CN116036356A

CN116036356A - Preparation method and application of glycosylated curcumin hydrogel dressing for promoting wound healing

Info

Publication number: CN116036356A
Application number: CN202211500195.7A
Authority: CN
Inventors: 朱道辰; 李奕萱; 姚明迅; 陈焱; 王倩茹; 孙亚如
Original assignee: Jiangsu University
Current assignee: Jiangsu University
Priority date: 2022-11-28
Filing date: 2022-11-28
Publication date: 2023-05-02

Abstract

The invention belongs to the field of bioengineering technology and additive manufacturing, and particularly relates to a preparation method and application of glycosylated curcumin hydrogel dressing for wound healing. The glycosyltransferase gene from Bacillus subtilis and 168 is constructed on an expression vector, inserted into NdeI and XhoI enzyme cutting sites to form a co-expression recombinant plasmid which is pET_YjiC, and the co-expression recombinant plasmid is transformed into competent cells of escherichia coli BL21 (DE 3) to obtain a recombinant strain, and the recombinant strain can be used for transforming curcumin into glycosylated products after induced expression, wherein the water solubility of the glycosylated curcumin of the obtained products can reach 49.60mg/L and 15.31mg/L. The hydrogel dressing prepared from glycosylated curcumin, enzymatically modified lignin, polycaprolactone, polyethylene glycol and keratin has the advantages of immunoregulation function and complete degradation, and the application is suitable for aseptic maintenance of skin with damaged skin trauma barrier and wound healing of postoperative inpatients.

Description

Preparation method and application of glycosylated curcumin hydrogel dressing for promoting wound healing

Technical Field

The invention belongs to the field of bioengineering technology and additive manufacturing, and particularly relates to a preparation method and application of a glycosylated curcumin hydrogel dressing for promoting wound healing.

Background

Accidental trauma, tumor surgery, and chronic trauma caused by diabetes are major problems of clinical concern. The main reason for the difficulty in healing chronic wounds is that oxygen and nutrient supply are insufficient due to poor blood circulation, the generation of immunocyte active oxygen is increased, and degradation of extracellular matrix and damage of skin tissues are caused. In clinical treatment of patient wounds, it is generally necessary to cover the wound site with a dressing to prevent bacterial invasion and maintain a moist environment for the wound surface. Although the traditional dressing such as gauze, cotton pad and the like has a protective effect, the traditional dressing has no performance of treating and repairing wounds, and the traditional dressing has poor biodegradability and is easy to adhere to wound tissues, so that secondary damage is caused. The hydrogel dressing is a novel wound dressing, is a gelatinous substance which is formed by taking high polymer materials such as polyacrylic acid, polyvinyl alcohol and the like as base materials, has a three-dimensional network structure and is insoluble in water, has various surface functional groups, has a certain slow release effect, and is widely applied to the field of biomedical engineering.

However, most hydrogels are prepared from petroleum-based chemicals, which are expensive to manufacture and adversely affect the environment. The lignin structure contains a large number of functional groups and chemical reaction active sites, and can be subjected to chemical modification and graft copolymerization to prepare lignin composite hydrogel. The lignin is introduced into the hydrogel, so that the strength of the hydrogel can be improved, the hydrogel is easy to degrade in water environment, and the high water content, good biocompatibility and special surface characteristics of the hydrogel make the hydrogel one of the most popular materials in biological, medical and environmental applications. Due to the good biocompatibility, the hydrogel dressing can absorb wound exudates to prevent wound infection, and the dressing does not need to be replaced frequently in a short time; dressing gels that absorb large amounts of exudates, however, may separate from the wound and may not effectively block bacterial invasion. Therefore, in the practical application process, medicines such as different antibacterial agents, bioactive components and the like are often embedded in the hydrogel according to the needs, and the medicines can be continuously released to a lesion area through body fluid exchange, so that bacterial growth is reduced, wound infection is prevented, and wound healing is promoted.

Curcumin is a polyphenol compound extracted from rhizomes of plants in the family of zingiberaceae, is a green, safe and nontoxic natural antibacterial agent, has good biological activity, and has pharmacological activities of anti-inflammatory, antioxidant, antitumor, anticancer, wound repair and the like, and is widely applied to the fields of medical treatment and cosmetics. However, because curcumin has large molecular weight, unstable structure and poor water solubility, and has hydroxyl groups at two ends of the molecule, conjugate effect is easy to occur under alkaline condition, and the curcumin is easy to dissolve in glacial acetic acid and alkaline solution, the bioavailability of the curcumin is low, and the application of the curcumin on wound dressing is limited.

Disclosure of Invention

The present invention has been made keeping in mind the above problems occurring in the prior art, and an object of the present invention is to solve one of the problems; the hydrogel dressing for promoting wound healing by glycosylated curcumin can improve the water solubility of the curcumin, improve the bioavailability of the curcumin, stably load the curcumin, play a role in long-acting anti-inflammation and sterilization, prevent bacterial infection of wound surfaces and help wound healing.

The invention firstly provides a glycosylated curcumin hydrogel dressing for promoting wound healing, which comprises the following components in percentage by mass: alkali lignin or enzyme modified alkali lignin, 0.1-15%;

glycosylated curcumin 0.01-5%;

polyethylene glycol (PEG), 0.1-2%;

polycaprolactone (PCL), 0.1-10%;

keratin, 0.1-20%;

0.01-1% of glycerol;

the balance being water.

The preparation method of the enzyme modified lignin comprises the following steps:

(1) Firstly preparing an enzymolysis system, and then reacting the enzymolysis system in a water bath shaking table of 120r/min at 37 ℃ for 12-24 hours to obtain a reaction liquid;

the enzymolysis system consists of laccase or dye decolorization peroxidase, tris-HCl buffer solution, 2-hydrazine-bis (3-ethyl-benzothiazole-6-sulfonic Acid) (ABTS) and lignin, wherein the dosage relationship of the laccase or dye decolorization peroxidase, the Tris-HCl buffer solution and the lignin is 5mL:20mL:1g; the final concentration of the ABTS in the enzymolysis system is 0.2mM; the concentration of laccase or dye decolorization peroxidase in an enzymolysis system is 10000U/L; the concentration of the Tris-HCl buffer solution is 50mM, and the pH value is 7; the lignin includes, but is not limited to, lignosulfonate, alkali lignin, ground lignin, or solvent type alcoholysis lignin.

(2) Centrifuging the reaction solution obtained in the step (1) to remove insoluble lignin in the solution, collecting supernatant, boiling for 10-15min, centrifuging to remove protein, adjusting the pH of the supernatant to 3 by HCl, centrifuging to collect precipitate;

(3) And (3) performing vacuum freeze drying on the collected precipitate for 48 hours to obtain a final product, namely the enzyme modified alkali lignin for later use.

The preparation method of the keratin comprises the following steps:

cleaning keratin raw materials with clear water, adding ethanol for treatment for a period of time, filtering, adding dilute HCl into the precipitate for treatment, and adding mercaptoethanol for reduction reaction after treatment; centrifuging to obtain precipitate after reaction, and lyophilizing to obtain keratin; the mass ratio of the keratin raw material to the ethanol to the dilute HCl to the mercaptoethanol is 1:2:2:1;

preferably, the keratin materials include, but are not limited to, animal hair, poultry feathers, or human hair.

Preferably, the mass fraction of the ethanol is 50%; the mass fraction of the dilute HCl is 5%; the ethanol is added for treatment for 30min, and the dilute HCl is added for treatment for 1h; the time of the reduction reaction is 1h.

The biological preparation method of the glycosylated curcumin comprises the following steps:

(1) Firstly preparing an enzymolysis system, and then carrying out water bath reaction on the enzymolysis system to obtain a reaction solution; wherein the enzymolysis system comprises purified protein, tris-HCl buffer solution, uridine diphosphate glucose (UDP-Glc) and curcumin; the dosage relationship of the purified protein, the Tris-HCl buffer solution and the curcumin is 5 mug: 300. Mu.L: 2. Mu.L; the final concentration of UDP-Glc in the enzymatic hydrolysis system is 1.37mM; the concentration of the curcumin is 20mg/mL; the concentration of the Tris-HCl buffer solution is 50mM, and the pH value is 7;

the purified protein is glucosyltransferase (named as Bs-YjiC) from Bacillus subtilits 168, the DNA sequence of the purified protein is shown as SEQ ID NO.1, and the amino acid sequence of the purified protein is shown as SEQ ID NO. 2;

(2) Adding ethyl acetate into the reaction solution obtained in the step (1) for extraction to obtain a mixed reaction solution, evaporating in a water bath to 1% -5% of the volume of the mixed reaction solution, and adding chromatographic pure ethanol into the evaporated mixed reaction solution to obtain glycosylated curcumin, wherein the glycosylated curcumin is stored at a low level Wen Biguang; the volume ratio of the reaction solution to the ethyl acetate to the chromatographic pure ethanol is 3:6:2.

preferably, the temperature of the water bath reaction in the step (1) is 37 ℃ and the reaction time is 1h.

Preferably, the temperature of evaporation of the water bath in step (2) is 55 ℃.

The invention also provides a preparation method of the glycosylated curcumin hydrogel dressing for promoting wound healing, which comprises the following steps:

(1) Polycaprolactone (PCL), polyethylene glycol (PEG) and stannous octoate (Sn (Oct) ₂ ) After mixing, carrying out reaction under the condition of introducing nitrogen, after the reaction is reduced to a certain temperature, adding keratin and glycosylated curcumin for continuous second reaction, after the reaction, adding deionized water and glycerol, and stirring to uniformly disperse the mixture to obtain aqueous emulsion;

(2) And (3) mixing the aqueous emulsion obtained in the step (1) with alkali lignin or enzyme modified alkali lignin at room temperature to obtain a mixture, performing pre-freezing treatment on the mixture, and then performing freeze drying to obtain the glycosylated curcumin hydrogel dressing.

Preferably, the PCL, PEG, sn (Oct) of step (1) ₂ The amounts of keratin, glycosylated curcumin, deionized water and glycerol were related to 10g:3g:0.1g:4.5g:1g:36g:0.21g; the reaction temperature is 75 ℃, and the reaction time is 3 hours; said cooling to a certain temperature of 50 ℃, said second reactionThe time to be taken was 1h.

Preferably, the pre-freezing treatment in the step (2) is carried out at a temperature of-20 ℃ for 24 hours; the dosage relation of the alkali lignin or the enzyme modified alkali lignin and the aqueous emulsion is 2g:1.7mL.

Compared with the prior art, the invention has the following beneficial effects:

the glycosylated curcumin hydrogel dressing for promoting wound healing has good antibacterial property and drug slow release effect, can continuously act on wound sites, and can be applied to wounds such as trauma, burn, scald, ulcer, bedsore and the like.

(1) The lignin selected by the invention contains a large number of functional groups and chemical reaction active sites, has good biocompatibility, can increase the strength of the hydrogel, is easy to degrade in water environment, and has the advantages of low toxicity, environmental friendliness and the like. The color of the enzyme modified lignin after laccase or dye decolorization peroxidase treatment is lighter and more attractive.

(2) According to the invention, polycaprolactone, polyethylene glycol, keratin, glycosylated curcumin and the like are dissolved in deionized water to obtain aqueous emulsion, and then the aqueous emulsion is polymerized with alkali lignin or enzyme modified alkali lignin to prepare the enzyme modified lignin hydrogel dressing for stably loading curcumin.

(3) Compared with natural curcumin, the glycosylated curcumin used in the invention has the advantages that the water solubility of the curcumin glucoside products can reach 49.60mg/L and 15.31mg/L respectively, the water solubility of curcumin in the hydrogel dressing is obviously improved, and the bioavailability and the slow release effect of the curcumin are improved.

(4) The glycosylated curcumin hydrogel dressing has good mechanical properties, good flexibility and elasticity, can form good adhesion with skin, and is suitable for wound surfaces of different parts of a body, in particular to frequent movement parts such as elbows, wrists, knees, ankles and the like.

Drawings

FIG. 1 is a graph showing the results of gel electrophoresis on a protein nickel column purification, wherein A: marker, negative control, C0 mM imidazole elution, D250 mM imidazole elution.

FIG. 2 is a graph of glycosylated curcumin preparation conditions, wherein A is a graph of HPLC before and after reaction; b is the influence of reaction time on the curcumin conversion effect; c is the influence of pH on the curcumin conversion effect; d is the influence of the reaction temperature on the curcumin conversion effect.

Fig. 3 is a photograph of a gel sample applied to the skin surface.

Fig. 4 is a graph of in vitro release test of curcumin in a gel sample.

FIG. 5 is a graph showing the antibacterial effect of gel samples on E.coli, wherein 1 and 2 in the graph correspond to control samples and gel samples, respectively.

Detailed Description

The invention will be better understood from the following examples. However, it is readily understood by those skilled in the art that the examples are only for illustrating the present invention and should not limit the invention described in the claims.

Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.

Strain description: the purified protein is glucosyltransferase (named as Bs-YjiC (NP-389104.1)) from Bacillus subtilits 168, the DNA sequence of the purified protein is shown as SEQ ID NO.1, and the amino acid sequence of the purified protein is shown as SEQ ID NO. 2; the Bacillus subtilis is derived from China general microbiological culture Collection center, and has a preservation number of: CGMCCNO.1.1391.

The scheme is as follows: the invention firstly provides a glycosylated curcumin hydrogel dressing for promoting wound healing, which comprises the following components in percentage by mass: alkali lignin or enzyme modified alkali lignin, 0.1-15%;

glycosylated curcumin 0.01-5%;

polyethylene glycol (PEG), 0.1-2%;

polycaprolactone (PCL), 0.1-10%;

keratin, 0.1-20%;

0.01-1% of glycerol;

the balance being water.

the enzymolysis system consists of laccase or dye decolorization peroxidase, tris-HCl buffer solution, 2-hydrazine-bis (3-ethyl-benzothiazole-6-sulfonic Acid) (ABTS) and alkali lignin, wherein the dosage relationship of the laccase or dye decolorization peroxidase, the Tris-HCl buffer solution and the alkali lignin is 5mL:20mL:1g; the final concentration of the ABTS in the enzymolysis system is 0.2mM; the concentration of laccase or dye decolorization peroxidase in an enzymolysis system is 10000U/L; the Tris-HCl buffer has a concentration of 50mM and a pH of 7.

(2) Centrifuging the reaction solution obtained in the step (1) to remove insoluble lignin in the solution, collecting supernatant, boiling for 10-15min, centrifuging to remove protein, regulating the pH of the supernatant to 3 by HCl, centrifuging, and collecting precipitate;

(3) And (3) performing vacuum freeze drying on the collected precipitate for 48 hours to obtain a final product, namely the enzyme modified alkali lignin for standby.

The keratin treatment method comprises the following steps:

cleaning keratin raw materials with clear water, adding ethanol with the mass fraction of 50% for 30min, filtering, adding dilute HCl (with the mass fraction of 5%) into the precipitate for treatment for 1h, and then adding mercaptoethanol for reduction for 1h; finally, obtaining precipitate after centrifugation, and obtaining keratin after freeze drying (-20 ℃); wherein the mass ratio of the keratin raw material to the ethanol to the dilute HCl to the mercaptoethanol is 1:2:2:1; the keratin materials include, but are not limited to: animal hair, poultry feathers and human hair.

(1) Firstly preparing an enzymolysis system, and then reacting the enzymolysis system in a water bath kettle at 37 ℃ for 1h to obtain a reaction liquid; the enzymolysis system consists of purified protein, tris-HCl buffer solution, uridine diphosphate glucose (UDP-Glc) and curcumin, wherein the dosage relationship of the purified protein, the Tris-HCl buffer solution and the curcumin is 5 mug: 300. Mu.L: 2. Mu.L; the final concentration of UDP-Glc in the enzymatic hydrolysis system is 1.37mM; the concentration of the curcumin is 20mg/mL; the concentration of the Tris-HCl buffer solution is 50mM, and the pH value is 7; wherein the purified protein is glucosyltransferase (named as Bs-YjiC) from Bacillus subtilits 168, the DNA sequence is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2.

(2) Adding ethyl acetate into the reaction solution obtained in the step (1) for extraction to obtain a mixed reaction solution, evaporating the mixed reaction solution to 1-5% of the volume of the mixed reaction solution in a water bath kettle at 55 ℃, and then adding chromatographic pure ethanol into the evaporated mixed reaction solution to obtain glycosylated curcumin, wherein the glycosylated curcumin is stored at a low temperature of Wen Biguang; the volume ratio of the reaction solution to the ethyl acetate to the chromatographic pure ethanol is 3:6:2.

a method for preparing a glycosylated curcumin hydrogel dressing for promoting wound healing, comprising the following steps of:

(1) Polycaprolactone (PCL), polyethylene glycol (PEG) and stannous octoate (Sn (Oct) ₂ ) After mixing, carrying out reaction under the condition of introducing nitrogen, wherein the reaction temperature is 75 ℃, and the reaction time is 3 hours; after the reaction, after the temperature is reduced by 50 ℃, keratin and glycosylated curcumin are added for continuous second reaction, wherein the reaction time is 1h; adding deionized water and glycerol after the reaction, and stirring to uniformly disperse the components to obtain the aqueous emulsion.

(2) Mixing the aqueous emulsion obtained in the step (1) with alkali lignin or enzyme modified alkali lignin under the condition of room temperature to obtain a mixture, and performing pre-freezing treatment on the mixture at the temperature of-20 ℃ for 24 hours; then freeze-drying to obtain glycosylated curcumin hydrogel dressing; the dosage relation of the alkali lignin or the enzyme modified alkali lignin and the aqueous emulsion is 2g:1.7mL.

Example 1: biological preparation of glycosylated curcumin and solubility test thereof

1. Construction of glycosyltransferase (Bs-YjiC) engineering bacteria

(1) Glycosyltransferase (Bs-YjiC) gene synthesis

The full-length gene of glycosyltransferase Bs-YjiC (synthesized by Suzhou Hongxun biotechnology Co., ltd.) is synthesized by an in vitro total gene synthesis method, and the DNA sequence is shown as SEQ ID NO. 1;

(2) Treatment and ligation of glycosyltransferase (Bs-YjiC) genes

T is used for the fragment of the glycosyltransferase (Bs-YjiC) gene synthesized in the step (1) ₄ Post-treatment with DNA PolymeraseThe resulting mixture was ligated with plasmid pET-28a (+) (Hongxun Biotech Co., ltd.) at room temperature for 20 minutes to obtain ligated plasmid pET-YjiC.

(3) Transformation of recombinant plasmid in E.coli BL21 (DE 3)

Adding 0.5uL of plasmid pET-YjiC obtained in the step (2) into 100uL E.coli BL21 (DE 3) competent cells, placing on ice for 20min, then carrying out heat shock at 42 ℃ for 90s, then carrying out ice bath for 2min, adding 250 mu L of LB culture medium after the ice bath, and culturing at 37 ℃ for 30min to obtain a culture solution; subsequently, 150. Mu.L of the culture broth was applied to LB plates containing 50. Mu.g/mL kanamycin sulfate (Kan), and the plates were incubated at 37℃overnight with inversion to give engineering bacteria containing the glycosyltransferase (Bs-YjiC) gene.

(4) Recombinant engineering bacteria small quantity induction expression

Picking engineering bacteria containing glycosyltransferase (Bs-YjiC) genes after recombination on the flat plate in the step (3), inoculating the engineering bacteria into a test tube containing 1mL LB culture solution of 50 mug/mL kanamycin sulfate (Kan), and shaking and culturing at 37 ℃ and 220rpm until OD ₆₀₀ When the concentration is 0.8, 500 mu L of culture bacteria liquid is taken out, IPTG is added to the culture bacteria liquid till the final concentration is 1mM, and the culture is continued for 3 hours at 220rpm and 37 ℃; the bacterial cells were collected by centrifugation at 10000rpm for 2min, 50. Mu.L of Tris-HCl buffer (50 mM, pH 7) was added to the bacterial cells to resuspend the bacterial cells, to obtain bacterial suspension, and protein expression was detected by electrophoresis.

2. Purification of proteins

Engineering bacteria of glycosyltransferase (Bs-YjiC) gene were inoculated into 600mL LB medium (1% of the inoculum size) containing 50. Mu.g/mL Kan, and shaken at 37℃and 220rpm to the cell OD ₆₀₀ Adding inducer IPTG to a final concentration of 1mM at 0.8-1.0, continuously culturing at 37deg.C and 220rpm for 4 hr, centrifuging at 5000rpm and 4deg.C for 5min to collect thallus, ultrasonic crushing, centrifuging at 12000rpm and 4deg.C for 10min, centrifuging, collecting supernatant, and purifying with Smart-Ni as filler; wherein the balancing liquid is as follows: tris-HCl buffer, pH 7.4, eluent: tris-HCl buffer, pH 7.4; gradient elution was performed with 20mM, 50mM, 250mM imidazole gradient, respectively, to give purified proteins, the results are shown in FIG. 1.

3. Glycosyltransferase Bs-YjiC enzyme activity assay

With curcumin as a substrate, the assay system is 300 mu L, and contains 50mM Tris-HCl buffer solution with pH value of 8.0, 5 mu g purified protein and 1.37mM UDP-Glc with detection wavelength of 425nm. The amount of enzyme required for converting 1 mu mol of substrate into a product is an international enzyme activity unit, and the maximum specific activity is 144.77 +/-0.53U/mg.

4. Optimal condition test for converting glycosyltransferase (Bs-YjiC) into curcumin

The assay system was 300. Mu.L and contained 5. Mu.g of purified protein, 50mM Tris-HCl,1.37mM UDP-Glc and 2. Mu.L curcumin. After the reaction, 600. Mu.L of ethyl acetate was added for extraction, 500. Mu.L of water bath at 55℃was taken for volatilization, 200. Mu.L of chromatographic pure ethanol was added for HPLC detection, and the analytical detection wavelength was 425nm. The results are shown in FIG. 2, panel A is a HPLC plot before and after the reaction; figure B, C, D shows the effect of reaction time, pH and reaction temperature on curcumin conversion effect, respectively. FIG. 2A illustrates the two new products produced after glycosyltransferase conversion and FIGS. 2B-2D illustrate that the conversion is best at pH 8, temperature 40℃and reaction time 30 min.

5. Glycosylated curcumin solubility assay

The glycosylated curcumin is dissolved in 1mL of Tris-HCl buffer solution (pH 8.0) and stirred for 30min, the mixture is centrifuged for 15min at 12000r/min, filtered by a 0.45 mu m filter membrane, and added with equal volume of methanol, the glycosylated curcumin solubility is measured by HPLC by taking natural curcumin as a reference, and the water solubility of the curcumin glucoside products can reach 49.60mg/L and 15.31mg/L respectively.

Example 2: preparation of glycosylated curcumin hydrogel dressing for promoting wound healing

The glycosylated curcumin hydrogel dressing for promoting wound healing comprises the following components in percentage by mass:

15% lignin, 5% glycosylated curcumin, 0.1% polyethylene glycol (PEG), 10% Polycaprolactone (PCL), 20% keratin, glycerol 1% and the balance water;

the following raw materials are used for the components:

(1) Preparation of modified lignin

The enzymolysis system is 500mL, the system contains 100mL of laccase with 10000U/L, 400mL of Tris-HCl buffer with pH of 7, ABTS with final concentration of 0.2mM and 20g of alkali lignin, and the reaction is carried out for 12-24h in a water bath shaking table with the temperature of 120r/min to obtain reaction liquid;

centrifuging the reaction solution to remove insoluble lignin in the solution, collecting supernatant, boiling for 10min, centrifuging to remove protein, regulating pH of the supernatant to 3 with HCl, centrifuging, and collecting precipitate; and (3) performing vacuum freeze drying on the collected precipitate for 48 hours to obtain a final product, namely the enzyme modified alkali lignin.

(2) Pretreatment of keratin

Animal wool is selected as keratin raw material, in particular wool;

cleaning with clear water, adding ethanol with the mass fraction of 50% for 30min, filtering, adding dilute HCl (with the mass fraction of 5%) for 1h, and then reducing with mercaptoethanol for 1h; centrifuging, and freeze drying (-20deg.C) to obtain keratin; the mass ratio of the keratin raw material to the ethanol to the dilute HCl to the mercaptoethanol is 1:2:2:1.

The preparation of the glycosylated curcumin hydrogel dressing for promoting wound healing comprises the following specific steps:

10g Polycaprolactone (PCL), 3g polyethylene glycol (PEG) and 0.1g stannous octoate Sn (Oct) ₂ After mixing, introducing nitrogen to react for 3 hours at 75 ℃; then the temperature is reduced to 50 ℃, 4.5g of keratin and 1g of glycosylated curcumin are added for continuous reaction for 1h, 36g of deionized water and 0.21g of glycerin are added after the reaction, and the mixture is stirred to be uniformly dispersed, so as to obtain aqueous emulsion;

finally, 2g of enzyme modified alkali lignin and 1.7mL of aqueous emulsion are mixed at room temperature to obtain a mixture, the mixture is placed at the temperature of minus 20 ℃ for pre-freezing for 24 hours, and the mixture is frozen and dried to obtain the glycosylated curcumin hydrogel dressing.

Example 3: performance verification of glycosylated curcumin-promoted wound healing hydrogel dressing

(1) Adhesion test:

the gel sample was applied to the arm as shown in fig. 3. From the figure, it can be seen that the gel sample forms a good bond with the skin and is not easily detached.

(2) Curcumin in vitro release assay:

cutting the gel sample into round sample pieces with the diameter of 25mm and the thickness of 2mm, and weighing and marking as m ₀ Curcumin in vitro release assay was performed at 37℃in 50mL of 50mM Tris-HCl buffer (pH 7.2). Absorbing proper amount of buffer solution at each interval of 12h, diluting with absolute ethanol, measuring absorbance at 425nm, and calculating curcumin release amount m ₁ . The cumulative release rate R (%) of curcumin over time was calculated according to the formula based on the average loading amount 536. Mu.g/g of curcumin in the gel sample, and the result is shown in FIG. 4. The curcumin loaded on the gel sample is released quickly in the initial 24 hours, the release rate is gradually slowed down after 24 hours, but the curcumin is basically unchanged until 96 hours, and the release rate reaches more than 50%. The glycosylated curcumin hydrogel dressing disclosed by the invention can slowly release curcumin for a long time and maintain the pharmacodynamic effect.

(3) Antibacterial test:

1mL of E.coli suspension (OD ₆₀₀ 1.0) was spread on LB agar plates, hydrogel samples were covered on the surfaces of the LB agar plates, and after 48 hours of culture at 37℃the LB plates were observed for the presence of a distinct zone of inhibition. Gel samples and control samples (without glycosylated curcumin added) were selected for bacteriostasis test, and the experimental results are shown in fig. 5. From the figure, after 48 hours of culture, the control sample has no obvious inhibition zone on the escherichia coli, which indicates that the control sample has no antibacterial activity on the escherichia coli; the gel sample generates a bacteriostasis zone of 3.1+9.7mm for the escherichia coli, which proves that the glycosylated curcumin-loaded hydrogel dressing has obvious antibacterial activity for the escherichia coli.

Description: the above embodiments are only for illustrating the present invention and not for limiting the technical solution described in the present invention; thus, while the invention has been described in detail with reference to the various embodiments described above, it will be understood by those skilled in the art that the invention may be modified or equivalents; all technical solutions and modifications thereof that do not depart from the spirit and scope of the present invention are intended to be included in the scope of the appended claims.

SEQ ID NO.1 Gene sequence (DNA):

AAAAAGTATCATATTAGCATGATTAACATTCCGGCGTACGGCCATGTGAACCCGACCCTGGCGCTGGTTGAAAAACTGTGCGAAAAAGGCCATCGCGTGACCTATGCGACCACTGAAGAATTTGCGCCGGCGGTGCAGCAAGCGGGTGGTGAAGCGCTGATCTACCATACTAGCCTGAACATTGATCCGAAACAGATTCGCGAAATGATGGAAAAGAACGATGCGCCGCTGAGCCTGCTGAAAGAAAGCCTGAGCATTCTGCCGCAGCTGGAGGAGCTGTATAAAGATGATCAGCCGGATCTGATTATTTATGACTTTGTGGCGCTGGCGGGCAAACTGTTTGCGGAAAAACTGAACGTGCCGGTGATTAAACTGTGCAGCAGCTATGCGCAGAACGAAAGCTTTCAGCTGGGCAACGAAGATATGCTGAAAAAGATTCGCGAAGCGGAAGCCGAATTTAAAGCGTATCTGGAACAGGAAAAACTGCCGGCGGTGAGCTTTGAACAGCTGGCGGTGCCGGAAGCGCTGAATATTGTGTTTATGCCGAAAAGCTTTCAGATTCAGCATGAAACCTTCGATGATCGCTTTTGCTTTGTGGGCCCGAGCCTGGGCGAACGCAAGGAAAAAGAAAGCCTGCTGATTGATAAAGATGATCGCCCGTTAATGCTGATTAGCCTGGGCACCGCGTTTAACGCGTGGCCGGAATTTTATAAAATGTGCATCAAGGCGTTTCGCGATAGCAGCTGGCAGGTGATTATGAGCGTGGGCAAAACCATTGATCCGGAAAGCCTGGAAGATATTCCGGCGAACTTTACCATTCGCCAGAGCGTGCCGCAGCTGGAAGTGCTGGAAAAAGCGGATCTGTTTATTAGCCATGGCGGCATGAACAGCACCATGGAAGCGATGAACGCGGGTGTGCCGCTGGTGGTGATTCCGCAGATGTATGAACAGGAACTGACCGCGAACCGCGTGGATGAACTGGGCTTAGGCGTGTATCTGCCGAAAGAAGAAGTGACCGTGAGCAGCCTGCAGGAAGCGGTGCAGGCGGTTAGCAGTGATCAAGAACTGCTGAGCCGCGTGAAAAACATGCAGAAAGATGTGAAAGAAGCGGGCGGCGCGGAACGCGCGGCAGCAGAAATTGAAGCGTTTATGAAAAAGAGCGCGGTGCCGCAGTAA

SEQ ID NO.2 amino acid sequence:

MKKYHISMINIPAYGHVNPTLALVEKLCEKGHRVTYATTEEFAPAVQQAGGEALIYHTSLNIDPKQIREMMEKNDAPLSLLKESLSILPQLEELYKDDQPDLIIYDFVALAGKLFAEKLNVPVIKLCSSYAQNESFQLGNEDMLKKIREAEAEFKAYLEQEKLPAVSFEQLAVPEALNIVFMPKSFQIQHETFDDRFCFVGPSLGERKEKESLLIDKDDRPLMLISLGTAFNAWPEFYKMCIKAFRDSSWQVIMSVGKTIDPESLEDIPANFTIRQSVPQLEVLEKADLFISHGGMNSTMEAMNAGVPLVVIPQMYEQELTANRVDELGLGVYLPKEEVTVSSLQEAVQAVSSDQELLSRVKNMQKDVKEAGGAERAAAEIEAFMKKSAVPQ

Claims

1. the glycosylated curcumin hydrogel dressing for promoting wound healing is characterized by comprising the following components in percentage by mass: alkali lignin or enzyme modified alkali lignin, 0.1-15%;

glycosylated curcumin 0.01-5%;

polyethylene glycol, 0.1-2%;

polycaprolactone 0.1-10%;

keratin, 0.1-20%;

0.01-1% of glycerol;

the balance being water.

2. The glycosylated curcumin promoted wound healing hydrogel dressing according to claim 1, wherein the enzyme-modified lignin is obtained by a preparation method comprising the following steps:

the enzymolysis system consists of laccase or dye decolorization peroxidase, tris-HCl buffer solution, ABTS and lignin, wherein the dosage relationship of the laccase or dye decolorization peroxidase, the Tris-HCl buffer solution and the lignin is 5mL:20mL:1g; the final concentration of the ABTS in the enzymolysis system is 0.2mM; the concentration of laccase or dye decolorization peroxidase in an enzymolysis system is 10000U/L; the concentration of the Tris-HCl buffer solution is 50mM, and the pH value is 7; the lignin includes, but is not limited to, lignosulfonate, alkaline lignin, ground lignin, or solvent-type alcoholysis lignin;

(3) And (3) performing vacuum freeze drying on the collected precipitate for 48 hours to obtain a final product, namely the enzyme modified alkali lignin.

3. The glycosylated curcumin promoted wound healing hydrogel dressing according to claim 1, wherein the keratin is obtained by a preparation method comprising the following steps:

cleaning keratin raw materials with clear water, adding ethanol for treatment for a period of time, filtering, adding dilute HCl into the precipitate for treatment, and adding mercaptoethanol for reduction reaction after treatment; centrifuging to obtain precipitate after reaction, and lyophilizing to obtain keratin; the mass ratio of the keratin raw material to the ethanol to the dilute HCl to the mercaptoethanol is 1:2:2:1.

4. A glycosylated curcumin promoted wound healing hydrogel dressing according to claim 3, wherein said keratin materials include but are not limited to animal hair, poultry feathers or human hair.

5. A glycosylated curcumin promoted wound healing hydrogel dressing according to claim 3, wherein the mass fraction of said ethanol is 50%; the mass fraction of the dilute HCl is 5%; the ethanol is added for treatment for 30min, and the dilute HCl is added for treatment for 1h; the time of the reduction reaction is 1h.

6. The glycosylated curcumin hydrogel dressing for promoting wound healing according to claim 1, wherein the glycosylated curcumin is obtained by a preparation method comprising the following preparation steps:

(1) Firstly preparing an enzymolysis system, and then carrying out water bath reaction on the enzymolysis system to obtain a reaction solution; the enzymolysis system consists of purified protein, tris-HCl buffer solution, UDP-Glc and curcumin, wherein the dosage relationship of the purified protein, the Tris-HCl buffer solution and the curcumin is 5 mug: 300. Mu.L: 2. Mu.L; the final concentration of UDP-Glc in the enzymatic hydrolysis system is 1.37mM; the concentration of the curcumin is 20mg/mL; the concentration of the Tris-HCl buffer solution is 50mM, and the pH value is 7;

the purified protein is glucosyltransferase from Bacillus subtilits 168, the DNA sequence of the purified protein is shown as SEQ ID NO.1, and the amino acid sequence of the purified protein is shown as SEQ ID NO. 2;

7. the glycosylated curcumin promoted wound healing hydrogel dressing according to claim 6, wherein the water bath reaction temperature in the step (1) is 37 ℃ and the reaction time is 1h; the temperature of the water bath evaporation in step (2) was 55 ℃.

8. A method of preparing a glycosylated curcumin promoted wound healing hydrogel dressing in accordance with any one of claims 1 to 7, comprising the steps of:

(1) PCL, PEG and Sn (Oct) ₂ After mixing, carrying out reaction under the condition of introducing nitrogen, after the reaction is reduced to a certain temperature, adding keratin and glycosylated curcumin for continuous second reaction, after the reaction, adding deionized water and glycerol, and stirring to uniformly disperse the mixture to obtain aqueous emulsion;

9. The method of preparing a glycosylated curcumin enhanced wound healing hydrogel dressing according to claim 8, wherein in step (1), the PCL, PEG, sn (Oct) ₂ The amounts of keratin, glycosylated curcumin, deionized water and glycerol were related to 10g:3g:0.1g:4.5g:1g:36g:0.21g; the reaction temperature is 75 ℃, and the reaction time is 3 hours; the temperature is reduced to 50 ℃, and the time of the second reaction is 1h.

10. The method for preparing a glycosylated curcumin hydrogel dressing for promoting wound healing according to claim 8, wherein the pre-freezing treatment in the step (2) is performed at a temperature of-20 ℃ for 24 hours; the dosage relation of the alkali lignin or the enzyme modified alkali lignin and the aqueous emulsion is 2g:1.7mL.