CN115246938A - Silk fibroin hydrogel with traditional Chinese medicine polysaccharide activity, and preparation method and application thereof - Google Patents
Silk fibroin hydrogel with traditional Chinese medicine polysaccharide activity, and preparation method and application thereof Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 22
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- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 8
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- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J3/00—Processes of treating or compounding macromolecular substances
- C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
- C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
- C08J3/075—Macromolecular gels
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
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- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
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- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2489/00—Characterised by the use of proteins; Derivatives thereof
Abstract
The invention discloses silk fibroin hydrogel with traditional Chinese medicine polysaccharide activity, and a preparation method and application thereof. The preparation method comprises the following steps: adding traditional Chinese medicine polysaccharide into the silk fibroin solution, and adding ethanol to adjust the pH value to acidity, thereby preparing the silk fibroin hydrogel with traditional Chinese medicine polysaccharide activity. The method can shorten the gelling time of the silk fibroin solution to be within 5min, not only improves the working efficiency, but also ensures that the prepared hydrogel has good biocompatibility and is green and safe, and also has the advantages of simple preparation process, lower cost, rapidness and high efficiency.
Description
Technical Field
The invention relates to the technical field of hydrogel preparation, in particular to silk fibroin hydrogel with traditional Chinese medicine polysaccharide activity, and a preparation method and application thereof.
Background
The silk fibroin is prepared from silk by degumming, is a natural high-molecular fiber protein without physiological activity, accounts for 70-80% of the silk, is a natural protein containing amino acids essential to human bodies, and has good biocompatibility. Silk fibroin exists in two different conformations of silk I and silk II, wherein the conformation of silk I comprises a random coil and a-helix, and the conformation of silk II is antiparallel beta-sheet. Wherein, the structure of the SilkI is not stable enough and can be converted into a stable SilkII structure by polar solvent, heat treatment and the like. Silk fibroin can be processed relatively simply to obtain a variety of different morphologies that facilitate tissue cell ingrowth.
The polysaccharide is the main component of the plant, a plurality of polysaccharides have important pharmacological activity, and the traditional Chinese medicine polysaccharide hydrogel is nontoxic, has good water retention property, biocompatibility and biodegradability, and has good application prospect in a drug release system.
The silk fibroin solution gradually changes to gelation when standing, however, the conversion time is long, and the mass concentration of silk fibroin has an influence on the gelation time, for example, silk fibroin solution is relatively easy to form gel when the mass concentration is more than 10%, but takes a long time to form gel when the mass concentration is less than 10%. And the silk fibroin solution with the mass concentration higher than 10% forms gel with low water content and low porosity, and is limited in application. The silk fibroin hydrogel with the mass concentration of less than 5 percent has a restriction on the use of the silk fibroin hydrogel due to too long gelation time.
At present, the main method for shortening the silk fibroin hydrogel time is as follows: the chemical cross-linking agent is added into the silk fibroin aqueous solution, the method can greatly shorten the gelation time of the silk fibroin aqueous solution, but a certain amount of the chemical cross-linking agent is remained in the prepared gel, so that certain toxicity is brought, and the method also reduces the biocompatibility of the silk fibroin hydrogel.
Therefore, how to gelatinize a low-concentration silk fibroin solution in a short time becomes a focus of current research.
Disclosure of Invention
Based on the above, the invention aims to provide silk fibroin hydrogel with traditional Chinese medicine polysaccharide activity, and a preparation method and application thereof. According to the invention, a certain volume of traditional Chinese medicine polysaccharide and ethanol are added to adjust the pH value to acidity, so that the gelling time of the silk fibroin solution is shortened to be within 5min, and the working efficiency is improved; the prepared hydrogel has good biocompatibility, does not contain a chemical cross-linking agent, and is green and safe; the invention has the advantages of simple preparation process, lower cost, rapidness, high efficiency and the like, and can be widely applied to the pharmaceutical material industry.
The above purpose of the invention is realized by the following technical scheme:
according to one aspect of the invention, the preparation method of the silk fibroin hydrogel with traditional Chinese medicine polysaccharide activity provided by the invention comprises the following steps:
mixing 1-6% silk fibroin solution with 0.2-4% traditional Chinese medicine polysaccharide to obtain mixed solution;
adding ethanol to adjust the pH value of the mixed solution to acidity, and gelling to obtain the silk fibroin hydrogel with traditional Chinese medicine polysaccharide activity.
Preferably, the concentration of the silk fibroin solution is 1% -4%.
Preferably, the concentration of the traditional Chinese medicine polysaccharide is 0.2-2%.
Preferably, the volume ratio of the silk fibroin solution to the traditional Chinese medicine polysaccharide is 1 (0.25-4). More preferably, the volume ratio of the silk fibroin solution to the traditional Chinese medicine polysaccharide can be 1 (0.25-3).
Preferably, the concentration of the added ethanol is 70 to 100 percent.
Preferably, the pH value of the mixed solution is adjusted to be 4-6.
Preferably, the temperature for forming the gel is 25-50 ℃.
Preferably, the preparation method of the silk fibroin solution comprises the following steps: placing the degummed silk in a 9.3M lithium bromide solution, and heating and dissolving in water bath; cooling the solution to room temperature, and dialyzing; and then centrifuging to remove impurities to obtain the silk fibroin solution.
Further, when the degumming silk and the lithium bromide solution are dissolved by heating in water bath, the water bath ratio of the degumming silk to the lithium bromide solution is 1 (5-10), and the water bath temperature is 65 ℃. During dialysis, a dialysis bag with a molecular weight cut-off of 14000 is adopted for dialysis at 4 ℃ for 3d. The centrifugation conditions were: the temperature is 4 ℃, the rotating speed is 9000r/min, and the centrifugation time is 20min.
Preferably, the traditional Chinese medicine polysaccharide is obtained by extracting the traditional Chinese medicine polysaccharide by a water extraction and alcohol precipitation method and removing protein by a Sevag method, wherein the extracting solution comprises the following components: chloroform: n-butanol = 25.
Further, the preparation method of the traditional Chinese medicine polysaccharide comprises the following steps: taking the mechanically crushed Chinese medicinal materials, placing the Chinese medicinal materials in a water bath according to the material-liquid ratio, extracting for multiple times, combining the filtrates, concentrating, and removing protein by adopting a Sevag method; precipitating the deproteinized polysaccharide solution with ethanol, filtering, and drying to obtain crude polysaccharide; dialyzing the traditional Chinese medicine crude polysaccharide in deionized water, and freeze-drying to obtain the traditional Chinese medicine polysaccharide. Wherein the volume of ethanol used in alcohol precipitation is about 4 times of that of polysaccharide solution after protein removal. Wherein the Chinese medicinal materials can be radix astragali, rhizoma Polygonati, radix Angelicae sinensis, radix et rhizoma Rhei, herba Epimedii, etc.
According to another aspect of the invention, the silk fibroin hydrogel with traditional Chinese medicine polysaccharide activity is prepared by adopting the preparation method.
According to another aspect of the invention, the silk fibroin hydrogel with traditional Chinese medicine polysaccharide activity provided by the invention is applied to medical materials, and the hydrogel is prepared by adopting the preparation method. Specifically, the medical material may be, for example, an enteric material, a drug release and cell culture material, an injectable hydrogel, or the like.
Compared with the prior art, the silk fibroin hydrogel is obtained by adding the traditional Chinese medicine polysaccharide and the ethanol into the silk fibroin solution with the concentration of 1-6% to adjust the pH value to be acidic, the gelling time of the low-concentration silk fibroin solution is shortened, the time can be shortened to be within 5min, the working efficiency is improved, the problem that the silk fibroin hydrogel is restricted to be used due to too long gelling time of the silk fibroin solution in the prior art is solved, and the problems that the toxicity is caused by adding the chemical cross-linking agent into the silk fibroin hydrogel and the biocompatibility of the hydrogel is reduced are also avoided. Moreover, the hydrogel has polysaccharide activity by adding the traditional Chinese medicine polysaccharide, so that the hydrogel has the effect of promoting cell proliferation. The hydrogel prepared by the invention has an obvious three-dimensional network structure, a porous internal structure and interconnection among pores, can be applied to drug release, and is beneficial to cell adhesion and migration.
The invention has the advantages of simple preparation process, lower cost, rapidness, high efficiency and the like; it also has the advantages of temperature sensitivity, acidity sensitivity, and injectability. The hydrogel prepared by the invention has good biocompatibility, does not contain chemical cross-linking agents and the like, is green and safe, and can be widely applied to the pharmaceutical material industry.
In addition, the optional and preferred ranges of the invention are finally obtained through continuously researching the parameter ranges of the proportion, the concentration and the like, and the low-concentration polysaccharide, the low-concentration silk fibroin and the absolute ethyl alcohol can gel more quickly. The pH value is adjusted to be acidic by adding the traditional Chinese medicine polysaccharide with the volume and the ethanol with the concentration range, so that the gelling time of the silk fibroin solution is shortened to be within 5min, even to be about 1min, and the working efficiency is greatly improved; in addition, the hydrogel has polysaccharide activity by adding the traditional Chinese medicine polysaccharide, so that the hydrogel has the effect of promoting cell proliferation.
Drawings
FIG. 1 shows silk fibroin hydrogel with Chinese medicinal polysaccharide activity prepared by the present invention;
FIG. 2 is a graph showing the release of bovine serum albumin coated with silk fibroin hydrogel having activity of polysaccharides from Chinese medicinal materials prepared in example 1 of the present invention;
fig. 3 is a scanning electron microscope image of silk fibroin hydrogel with traditional Chinese medicine polysaccharide activity prepared in example 2 of the present invention.
Detailed Description
The technical solutions of the present invention are described below clearly and completely with reference to the following embodiments, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. The following description of at least one exemplary embodiment is merely illustrative in nature and is in no way intended to limit the invention, its application, or uses. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides a preparation method of silk fibroin hydrogel with traditional Chinese medicine polysaccharide activity, which comprises the following steps:
1. extracting crude polysaccharide from traditional Chinese medicine by water extraction and alcohol precipitation:
1) Taking a certain mass of mechanically crushed traditional Chinese medicinal materials, placing the traditional Chinese medicinal materials in a water bath kettle at 80 ℃ for extraction for 3 times according to the material-liquid ratio of 1. Wherein, the extracting solution: chloroform: n-butanol = 25.
2) Precipitating the deproteinized polysaccharide solution with a certain volume of 95% ethanol, filtering, and drying to obtain crude polysaccharide of traditional Chinese medicine. Wherein, the volume dosage of the ethanol can be 4 times of that of the polysaccharide solution after protein removal.
2. Preparing traditional Chinese medicine polysaccharide:
3) Dialyzing the dried crude polysaccharide of the traditional Chinese medicine in deionized water for 3d, and freeze-drying to obtain traditional Chinese medicine polysaccharide for later use; during dialysis, the cut-off molecular weight of the dialysis bag is 3500, and the dialysis time is 3d.
3. Preparing a silk fibroin solution:
4) Placing a certain mass of degummed silk in a 9.3M lithium bromide solution, heating in a 65 ℃ water bath kettle for full dissolution, cooling the solution to room temperature, transferring into a dialysis bag, and dialyzing in distilled water at low temperature for 3d. And after the dialysis is finished, placing the solution in a centrifuge for centrifugation, and removing insoluble impurities to obtain a clear silk fibroin solution. Wherein the water bath ratio of the degummed silk to the lithium bromide solution is 1 (5-10). During dialysis, a dialysis bag with a molecular weight cut-off of 14000 is adopted for dialysis at 4 ℃ for 3d. The centrifugation conditions were: the temperature is 4 ℃, the rotating speed is 9000r/min, and the centrifugation time is 20min.
4. Preparing silk fibroin hydrogel with traditional Chinese medicine polysaccharide activity:
5) Mixing 1-6% silk fibroin solution and 0.2-4% Chinese medicinal polysaccharide at different volume ratios, adding ethanol with a certain volume, such as 0.2-1 mL, adjusting pH of the mixture to acidity, such as pH 4-6, standing at different temperatures, such as room temperature, 37 deg.C, and 50 deg.C, and observing gelling time. The volume ratio of the silk fibroin solution to the traditional Chinese medicine polysaccharide can be 1 (0.25-4), preferably 1 (0.25-3), and for example, the volume ratio of the silk fibroin solution to the traditional Chinese medicine polysaccharide can be 1: 4. 2: 3. 1:1, 3:2, 4:1. The concentration of the ethanol is 70-100%. Further, the concentration of the silk fibroin solution is preferably 1% -4%. The concentration of the traditional Chinese medicine polysaccharide is preferably 0.2-2%. The ethanol is preferably absolute ethanol. Low concentrations of polysaccharide, low concentrations of silk fibroin and absolute ethanol will gel faster.
6) Determining the gelling time: inverting the centrifuge tube, and keeping the solution from flowing to obtain silk fibroin hydrogel (i.e. silk fibroin-Chinese medicinal polysaccharide hydrogel) with Chinese medicinal polysaccharide activity.
Statistics shows that the preparation method of the silk fibroin hydrogel can shorten the gelling time of the silk fibroin solution to within 5 min.
The present invention will be described in further detail with reference to specific examples.
Example 1:
1) 400g of mechanically crushed astragalus medicinal material is taken, and crude astragalus polysaccharide is extracted by a water extraction and alcohol precipitation method. Wherein, the step of extracting the crude astragalus polysaccharide by using a water extraction and alcohol precipitation method specifically comprises the following steps: extracting in a water bath at 80 ℃ for 3 times according to the ratio of material to liquid of 1. Precipitating the deproteinized polysaccharide solution with 4 times volume of 95% ethanol, filtering, and drying to obtain crude polysaccharide of radix astragali.
2) Dialyzing the dried crude radix astragali polysaccharide in deionized water for 3d, and freeze drying to obtain radix astragali polysaccharide.
3) Placing a certain mass of degummed silk in a 9.3M lithium bromide solution, heating in a 65 ℃ water bath kettle for full dissolution, cooling the solution to room temperature, transferring into a dialysis bag, and dialyzing with distilled water at 4 ℃ for 3d. Centrifuging the solution in a centrifuge at 4 deg.C for 20min and 9000r/min to remove insoluble impurities; a clear silk fibroin solution was obtained.
4) Mixing a silk fibroin solution with the concentration of 2% and astragalus polysaccharide with the concentration of 0.6% according to the volume ratio of 1:4, 3:2, 1:1, 2:3 and 4:1, adding 0.2-1 mL of ethanol with the concentration of 100%, adjusting the pH value of the mixed solution to 4-6, and placing the mixed solution at 37 ℃ to observe gelling time.
5) Inverting the centrifuge tube, and keeping the solution from flowing to obtain silk fibroin astragalus polysaccharide hydrogel as shown in figure 1.
In this example, the gel forming time at each volume ratio is: within 1h, no gel is formed, 1min30s, 1min47s, 2min30s and 1min54s.
Fig. 2 is a release curve diagram of bovine serum albumin coated with silk fibroin hydrogel with traditional Chinese medicine polysaccharide activity prepared in example 1.
The test process comprises the following steps: preparing a polysaccharide solution containing 0.01g/mL bovine serum albumin to prepare hydrogel, adding 10mL PBS serving as a release medium into an EP tube filled with the hydrogel, incubating in a shaker at 37 ℃, and measuring the absorbance of the solution at intervals.
As can be seen from fig. 2: PBS with pH =7.4 is used as a release medium, sample points are taken at intervals of 4h, and the release rate reaches over 80% when 24h and 24h are measured in total.
Example 2:
1) Taking 400g of a mechanically-crushed rhizoma polygonati medicinal material, placing the mechanically-crushed rhizoma polygonati medicinal material in a water bath kettle at 80 ℃ for extraction for 3 times according to the material-liquid ratio of 1: chloroform: n-butanol = 25. Precipitating the polysaccharide solution after protein removal with 4 times volume of 95% ethanol, filtering, and drying to obtain rhizoma Polygonati crude polysaccharide.
2) Dialyzing the dried crude rhizoma Polygonati polysaccharide in deionized water for 3d, and freeze drying to obtain rhizoma Polygonati polysaccharide.
3) Placing a certain mass of degummed silk in a 9.3M lithium bromide solution, heating in a 65 ℃ water bath kettle for full dissolution, cooling the solution to room temperature, transferring into a dialysis bag, and dialyzing with distilled water at 4 ℃ for 3d. Centrifuging the solution in a centrifuge at 4 deg.C for 20min and 9000r/min to remove insoluble impurities; a clear silk fibroin solution was obtained.
4) Mixing a silk fibroin solution with the concentration of 4% and polygonatum polysaccharide with the concentration of 1.6% according to the volume ratio of 1:4, 3:2, 1:1, 2:3 and 4:1, adding 0.2-1 mL of ethanol with the concentration of 85%, adjusting the pH value of the mixed solution to 4-6, and standing at room temperature to observe the gelling time.
5) Inverting the centrifugal tube, and keeping the solution not flowing to obtain the silk fibroin rhizoma polygonati polysaccharide hydrogel.
In this example, the gel forming time at each volume ratio is: no gelling, 4min10s, 3min30s and 3min56s in1 h.
Fig. 3 is a scanning electron microscope image of silk fibroin hydrogel with traditional Chinese medicine polysaccharide activity prepared in the above example 2 of the present invention, and it can be known from fig. 3 that: the prepared hydrogel has an obvious three-dimensional network structure, a porous internal structure and the mutual connection among pores, can be applied to drug release, and is beneficial to the adhesion and migration of cells.
Claims (10)
1. A preparation method of silk fibroin hydrogel with traditional Chinese medicine polysaccharide activity is characterized by comprising the following steps:
mixing 1-6% silk fibroin solution with 0.2-4% traditional Chinese medicine polysaccharide to obtain mixed solution;
and adding ethanol into the mixed solution, adjusting the pH value of the mixed solution to acidity, and gelling to obtain the silk fibroin hydrogel with the traditional Chinese medicine polysaccharide activity.
2. The preparation method of claim 1, wherein the volume ratio of the silk fibroin solution to the traditional Chinese medicine polysaccharide is 1 (0.25-3).
3. The method according to claim 1, wherein the concentration of ethanol is 70% to 100%; and adjusting the pH value of the mixed solution to 4-6.
4. The method of claim 1, wherein the temperature of the gel is 25 ℃ to 50 ℃.
5. The method of claim 1, wherein the method comprises:
placing the degummed silk in a 9.3M lithium bromide solution, and heating and dissolving in water bath;
cooling the solution to room temperature, and dialyzing;
and then centrifuging to remove impurities to obtain the silk fibroin solution.
6. The production method according to claim 5,
when the degummed silk and the lithium bromide solution are heated and dissolved in water bath, the water bath ratio of the degummed silk to the lithium bromide solution is 1 (5-10), and the water bath temperature is 65 ℃;
dialyzing at 4 deg.C for 3d with dialysis bag with molecular weight cutoff of 14000;
the centrifugation conditions were: the temperature is 4 ℃, the rotating speed is 9000r/min, and the centrifugation time is 20min.
7. The preparation method of claim 1, wherein the Chinese medicinal polysaccharide is obtained by extracting the Chinese medicinal polysaccharide by a water extraction and alcohol precipitation method and removing protein by a Sevag method, wherein an extracting solution: chloroform: n-butanol = 25.
8. The method for preparing the Chinese medicinal polysaccharide according to claim 7, wherein the method for preparing the Chinese medicinal polysaccharide comprises the following steps:
taking the mechanically crushed Chinese medicinal materials, placing the Chinese medicinal materials in a water bath according to the material-liquid ratio, extracting for multiple times, combining filtrates, concentrating, and removing protein by adopting a Sevag method;
precipitating the deproteinized polysaccharide solution with ethanol, filtering, and drying to obtain crude polysaccharide;
dialyzing the crude polysaccharide of the traditional Chinese medicine in deionized water, and freeze-drying to obtain the polysaccharide of the traditional Chinese medicine.
9. A silk fibroin hydrogel with traditional Chinese medicine polysaccharide activity is characterized by being prepared by the preparation method of any one of claims 1-8.
10. An application of silk fibroin hydrogel with traditional Chinese medicine polysaccharide activity prepared by the preparation method of any one of claims 1-8 in medical materials.
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| CN116808111A (en) * | 2023-08-21 | 2023-09-29 | 长春中医药大学 | Hydrogel film loaded with traditional Chinese medicine, preparation method thereof and application of hydrogel film in resisting osteosarcoma |
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