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

Abstract

The present invention belongs to the fields of bioengineering technology and additive manufacturing, and particularly relates to a preparation method and application of a glycosylated curcumin wound healing hydrogel dressing. By constructing the glycosyltransferase gene derived from Bacillus subtilis 168 onto an expression vector and inserting it at the NdeI and XhoI restriction enzyme sites, a co-expression recombinant plasmid pET_YjiC is formed. This plasmid is transformed into Escherichia coli BL21(DE3) competent cells to obtain a recombinant strain. After induction expression of this recombinant strain, it can be used to transform curcumin into a glycosylated product, and the water solubility of the obtained glycosylated curcumin can reach 49.60 mg / L and 15.31 mg / L. The glycosylated curcumin is prepared with enzyme-modified lignin, polycaprolactone, polyethylene glycol, and keratin to obtain a hydrogel dressing, which has the advantages of immunomodulatory function and complete degradability, and its use is suitable for the aseptic maintenance of damaged skin barriers of skin trauma and the wound healing of postoperative inpatients.

Description

Technical Field

[0001] The present invention belongs to the fields 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 Art

[0002] Accidental trauma, tumor surgery, and chronic wounds caused by diabetes are the main issues of clinical concern. The main reason for the difficulty in healing chronic wounds is the insufficient supply of oxygen and nutrients due to poor blood circulation, which increases the generation of reactive oxygen species by immune cells, leading to the degradation of the extracellular matrix and damage to skin tissues. When clinically treating a patient's wound, a dressing is generally required to cover the wound to prevent bacteria from invading and maintain a moist environment on the wound surface. Although traditional dressings such as gauze and cotton pads have a protective effect, they do not have the performance of treating and repairing wounds, and traditional dressings have poor biodegradability and are prone to adhering to wound tissues, causing secondary damage. As a type of wound dressing, hydrogel dressings are a colloidal substance with a three-dimensional network structure and insoluble in water, formed from polymer materials such as polyacrylic acid and polyvinyl alcohol. They have a variety of surface functional groups and have a certain slow-release effect, and have been widely used in the field of biomedical engineering.

[0003] However, most hydrogels use petroleum-based chemicals as raw materials, with high production costs and adverse effects on the environment. Lignin contains a large number of functional groups and chemical reaction active sites in its structure, and can be chemically modified and graft copolymerized to prepare lignin composite hydrogels. Introducing lignin into the hydrogel can improve the strength of the hydrogel, and this hydrogel is easily degraded in an aqueous environment. Its high water content, good biocompatibility, and special surface properties make it one of the most popular materials in biological, medical, and environmental applications. Due to its good biocompatibility, hydrogel dressings can absorb wound exudate to prevent wound infection, and there is no need to frequently change the dressing in a short time; however, the dressing colloid that absorbs a large amount of exudate will separate from the wound and cannot effectively block the invasion of bacteria. Therefore, in actual applications, different antibacterial agents, bioactive components, and other drugs are often encapsulated in the hydrogel as needed, and the drugs can be continuously released into the lesion area through body fluid exchange, thereby reducing bacterial growth, preventing wound surface infection, and promoting wound healing.

[0004] Curcumin is a polyphenolic compound extracted from the rhizomes of Zingiberaceae plants. It is a green, safe, and non-toxic natural antibacterial agent with good biological activity. At the same time, it has pharmacological activities such as anti-inflammatory, antioxidant, anti-tumor, anti-cancer, and wound repair, and is widely used in the medical and cosmetic fields. However, due to the large molecular weight, unstable structure, poor water solubility of curcumin, and the presence of hydroxyl groups at both ends of its molecule, under alkaline conditions, it is prone to conjugate effects and is easily soluble in glacial acetic acid and alkaline solutions. This results in low bioavailability of curcumin, thus limiting its application in wound dressings. Summary of the Invention

[0005] In view of the problems existing in the above-mentioned prior art, the present invention aims to solve one of the problems; to provide a glycosylated curcumin wound healing hydrogel dressing, which can improve the water solubility of curcumin, enhance its bioavailability, stably load curcumin, play a long-term anti-inflammatory and bactericidal role, prevent wound surface bacterial infection, and help wound healing.

[0006] The present invention first provides a glycosylated curcumin wound healing hydrogel dressing, which comprises the following components in mass percentage: alkali lignin or enzyme-modified alkali lignin, 0.1-15%;

[0007] glycosylated curcumin, 0.01-5%;

[0008] polyethylene glycol (PEG), 0.1-2%;

[0009] polycaprolactone (PCL), 0.1-10%;

[0010] keratin, 0.1-20%;

[0011] glycerol 0.01-1%;

[0012] The balance is water.

[0013] The preparation method of the enzyme-modified lignin comprises the following steps:

[0014] (1) First, prepare an enzymatic hydrolysis system, and then react the enzymatic hydrolysis system in a water bath shaker at 120 r / min at 37 °C for 12-24 h to obtain a reaction solution;

[0015] The enzymatic hydrolysis system is composed of laccase or dye-decolorizing peroxidase, Tris-HCl buffer, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and lignin. The dosage relationship of laccase or dye-decolorizing peroxidase, Tris-HCl buffer and lignin is 5 mL: 20 mL: 1 g; the final concentration of ABTS in the enzymatic hydrolysis system is 0.2 mM; the concentration of laccase or dye-decolorizing peroxidase in the enzymatic hydrolysis system is 10000 U / L; the concentration of the Tris-HCl buffer is 50 mM and the pH value is 7; the lignin includes but is not limited to lignosulfonate, alkali lignin, groundwood lignin or solvent-based alcoholysis lignin.

[0016] (2) Centrifuge the reaction solution obtained in step (1) to remove the insoluble lignin in the solution, then collect the supernatant, boil it for 10-15 min and then centrifuge to remove the protein, and then adjust the pH of the supernatant to 3 with HCl, and centrifuge to collect the precipitate;

[0017] (3) After vacuum freeze-drying the collected precipitate for 48 h, the final product, namely enzyme-modified alkali lignin, is obtained and reserved for use.

[0018] The preparation method of the keratin comprises the following steps:

[0019] Clean the keratin raw material with clear water, add ethanol and treat for a period of time, filter, add dilute HCl to the precipitate for treatment, and add mercaptoethanol for reduction reaction after treatment; after the reaction, centrifuge to obtain a precipitate, and then freeze-dry to obtain keratin; the mass ratio of the keratin raw material, ethanol, dilute HCl, and mercaptoethanol is 1:2:2:1;

[0020] Preferably, the keratin raw material includes but is not limited to animal hair, poultry feathers or human hair.

[0021] Preferably, the mass fraction of the ethanol is 50%; the mass fraction of the dilute HCl is 5%; the time for adding ethanol and treating for a period of time is 30 min, the time for adding dilute HCl and treating is 1 h; the time for the reduction reaction is 1 h.

[0022] The biological preparation method of the glycosylated curcumin comprises the following steps:

[0023] (1) First, prepare an enzymatic hydrolysis system, and then obtain a reaction solution after a water bath reaction of the enzymatic hydrolysis system; the enzymatic hydrolysis system is composed of purified protein, Tris-HCl buffer, uridine diphosphate glucose (UDP-Glc) and curcumin; the dosage relationship of the purified protein, Tris-HCl buffer, and curcumin is 5 μg: 300 μL: 2 μL; the final concentration of UDP-Glc in the enzymatic hydrolysis system is 1.37 mM; the concentration of curcumin is 20 mg / mL; the concentration of the Tris-HCl buffer is 50 mM, and the pH value is 7;

[0024] The purified protein is a glucosyltransferase derived from Bacillus subtilits 168 (named Bs-YjiC), its DNA sequence is as shown in SEQ ID NO.1, and its amino acid sequence is as shown in SEQ ID NO.2;

[0025] (2) Add the reaction solution obtained in step (1) to ethyl acetate for extraction to obtain a mixed reaction solution, perform water bath evaporation until the volume of the mixed reaction solution is reduced to 1%-5%, and then add chromatographically pure ethanol to the evaporated mixed reaction solution to obtain glycosylated curcumin, which is stored at low temperature in the dark; the volume ratio of the reaction solution, ethyl acetate, and chromatographically pure ethanol is 3:6:2.

[0026] Preferably, in step (1), the temperature of the water bath reaction is 37 °C, and the reaction time is 1 h.

[0027] Preferably, the temperature of the water bath evaporation in step (2) is 55 °C.

[0028] The present invention also provides a preparation method of a glycosylated curcumin wound healing hydrogel dressing, comprising the following steps:

[0029] (1) After mixing polycaprolactone (PCL), polyethylene glycol (PEG) and stannous octoate (Sn(Oct)2), the reaction is carried out under the condition of introducing nitrogen. After the reaction, when the temperature drops to a certain temperature, keratin and glycosylated curcumin are added and the second reaction is continued. After the reaction, deionized water and glycerol are added and stirred to disperse them evenly to obtain an aqueous emulsion;

[0030] (2) Under room temperature conditions, the aqueous emulsion obtained in step (1) is mixed with alkali lignin or enzyme-modified alkali lignin to obtain a mixture. First, it is pre-frozen, and then freeze-dried to obtain a glycosylated curcumin hydrogel dressing.

[0031] Preferably, the dosage relationship of PCL, PEG, Sn(Oct)2, keratin, glycosylated curcumin, deionized water and glycerol in step (1) is 10 g: 3 g: 0.1 g: 4.5 g: 1 g: 36 g: 0.21 g; the temperature of the reaction is 75 °C, the reaction time is 3 h; the temperature drop to a certain temperature is 50 °C, and the time of the second reaction is 1 h.

[0032] Preferably, the temperature of the pre-freezing treatment in step (2) is -20 °C, and the time is 24 h; the dosage relationship between the alkali lignin or enzyme-modified alkali lignin and the aqueous emulsion is 2 g: 1.7 mL.

[0033] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0034] The glycosylated curcumin wound healing hydrogel dressing of the present invention has good antibacterial properties and drug slow-release effects, can continuously act on the wound area, and can be applied to wounds such as trauma, burns, scalds, ulcers, and pressure sores.

[0035] (1) The lignin selected in the present invention contains a large number of functional groups and chemical reaction active sites, has good biocompatibility, can increase the strength of the hydrogel, and the hydrogel is easily degraded in an aqueous environment, having the advantages of low toxicity and environmental friendliness. The chromaticity of the enzyme-modified lignin becomes lighter and more beautiful after being treated with laccase or dye-decolorizing peroxidase.

[0036] (2) In the present invention, polycaprolactone, polyethylene glycol, keratin, glycosylated curcumin, etc. are dissolved in deionized water to obtain an aqueous emulsion, and then polymerized with alkali lignin or enzyme-modified alkali lignin to prepare an enzyme-modified lignin hydrogel dressing with stable curcumin loading. The preparation method is simple, the raw materials are widely sourced, inexpensive, and the cost is controllable, enabling industrial production.

[0037] (3) For the glycosylated curcumin used in the present invention, compared with natural curcumin, the water solubilities of the curcumin glucoside products can reach 49.60 mg / L and 15.31 mg / L respectively, significantly improving the water solubility of curcumin in the hydrogel dressing, which is beneficial to enhancing the bioavailability and sustained-release effect of curcumin.

[0038] (4) The glycosylated curcumin hydrogel dressing of the present invention has good mechanical properties, good flexibility and elasticity, can form a good adhesion with the skin, and is suitable for wounds in different parts of the body, especially frequently moving parts such as elbows, wrists, knees, and ankles. Description of the Drawings

[0039] Figure 1 It is the result diagram of protein nickel column purification gel electrophoresis, where A: Marker, B: negative control, C: elution with 0 mM imidazole, D: elution with 250 mM imidazole.

[0040] Figure 2 It is the test diagram of the preparation conditions of glycosylated curcumin, where A is the HPLC diagram before and after the reaction; B is the influence of reaction time on the conversion effect of curcumin; C is the influence of pH on the conversion effect of curcumin; D is the influence of reaction temperature on the conversion effect of curcumin.

[0041] Figure 3 It is the photo of the gel sample pasted on the skin surface.

[0042] Figure 4 It is the in vitro release test curve diagram of curcumin in the gel sample.

[0043] Figure 5 It is the antibacterial effect diagram of the gel sample against Escherichia coli, where 1 and 2 in the figure correspond to the control sample and the gel sample respectively. Detailed Embodiments

[0044] The present invention can be better understood according to the following embodiments. However, those skilled in the art can easily understand that the embodiments are only used to illustrate the present invention and should not and will not limit the invention described in the claims.

[0045] Now, various exemplary embodiments of the present invention are described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0046] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0047] Unless otherwise specified, 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 pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0048] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0049] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0050] Strain description: The purified protein is a glucosyltransferase derived from Bacillus subtilits 168 (named Bs - YjiC (NP_389104.1)), its DNA sequence is shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.2; the Bacillus subtilis 168 is derived from the China General Microbiological Culture Collection Center, and its preservation number is: CGMCC NO.1.1391.

[0051] Scheme description: The present invention first provides a glycosylated curcumin - promoted wound - healing hydrogel dressing, which comprises the following components in mass percentage: alkali lignin or enzyme - modified alkali lignin, 0.1 - 15%;

[0052] Glycosylated curcumin, 0.01 - 5%;

[0053] Polyethylene glycol (PEG), 0.1 - 2%;

[0054] Polycaprolactone (PCL), 0.1 - 10%;

[0055] Keratin, 0.1 - 20%;

[0056] Glycerol 0.01 - 1%;

[0057] The balance is water.

[0058] The preparation method of the enzyme-modified lignin includes the following steps:

[0059] (1) First, prepare an enzymatic hydrolysis system, and then react the enzymatic hydrolysis system in a water bath shaker at 120 r / min at 37 °C for 12 - 24 h to obtain a reaction solution;

[0060] The enzymatic hydrolysis system consists of laccase or dye-decolorizing peroxidase, Tris-HCl buffer, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and alkali lignin. The dosage relationship of the laccase or dye-decolorizing peroxidase, Tris-HCl buffer and alkali lignin is 5 mL: 20 mL: 1 g; the final concentration of ABTS in the enzymatic hydrolysis system is 0.2 mM; the concentration of the laccase or dye-decolorizing peroxidase in the enzymatic hydrolysis system is 10000 U / L; the concentration of the Tris-HCl buffer is 50 mM, and the pH value is 7.

[0061] (2) Centrifuge the reaction solution obtained in step (1) to remove the insoluble lignin in the solution, then collect the supernatant, centrifuge to remove proteins after boiling for 10 - 15 min, and then adjust the pH of the supernatant to 3 with HCl and centrifuge to collect the precipitate;

[0062] (3) After vacuum freeze-drying the collected precipitate for 48 h, the final product is obtained, which is the enzyme-modified alkali lignin for standby.

[0063] The treatment method of the keratin includes the following steps:

[0064] Clean the keratin raw material with clean water, add ethanol with a mass fraction of 50% and treat for 30 min. After filtration, add dilute HCl (mass fraction 5%) to the precipitate and treat for 1 h, and then add mercaptoethanol to reduce for 1 h; finally, centrifuge to obtain a precipitate, and then obtain keratin through freeze-drying (-20 °C); the mass ratio of the keratin raw material, ethanol, dilute HCl, and mercaptoethanol is 1:2:2:1; the keratin raw material includes but is not limited to: animal hair, poultry feathers, and human hair.

[0065] The biological preparation method of the glycosylated curcumin includes the following steps:

[0066] (1) First, prepare the enzymatic hydrolysis system, and then react the enzymatic hydrolysis system in a water bath at 37 °C for 1 h to obtain a reaction solution. The enzymatic hydrolysis system consists of purified protein, Tris-HCl buffer, uridine diphosphate glucose (UDP-Glc), and curcumin. The dosage relationship of the purified protein, Tris-HCl buffer, and curcumin is 5 μg: 300 μL: 2 μL. The final concentration of UDP-Glc in the enzymatic hydrolysis system is 1.37 mM. The concentration of curcumin is 20 mg / mL. The concentration of the Tris-HCl buffer is 50 mM, and the pH value is 7. The purified protein is a glucosyltransferase derived from Bacillus subtilits 168 (named Bs-YjiC), and its DNA sequence is as shown in SEQ ID NO.1, and the amino acid sequence is as shown in SEQ ID NO.2.

[0067] (2) Add the reaction solution obtained in step (1) to ethyl acetate for extraction to obtain a mixed reaction solution, evaporate it in a water bath at 55 °C to 1% - 5% of the volume of the mixed reaction solution, and then add chromatographically pure ethanol to the evaporated mixed reaction solution to obtain glycosylated curcumin, which is stored at low temperature in the dark. The volume ratio of the reaction solution, ethyl acetate, and chromatographically pure ethanol is 3: 6: 2.

[0068] A preparation method of a glycosylated curcumin-promoted wound healing hydrogel dressing, comprising the following steps:

[0069] (1) Mix polycaprolactone (PCL), polyethylene glycol (PEG), and stannous octanoate (Sn(Oct)2), and react under a nitrogen atmosphere. The reaction temperature is 75 °C, and the reaction time is 3 h. After the reaction, wait until the temperature drops to 50 °C, and then add keratin and glycosylated curcumin to continue the second reaction for 1 h. After the reaction, add deionized water and glycerol, and stir to disperse them evenly to obtain an aqueous emulsion.

[0070] (2) Under room temperature conditions, mix the aqueous emulsion obtained in step (1) with alkali lignin or enzyme-modified alkali lignin to obtain a mixture. First, perform a pre-freezing treatment on it at a temperature of -20 °C for 24 h. Then, obtain the glycosylated curcumin hydrogel dressing by freeze-drying. The dosage relationship between the alkali lignin or enzyme-modified alkali lignin and the aqueous emulsion is 2 g: 1.7 mL.

[0071] Example 1: Biological preparation of glycosylated curcumin and its solubility test

[0072] 1. Construction of glycosyltransferase (Bs-YjiC) engineering bacteria

[0073] (1) Synthesis of glycosyltransferase (Bs-YjiC) gene

[0074] The full-length gene of glycosyltransferase Bs-YjiC was synthesized by in vitro total gene synthesis method (synthesized by Suzhou Hongxun Biotechnology Co., Ltd.), and its DNA sequence is shown in SEQ ID NO.1;

[0075] (2) Treatment and ligation of the glycosyltransferase (Bs-YjiC) gene

[0076] The fragment of the glycosyltransferase (Bs-YjiC) gene synthesized in step (1) was treated with T4 DNA Polymerase and then ligated with plasmid pET-28a(+) (Suzhou Hongxun Biotechnology Co., Ltd.) at room temperature for 20 min to obtain the ligated plasmid pET-YjiC.

[0077] (3) Transformation of the recombinant plasmid into E. coli BL21(DE3)

[0078] 0.5 μL of the plasmid pET-YjiC from step (2) was added to 100 μL of E. coli BL21(DE3) competent cells, placed on ice for 20 min, then heat-shocked at 42 °C for 90 s and then ice-bathed for 2 min. After ice-bathing, 250 μL of LB medium was added, and the mixture was cultured at 37 °C for 30 min to obtain a culture solution; subsequently, 150 μL of the culture solution was spread on an LB plate containing 50 μg / mL kanamycin sulfate (Kan) and cultured overnight at 37 °C in an inverted position to obtain an engineered bacterium containing the glycosyltransferase (Bs-YjiC) gene.

[0079] (4) Small-scale induction expression of the recombinant engineered bacterium

[0080] The engineered bacterium containing the glycosyltransferase (Bs-YjiC) gene after recombination on the plate in step (3) was picked and inoculated into a test tube containing 1 mL of LB culture solution with 50 μg / mL kanamycin sulfate (Kan), and cultured with shaking at 37 °C and 220 rpm until the OD 600 reached 0.8. Then, 500 μL of the cultured bacterial solution was taken out, IPTG was added to a final concentration of 1 mM, and the culture was continued at 220 rpm and 37 °C for 3 h; after centrifugation at 10000 rpm for 2 min to remove the supernatant, the cells were collected, and 50 μL of Tris-HCl buffer (50 mM, pH 7) was added to the cells to resuspend the cells to obtain a cell suspension, and the protein expression was detected by electrophoresis.

[0081] 2. Protein purification

[0082] The engineered bacterium containing the glycosyltransferase (Bs-YjiC) gene was inoculated into 600 mL of LB culture solution containing 50 μg / mL Kan (inoculation amount was 1%), and cultured with shaking at 37 °C and 220 rpm until the OD of the cells 600It was 0.8 - 1.0, then IPTG as an inducer was added to a final concentration of 1 mM, and the culture was continued for 4 h at 37 °C and 220 rpm. After cultivation, the cells were collected by centrifugation at 5000 rpm and 4 °C for 5 min, sonicated, and the sonicated cells were centrifuged at 12000 rpm and 4 °C for 10 min. After centrifugation, the supernatant was collected for nickel column purification, and the packing type was Smart-Ni; the equilibration buffer was: Tris-HCl buffer with a pH of 7.4, and the elution buffer: Tris-HCl buffer with a pH of 7.4; gradient elution was carried out according to the imidazole gradients of 20 mM, 50 mM, and 250 mM respectively to obtain the purified protein, and the results were as Figure 1 shown.

[0083] 3. Activity test of glycosyltransferase Bs-YjiC

[0084] Using curcumin as the substrate, the assay system was 300 μL, which contained 50 mM Tris-HCl buffer with a pH of 8.0, 5 μg of purified protein, and 1.37 mM UDP-Glc. The detection wavelength was 425 nm. The amount of enzyme required to convert 1 μmol of substrate into product was one international unit of enzyme activity, and the maximum specific activity was 144.77 ± 0.53 U / mg.

[0085] 4. Test for the optimal conditions of curcumin conversion by glycosyltransferase (Bs-YjiC)

[0086] The assay system was 300 μL, which contained 5 μg of purified protein, 50 mM Tris-HCl, 1.37 mM UDP-Glc, and 2 μL of curcumin. After the reaction, 600 μL of ethyl acetate was added for extraction, 500 μL was taken and volatilized in a water bath at 55 °C, and 200 μL of chromatographically pure ethanol was added for HPLC detection. The analysis detection wavelength was 425 nm. The results were as Figure 2 shown. Figure A was the HPLC chromatogram before and after the reaction; Figures B, C, and D were the effects of reaction time, pH, and reaction temperature on the curcumin conversion effect respectively. Figure 2 A in it showed that two new products were produced after the conversion by glycosyltransferase, Figure 2 B - 2D showed that when the pH was 8, the temperature was 40 °C, and the reaction time was 30 min, the conversion effect was the best.

[0087] 5. Solubility determination of glycosylated curcumin

[0088] Dissolve glycosylated curcumin in 1 mL of Tris-HCl buffer (pH 8.0), stir for 30 min, centrifuge at 12,000 r / min for 15 min, filter through a 0.45-μm filter membrane, add an equal volume of methanol, use natural curcumin as a control, and determine the solubility of glycosylated curcumin by HPLC. The water solubilities of the curcumin glucoside products were measured to be 49.60 mg / L and 15.31 mg / L, respectively.

[0089] Example 2: Preparation of a hydrogel dressing for promoting wound healing with glycosylated curcumin

[0090] The hydrogel dressing for promoting wound healing with glycosylated curcumin comprises the following components by mass percentage:

[0091] 15% lignin, 5% glycosylated curcumin, 0.1% polyethylene glycol (PEG), 10% polycaprolactone (PCL), 20% keratin, 1% glycerol, and the balance is water;

[0092] The description of the component raw materials is as follows:

[0093] (1) Preparation of modified lignin

[0094] The enzymatic hydrolysis system is 500 mL, containing 100 mL of laccase at 10,000 U / L, 400 mL of Tris-HCl buffer at pH 7, ABTS at a final concentration of 0.2 mM, and 20 g of alkali lignin. React at 37 °C for 12 - 24 h in a water bath shaker at 120 r / min to obtain a reaction solution;

[0095] Centrifuge the reaction solution to remove the insoluble lignin in the solution, then collect the supernatant. After boiling for 10 min, centrifuge to remove the protein, then adjust the pH of the supernatant to 3 with HCl, and centrifuge to collect the precipitate; After vacuum freeze-drying the collected precipitate for 48 h, the final product, namely enzyme-modified alkali lignin, is obtained.

[0096] (2) Pretreatment of keratin

[0097] Select animal hair as the keratin raw material, specifically wool;

[0098] Wash it clean with water, then add ethanol with a mass fraction of 50% and treat for 30 min. After filtration, add dilute HCl (mass fraction 5%) and treat for 1 h, then use mercaptoethanol to reduce for 1 h; After centrifugation and freeze-drying (-20 °C), keratin is obtained; The mass ratio of the keratin raw material, ethanol, dilute HCl, and mercaptoethanol is 1:2:2:1.

[0099] The preparation of the hydrogel dressing for promoting wound healing with glycosylated curcumin is as follows:

[0100] After mixing 10 g of polycaprolactone (PCL), 3 g of polyethylene glycol (PEG) and 0.1 g of stannous octoate Sn(Oct)₂, nitrogen was introduced and the reaction was carried out at 75 °C for 3 h; subsequently, the temperature was lowered to 50 °C, and then 4.5 g of keratin and 1 g of glycosylated curcumin were added and the reaction was continued for 1 h. After the reaction, 36 g of deionized water and 0.21 g of glycerol were added, and stirred to disperse evenly to obtain an aqueous emulsion;

[0101] Finally, at room temperature, 2 g of enzyme-modified alkali lignin was mixed with 1.7 mL of the aqueous emulsion to obtain a mixture, which was pre-frozen at -20 °C for 24 h, and then freeze-dried to obtain a glycosylated curcumin hydrogel dressing.

[0102] Example 3: Performance verification of glycosylated curcumin hydrogel dressing for promoting wound healing

[0103] (1) Adhesion test:

[0104] The gel sample was attached to the arm as Figure 3 shown. It can be observed from the figure that the gel sample can form good adhesion with the skin and is not easy to fall off.

[0105] (2) In vitro release test of curcumin:

[0106] The gel sample was cut into circular slices with a diameter of 25 mm and a thickness of 2 mm, weighed and recorded as m0, placed in 50 mL of 50 mM Tris-HCl buffer (pH 7.2), and the in vitro release test of curcumin was carried out at 37 °C. Every 12 h, an appropriate amount of buffer was taken, diluted with absolute ethanol, and the absorbance was measured at 425 nm to calculate the release amount m1 of curcumin. Based on the average loading amount of curcumin in the gel sample of 536 μg / g, the cumulative release rate R(%) of curcumin over time was calculated according to the formula. The results are as Figure 4 shown. The curcumin loaded in the gel sample was released rapidly in the initial 24 h, and the release rate gradually slowed down after 24 h, but remained basically unchanged until 96 h, and the release rate reached more than 50%. It shows that the glycosylated curcumin hydrogel dressing of the present invention can release curcumin in a long-term and sustained manner and maintain the drug effect.

[0107]

[0108] (3) Antibacterial test:

[0109] 1 mL of Escherichia coli suspension (OD 600 was 1.0) was spread on an LB agar plate, and the hydrogel sample was covered on the surface of the LB agar plate. After culturing at 37 °C for 48 h, it was observed whether there was an obvious antibacterial zone on the LB plate. The gel sample and the control sample (without glycosylated curcumin) were selected for the antibacterial test, and the experimental results are as Figure 5As shown. It can be seen from the figure that after 48 hours of cultivation, there is no obvious inhibition zone for the control sample against Escherichia coli, indicating that the control sample has no antibacterial activity against Escherichia coli; while the gel sample has an inhibition zone of 3.1 + 9.7 mm against Escherichia coli, indicating that the hydrogel dressing loaded with glycosylated curcumin of the present invention has obvious antibacterial activity against Escherichia coli.

[0110] Note: The above embodiments are only used to illustrate the present invention and do not limit the technical solutions described in the present invention; therefore, although this specification has described the present invention in detail with reference to the above respective embodiments, those of ordinary skill in the art should understand that the present invention can still be modified or equivalently replaced; and all technical solutions and their improvements that do not depart from the spirit and scope of the present invention should be covered within the scope of the claims of the present invention.

[0111] SEQ ID NO.1 gene sequence (DNA):

[0112]

[0113] Amino acid sequence of SEQ ID NO.2:

[0114] MKKYHISMINIPAYGHVNPTLALVEKLCEKGHRVTYATTEEFAPAVQQAGGEALIYHTSLNIDPKQIREMMEKNDAPLSLLKESLSILPQLEELYKDDQPDLIIYDFVALAGKLFAEKLNVPVIKLCSSYAQNESFQLGNEDMLKKIREAEAEFKAYLEQEKLPAVSFEQLAVPEALNIVFMPKSFQIQHETFDDRFCFVGPSLGERKEKESLLIDKDDRPLMLISLGTAFNAWPEFYKMCIKAFRDSSWQVIMSVGKTIDPESLEDIPANFTIRQSVPQLEVLEKADLFISHGGMNSTMEAMNAGVPLVVIPQMYEQELTANRVDELGLGVYLPKEEVTVSSLQEAVQAVSSDQELLSRVKNMQKDVKEAGGAERAAAEIEAFMKKSAVPQ

Claims

1. A preparation method of a glycosylated curcumin hydrogel dressing for promoting wound healing, characterized in that It includes the following steps: (1) After mixing polycaprolactone, polyethylene glycol and stannous octoate, carry out the reaction under the condition of introducing nitrogen. After the reaction, wait until the temperature drops to a certain level, then add keratin and glycosylated curcumin and continue the second reaction. After the reaction, add deionized water and glycerol, and stir to disperse them evenly to obtain an aqueous emulsion; (2) Under room temperature conditions, mix the aqueous emulsion obtained in step (1) with alkali lignin or enzyme-modified alkali lignin to obtain a mixture. First, perform pre-freezing treatment on it, and then obtain a glycosylated curcumin hydrogel dressing by freeze-drying.

2. The preparation method of a glycosylated curcumin hydrogel dressing for promoting wound healing according to claim 1, characterized in that, The enzyme-modified lignin is obtained by the preparation method, and the preparation steps are as follows: (a) First, prepare an enzymatic hydrolysis system, and then react the enzymatic hydrolysis system in a water bath shaker at 120 r / min at 37 °C for 12 - 24 h to obtain a reaction solution; The enzymatic hydrolysis system consists of laccase or dye-decolorizing peroxidase, Tris-HCl buffer, ABTS and lignin. The dosage relationship of laccase or dye-decolorizing peroxidase, Tris-HCl buffer and lignin is 5 mL: 20 mL: 1 g; the final concentration of ABTS in the enzymatic hydrolysis system is 0.2 mM; the concentration of laccase or dye-decolorizing peroxidase in the enzymatic hydrolysis system is 10000 U / L; the concentration of the Tris-HCl buffer is 50 mM, and the pH value is 7; the lignin includes lignosulfonate, alkaline lignin, groundwood lignin or solvent-type alcoholysis lignin; (b) Centrifuge the reaction solution obtained in step (a) to remove the insoluble lignin in the solution, then collect the supernatant, centrifuge to remove proteins after boiling for 10 - 15 min, and then adjust the pH of the supernatant to 3 with HCl, and centrifuge to collect the precipitate; (c) After freeze-drying the collected precipitate under vacuum for 48 h, obtain the final product, which is enzyme-modified alkali lignin.

3. The preparation method of a glycosylated curcumin hydrogel dressing for promoting wound healing according to claim 1, characterized in that The keratin is obtained by the following preparation method, and the preparation steps are as follows: Wash the keratin raw material with clean water, add ethanol and treat for a period of time, filter, then add dilute HCl to the precipitate for treatment, and add mercaptoethanol for reduction reaction after treatment; after the reaction, centrifuge to obtain a precipitate, and then obtain keratin by freeze-drying; the mass ratio of the keratin raw material, ethanol, dilute HCl, and mercaptoethanol is 1: 2: 2:

1.

4. The preparation method of a glycosylated curcumin wound healing hydrogel dressing according to claim 3, characterized in that, The keratin raw material includes animal hair, poultry feathers or human hair.

5. The preparation method of a glycosylated curcumin hydrogel dressing for promoting wound healing according to claim 3, characterized in that, The mass fraction of the ethanol is 50%; the mass fraction of the dilute HCl is 5%; the time for adding ethanol and treating for a period of time is 30 min, the time for adding dilute HCl and treating is 1 h; the time for the reduction reaction is 1 h.

6. The preparation method of a glycosylated curcumin hydrogel dressing for promoting wound healing according to claim 1, characterized in that, The glycosylated curcumin is obtained by the preparation method, and the preparation steps are as follows: S1. First, prepare an enzymatic hydrolysis system, and then obtain a reaction solution after subjecting the enzymatic hydrolysis system to a water bath reaction. The enzymatic hydrolysis system consists of a purified protein, Tris-HCl buffer, UDP-Glc, and curcumin. The dosage relationship of the purified protein, Tris-HCl buffer, and curcumin is 5 μg: 300 μL: 2 μL. The final concentration of UDP-Glc in the enzymatic hydrolysis system is 1.37 mM. The concentration of curcumin is 20 mg / mL. The concentration of the Tris-HCl buffer is 50 mM, and the pH value is 7. The purified protein is a glucosyltransferase derived from Bacillus subtilits 168, and its DNA sequence is as shown in SEQ ID NO.1, and the amino acid sequence is as shown in SEQ ID NO.

2. S2. Add the reaction solution obtained in step S1 to ethyl acetate for extraction to obtain a mixed reaction solution, and perform water bath evaporation until the volume of the mixed reaction solution is reduced to 1%-5%. Then, add chromatographically pure ethanol to the evaporated mixed reaction solution to obtain glycosylated curcumin, and store it at low temperature in the dark. The volume ratio of the reaction solution, ethyl acetate, and chromatographically pure ethanol is 3:6:

2.

7. The preparation method of a glycosylated curcumin hydrogel dressing for promoting wound healing according to claim 6, characterized in that, In step S1, the temperature of the water bath reaction is 37°C, and the reaction time is 1 h. In step S2, the temperature of the water bath evaporation is 55°C.

8. The preparation method of a glycosylated curcumin hydrogel dressing for promoting wound healing according to claim 1, characterized in that, In step (1), the dosage relationship of polycaprolactone, polyethylene glycol, stannous octoate, keratin, glycosylated curcumin, deionized water, and glycerol is 10 g: 3 g: 0.1 g: 4.5 g: 1 g: 36 g: 0.21 g. The temperature of the reaction is 9. The preparation method of a glycosylated curcumin hydrogel dressing for promoting wound healing according to claim 1, characterized in that, ​

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