relatively preferred embodiment of the present invention, there is provided a method for preparing a hydrogel, comprising the steps of:
(1) mixing carboxymethyl chitosan with water at 50-60 deg.C to obtain carboxymethyl chitosan solution, and mixing Cordyceps polysaccharide with carboxymethyl chitosan solution to obtain mixed solution I;
(2) mixing the mixed solution I with the cordyceps sinensis fermentation alcohol extract to obtain a mixed solution II;
(3) heating the mixed solution II at the temperature of 45-55 ℃ for 15-20min, then adjusting the pH value to 6-7, and standing to obtain the hydrogel;
wherein the mass ratio of the cordyceps polysaccharide to the carboxymethyl chitosan to the cordyceps fermentation alcohol extract is 1:1.5-2.5:1.5-3, and the mass ratio of the total mass of the cordyceps polysaccharide to the carboxymethyl chitosan to the cordyceps fermentation alcohol extract to the mass of water is 1: 7-15;
the preparation method of the cordyceps sinensis fermentation alcohol extract and the cordyceps sinensis polysaccharide comprises the following steps: inoculating the fine cordyceps sinensis into a fermentation culture medium according to the inoculation amount of 3-8% in a shaking table for aerobic fermentation (the temperature is 18-25 ℃ and the time is 14-24 days), and then carrying out solid-liquid separation II on a fermentation product; mixing the collected supernatant II with 90-98 mass% of ethanol aqueous solution according to the volume ratio of 1:3-5 at the temperature of 2-6 ℃, standing, carrying out alcohol precipitation treatment, then carrying out solid-liquid separation I, collecting the supernatant I, and concentrating to 0.025-0.1 of the volume of the supernatant I to obtain cordyceps sinensis fermentation alcohol extract; and (2) carrying out freeze drying I treatment on the collected mycelia, grinding the product obtained after the freeze drying I treatment, mixing the product with water according to the mass ratio of 1:5-15, mixing the solution and 90-98 mass% of ethanol water solution according to the volume ratio of 1:3-5 at the temperature of 2-6 ℃, standing, carrying out solid-liquid separation III, and collecting the precipitate, namely the cordyceps polysaccharide.
The present invention will be described in detail below by way of examples. In the following examples of the present invention,
the scanning electron microscope was a 7539-H field emission scanning electron microscope available from Hitachi, Japan.
Example 1
(1) Mixing carboxymethyl chitosan with water at 50 ℃ to obtain a carboxymethyl chitosan solution, and then mixing cordyceps polysaccharide with the carboxymethyl chitosan solution to obtain a mixed solution I;
(2) mixing the mixed solution I with the cordyceps sinensis fermentation alcohol extract to obtain a mixed solution II;
(3) heating the mixed solution II at 50 ℃ for 15min, then adjusting the pH value to 6, and standing to obtain the hydrogel;
wherein the mass ratio of the cordyceps polysaccharide to the carboxymethyl chitosan to the cordyceps fermentation alcohol extract is 1:2.5:3, and the ratio of the total mass of the cordyceps polysaccharide to the carboxymethyl chitosan to the cordyceps fermentation alcohol extract to the mass of the water is 1: 10;
the preparation method of the cordyceps sinensis fermentation alcohol extract and the cordyceps sinensis polysaccharide comprises the following steps: inoculating the fine cordyceps sinensis into a fermentation culture medium according to the inoculation amount of 5% in a shaking table for aerobic fermentation (the temperature is 21 ℃ and the time is 16 days), and then carrying out solid-liquid separation on a fermentation product II; mixing the collected supernatant II with 95 mass percent ethanol aqueous solution according to the volume ratio of 1:4 at the temperature of 4 ℃, standing, carrying out alcohol precipitation treatment, then carrying out solid-liquid separation I, collecting the supernatant I, and concentrating to 0.05 volume of the supernatant I to obtain cordyceps sinensis fermentation alcohol extract; and (3) carrying out freeze drying I treatment on the collected mycelia, grinding the product obtained after the freeze drying I treatment, mixing the product with water according to the mass ratio of 1:10, mixing the solution and 95 mass% ethanol aqueous solution according to the volume ratio of 1:4 at the temperature of 4 ℃, standing, carrying out solid-liquid separation III, and collecting precipitates, namely cordyceps polysaccharide.
Example 2
(1) Mixing carboxymethyl chitosan with water at 55 ℃ to obtain a carboxymethyl chitosan solution, and then mixing cordyceps polysaccharide with the carboxymethyl chitosan solution to obtain a mixed solution I;
(2) mixing the mixed solution I with the cordyceps sinensis fermentation alcohol extract to obtain a mixed solution II;
(3) heating the mixed solution II at 45 ℃ for 18min, then adjusting the pH value to 6.5, and standing to obtain the hydrogel;
wherein the mass ratio of the cordyceps polysaccharide to the carboxymethyl chitosan to the cordyceps fermentation alcohol extract is 1:1.75:2, and the ratio of the total mass of the cordyceps polysaccharide to the carboxymethyl chitosan to the cordyceps fermentation alcohol extract to the mass of the water is 1: 15;
the preparation method of the cordyceps sinensis fermentation alcohol extract and the cordyceps sinensis polysaccharide comprises the following steps: inoculating the fine cordyceps sinensis into a fermentation culture medium according to the inoculation amount of 3% in a shaking table for aerobic fermentation (the temperature is 18 ℃ and the time is 24 days), and then carrying out solid-liquid separation on a fermentation product II; mixing the collected supernatant II with 98 mass percent ethanol water solution according to the volume ratio of 1:3 at the temperature of 2 ℃, standing, carrying out alcohol precipitation treatment, then carrying out solid-liquid separation I, collecting the supernatant I, and concentrating to 0.025 volume of the supernatant I to obtain cordyceps sinensis fermentation alcohol extract; and (2) carrying out freeze drying I treatment on the collected mycelia, grinding the product obtained after the freeze drying I treatment, mixing the product with water according to the mass ratio of 1:15, mixing the solution and 98 mass% of ethanol aqueous solution according to the volume ratio of 1:3 at the temperature of 2 ℃, standing, carrying out solid-liquid separation III, and collecting precipitates, namely cordyceps polysaccharide.
Example 3
(1) Mixing carboxymethyl chitosan with water at 60 ℃ to obtain a carboxymethyl chitosan solution, and then mixing cordyceps polysaccharide with the carboxymethyl chitosan solution to obtain a mixed solution I;
(2) mixing the mixed solution I with the cordyceps sinensis fermentation alcohol extract to obtain a mixed solution II;
(3) heating the mixed solution II for 20min at the temperature of 55 ℃, then adjusting the pH value to 7, and standing to obtain the hydrogel;
wherein the mass ratio of the cordyceps polysaccharide to the carboxymethyl chitosan to the cordyceps fermentation alcohol extract is 1:1.5:1.7, and the mass ratio of the total mass of the cordyceps polysaccharide to the carboxymethyl chitosan to the cordyceps fermentation alcohol extract to the mass of water is 1: 7;
the preparation method of the cordyceps sinensis fermentation alcohol extract and the cordyceps sinensis polysaccharide comprises the following steps: inoculating the fine cordyceps sinensis into a fermentation culture medium according to the inoculation amount of 8% in a shaking table for aerobic fermentation (the temperature is 25 ℃, the time is 14 days), and then carrying out solid-liquid separation on a fermentation product II; mixing the collected supernatant II with 90 mass percent ethanol aqueous solution according to the volume ratio of 1:5 at the temperature of 6 ℃, standing, carrying out alcohol precipitation treatment, then carrying out solid-liquid separation I, collecting the supernatant I, and concentrating to 0.1 of the volume of the supernatant I to obtain cordyceps sinensis fermentation alcohol extract; and (3) carrying out freeze drying I treatment on the collected mycelia, grinding the product obtained after the freeze drying I treatment, mixing the product with water according to the mass ratio of 1:7, mixing the solution and 90 mass% of ethanol aqueous solution according to the volume ratio of 1:5 at the temperature of 6 ℃, standing, carrying out solid-liquid separation III, and collecting precipitates, namely cordyceps polysaccharide.
Example 4
According to the method of the embodiment 1, except that the mass ratio of the cordyceps polysaccharide, the carboxymethyl chitosan and the cordyceps fermentation alcohol extract is 1:4.5: 5.
Example 5
According to the method of the embodiment 3, except that the mass ratio of the cordyceps polysaccharide, the carboxymethyl chitosan and the cordyceps fermentation alcohol extract is 1:0.8: 1.
Example 6
According to the method of the embodiment 1, except that the preparation method of the cordyceps sinensis fermentation alcohol extract and the cordyceps sinensis polysaccharide comprises the following steps:
inoculating the fine cordyceps sinensis into a fermentation culture medium according to the inoculation amount of 5% in a shaking table for aerobic fermentation (the temperature is 21 ℃ and the time is 16 days), and then carrying out solid-liquid separation on a fermentation product II; mixing the collected supernatant II with 80 mass percent of ethanol aqueous solution according to the volume ratio of 1:4 at the temperature of 10 ℃, standing, carrying out alcohol precipitation treatment, then carrying out solid-liquid separation I, collecting the supernatant I, and concentrating to 0.05 volume of the supernatant I to obtain cordyceps sinensis fermentation alcohol extract; and (3) carrying out freeze drying I treatment on the collected mycelia, grinding the product obtained after the freeze drying I treatment, mixing the product with water according to the mass ratio of 1:10, mixing the solution and 80 mass% of ethanol aqueous solution according to the volume ratio of 1:4 at the temperature of 4 ℃, standing, carrying out solid-liquid separation III, and collecting precipitates, namely cordyceps polysaccharide.
Example 7
According to the method of example 1, except that, in the step (1), carboxymethyl chitosan, cordyceps polysaccharide and water are directly mixed at 50 ℃, to obtain the mixed solution I.
Comparative example 1
(1) Mixing carboxymethyl chitosan with water at 50 ℃ to obtain a carboxymethyl chitosan solution;
(2) mixing the carboxymethyl chitosan solution and the cordyceps sinensis fermentation alcohol extract to obtain a mixed solution;
(3) heating the mixed solution at 50 ℃ for 15min, then adjusting the pH value to 6, and standing to obtain the hydrogel;
wherein the mass ratio of the carboxymethyl chitosan to the cordyceps sinensis fermentation alcohol extract is 2.5:3, and the ratio of the total mass of the carboxymethyl chitosan to the cordyceps sinensis fermentation alcohol extract to the mass of the water is 1: 10;
the preparation method of the cordyceps sinensis fermentation alcohol extract comprises the following steps: inoculating the fine cordyceps sinensis into a fermentation culture medium according to the inoculation amount of 5% in a shaking table for aerobic fermentation (the temperature is 21 ℃ and the time is 16 days), and then carrying out solid-liquid separation on a fermentation product II; mixing the collected supernatant II and 95 mass percent ethanol aqueous solution according to the volume ratio of 1:4 at the temperature of 4 ℃, standing, carrying out alcohol precipitation treatment, then carrying out solid-liquid separation I, collecting the supernatant I, and concentrating to 0.05 of the volume of the supernatant I to obtain the cordyceps sinensis fermentation alcohol extract.
Comparative example 2
(1) Mixing cordyceps polysaccharide with water to obtain a cordyceps polysaccharide solution;
(2) mixing the cordyceps polysaccharide solution and the cordyceps fermentation alcohol extract to obtain a mixed solution;
(3) heating the mixed solution at 50 ℃ for 15min, then adjusting the pH value to 6, and standing to obtain the hydrogel;
wherein the mass ratio of the cordyceps polysaccharide to the carboxymethyl chitosan to the cordyceps fermentation alcohol extract is 1:2.5:3, and the ratio of the total mass of the cordyceps polysaccharide to the carboxymethyl chitosan to the cordyceps fermentation alcohol extract to the mass of the water is 1: 10;
the preparation method of the cordyceps sinensis fermentation alcohol extract and the cordyceps sinensis polysaccharide comprises the following steps: inoculating the fine cordyceps sinensis into a fermentation culture medium according to the inoculation amount of 5% in a shaking table for aerobic fermentation (the temperature is 21 ℃ and the time is 16 days), and then carrying out solid-liquid separation on a fermentation product II; mixing the collected supernatant II with 95 mass percent ethanol aqueous solution according to the volume ratio of 1:4 at the temperature of 4 ℃, standing, carrying out alcohol precipitation treatment, then carrying out solid-liquid separation I, collecting the supernatant I, and concentrating to 0.05 volume of the supernatant I to obtain cordyceps sinensis fermentation alcohol extract; and (3) carrying out freeze drying I treatment on the collected mycelia, grinding the product obtained after the freeze drying I treatment, mixing the product with water according to the mass ratio of 1:10, mixing the solution and 95 mass% ethanol aqueous solution according to the volume ratio of 1:4 at the temperature of 4 ℃, standing, carrying out solid-liquid separation III, and collecting precipitates, namely cordyceps polysaccharide.
Comparative example 3
The procedure of example 1 was followed except that the carboxymethyl chitosan was replaced with chitosan.
Test example
And (3) detection by a scanning electron microscope: and (3) placing the prepared cordyceps natural component composite hydrogel sample in a vacuum freeze dryer for freeze drying, taking out the dried cordyceps natural component composite hydrogel sample, and cutting the sample in liquid nitrogen. Fixing the gel section with conductive adhesive, and spraying gold for 20 min. The microstructure of the cross section of the sample is observed by a scanning electron microscope under the voltage of 5 KV. The microstructure of the hydrogel prepared in example 2 is shown in FIG. 1. As can be seen from FIG. 1, the hydrogel obtained in the example of the present invention has a dense surface and a distinct pore structure. The presence of these porous structures provides a pathway for the ingress and egress of water molecules and drug delivery.
The hydrogels in the examples and comparative examples were subjected to in vitro bacteriostatic tests and the antibacterial properties of the hydrogels were determined according to GB/T20944.1-2007. Activated escherichia coli (e.coli) and staphylococcus aureus (s.aureus) suspensions were diluted to 106CFU/mL and coated on a solidified agar medium, the cordyceps natural component composite hydrogels in the above examples and comparative examples and the hydrogel without cordyceps fermentation alcohol extract (comparative example) were cut into circles (d ═ 20mm), and placed on a petri dish for incubation at 37 ℃ for 24 hours, and the size of the zone of inhibition H (zone width) was as shown in the following table.
| Coli middle zone width (mm) | Staphylococcus aureus middle zone width (mm) | |
| Example 1 | 2.5 | 3 |
| Example 2 | 4 | 3.5 |
| Example 3 | 1.5 | 2 |
| Example 4 | 3.3 | 1.5 |
| Example 5 | 2.6 | 3.1 |
| Example 6 | 2.2 | 2.8 |
| Example 7 | 1.5 | 3.4 |
| Comparative example 1 | 0.2 | 0.1 |
| Comparative example 2 | 0.3 | 0.1 |
| Comparative example 3 | 0.2 | 0.2 |
As can be seen from the results in the above table, the hydrogels of examples 1 to 7 of the present invention have better bacteriostatic properties as compared with comparative examples 1 to 3.
The forward osmosis membrane (cellulose triacetate film, available from HTI corporation, having a thickness of 10 μm) was immersed in 2000mg/L NaCl aqueous solution for 10 hours to activate the forward osmosis membrane. Then, 200mg of the dried hydrogel of example or comparative example was placed on the active layer side of the forward osmosis membrane having an effective membrane area of 1.77cm, and 2000mg/L of an aqueous NaCl solution as a raw material solution was placed on the other side of the forward osmosis membrane2. Water flux (F) (L.m) of hydrogel－2·h－1LMH, liters of liquid passed per square meter per hour) is calculated by the following formula: f ═ V/(a × t); in the formula: v-the volume (L) of water absorbed by the hydrogel through the forward osmosis membrane; a-effective Membrane area (m) of Forward osmosis Membrane2) (ii) a t-time (h) for forward osmosis testing. The data obtained are shown in FIG. 2.
As can be seen from FIG. 2, the hydrogel provided in the examples of the present invention has a higher positive permeability compared to the comparative examples, which indicates that the hydrogel provided in the present invention has a better permeability. And the water flux of example 2 was generally maintained at a higher level over 24h, indicating that the positive permeability of example 2 is optimal, and the comparative ratio of the other examples is also at a higher level.
The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.
Claims (10)
1. The composition for preparing the hydrogel is characterized by comprising cordyceps polysaccharide, carboxymethyl chitosan and cordyceps fermentation alcohol extract, wherein the mass ratio of the cordyceps polysaccharide to the carboxymethyl chitosan to the cordyceps fermentation alcohol extract is 1:0.8-4.5: 1-5.
2. The composition for preparing hydrogel according to claim 1, wherein the mass ratio of the cordyceps polysaccharide, the carboxymethyl chitosan and the cordyceps fermentation alcohol extract is 1:1.5-2.5: 1.5-3.
3. The composition for preparing hydrogel according to claim 1 or 2, wherein the preparation method of the cordyceps sinensis fermentation alcohol extract comprises the following steps: carrying out alcohol precipitation treatment on the cordyceps sinensis fermentation liquor I, then carrying out solid-liquid separation I, collecting supernatant I and concentrating.
4. The composition for preparing hydrogel according to claim 3, wherein the preparation method of the cordyceps sinensis fermentation broth comprises the following steps: inoculating the fine cordyceps sinensis into a fermentation culture medium for aerobic fermentation, then carrying out solid-liquid separation II on a fermentation product, and collecting supernatant II, namely cordyceps sinensis fermentation liquor;
the inoculation amount of the fine cordyceps is 3-8%;
the aerobic fermentation conditions include: the temperature is 18-25 ℃, and the time is 14-24 days;
preferably, the alcohol precipitation treatment I comprises: mixing the cordyceps sinensis fermentation liquor and an alcohol precipitation solvent I according to the volume ratio of 1:3-5, and standing, wherein the alcohol precipitation solvent I is 90-98 mass% of ethanol water solution, and the alcohol precipitation temperature is 2-6 ℃;
preferably, the concentrating comprises: concentrating the supernatant I to 0.025-0.1 of the volume of the supernatant I.
5. A hydrogel, comprising the composition for producing a hydrogel according to any one of claims 1 to 4 and water in a mass ratio of 1:7 to 15.
6. A method for preparing a hydrogel, comprising the steps of:
(1) mixing cordyceps polysaccharide, carboxymethyl chitosan and water to obtain a mixed solution I;
(2) mixing the mixed solution I with the cordyceps sinensis fermentation alcohol extract to obtain a mixed solution II;
(3) heating the mixed solution II, adjusting the pH value to 6-7, and standing to obtain the hydrogel;
wherein the mass ratio of the cordyceps polysaccharide to the carboxymethyl chitosan to the cordyceps fermentation alcohol extract is 1:0.8-4.5:1-5, and the ratio of the total mass of the cordyceps polysaccharide to the carboxymethyl chitosan to the cordyceps fermentation alcohol extract to the mass of water is 1: 7-15.
7. The preparation method of the hydrogel according to claim 6, wherein the mass ratio of the cordyceps polysaccharide, the carboxymethyl chitosan and the cordyceps fermentation alcohol extract is 1:1.5-2.5: 1.5-3.
8. The method for preparing the hydrogel according to claim 6 or 7, wherein the method for preparing the cordyceps sinensis fermentation alcoholic extract comprises the following steps: carrying out alcohol precipitation treatment on the cordyceps sinensis fermentation liquor I, then carrying out solid-liquid separation I, collecting supernatant I and concentrating;
preferably, the preparation method of the cordyceps sinensis fermentation liquid comprises the following steps: inoculating the fine cordyceps sinensis into a fermentation culture medium for aerobic fermentation, then carrying out solid-liquid separation II on a fermentation product, and collecting supernatant II, namely cordyceps sinensis fermentation liquor;
wherein the inoculation amount of the fine cordyceps is 3-8%;
the aerobic fermentation conditions include: the temperature is 18-25 ℃, and the time is 14-24 days;
preferably, the alcohol precipitation treatment I comprises: mixing the cordyceps sinensis fermentation liquor and an alcohol precipitation solvent I according to the volume ratio of 1:3-5, and standing, wherein the alcohol precipitation solvent I is 90-98 mass% of ethanol water solution, and the alcohol precipitation temperature is 2-6 ℃;
preferably, the concentrating comprises: concentrating the supernatant I to 0.025-0.1 of the volume of the supernatant I.
9. The method for preparing the hydrogel according to claim 6 or 7, wherein in the step (1), carboxymethyl chitosan is mixed with water to obtain a carboxymethyl chitosan solution, and then the carboxymethyl chitosan solution is mixed with the cordyceps polysaccharide;
in the step (3), the conditions of the heat treatment include: the temperature is 45-55 deg.C, and the time is 15-30 min.
10. Use of the hydrogel according to claim 5 or the hydrogel produced by the method according to any one of claims 6 to 9 as a dressing.
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