CN115006585A - Wound dressing with good hemostatic performance and preparation method thereof - Google Patents
Wound dressing with good hemostatic performance and preparation method thereof Download PDFInfo
- Publication number
- CN115006585A CN115006585A CN202210618054.9A CN202210618054A CN115006585A CN 115006585 A CN115006585 A CN 115006585A CN 202210618054 A CN202210618054 A CN 202210618054A CN 115006585 A CN115006585 A CN 115006585A
- Authority
- CN
- China
- Prior art keywords
- wound dressing
- codonopsis pilosula
- acetylated
- pseudo
- pilosula polysaccharide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
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Classifications
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Abstract
The invention belongs to the field of biomedical materials, and particularly relates to a wound dressing with good hemostatic performance and a preparation method thereof. A wound dressing with good hemostatic performance comprises Notoginseng radix extract as raw material. In the invention, in order to develop a special hydrogel with new functions, pseudo-ginseng extract is loaded to increase the hemostatic effect of the pseudo-ginseng extract, and experiments show that the pseudo-ginseng extract extracted by an enzymatic hydrolysis method has higher biological activity, and the wound dressing prepared by compounding the pseudo-ginseng extract with the hydrogel has good hemostatic effect, so that the wound dressing is applied to wound repair and treatment, and not only can promote the rapid coagulation and closure of wounds, but also can further increase the anti-inflammatory and healing promotion effects, and can accelerate the speed and quality of wound healing.
Description
Technical Field
The invention belongs to the field of biomedical materials, and particularly relates to a wound dressing with good hemostatic performance and a preparation method thereof.
Background
The most common skin injury repair process of the human body is a dynamic process, and in the first hemostasis stage of wound repair, especially for acute skin injury patients, bleeding can be rapidly controlled to avoid the body from suffering larger injuries. The history of using animal and plant derived medicines to treat skin wounds is long, and at present, domestic external medicines applied to various skin wounds are common in compound traditional Chinese medicine preparations in the form of paste, wherein various complex active ingredients play a role in coordination on the wounds. The animal and plant components have the curative effects of promoting blood circulation, removing blood stasis, astringing the mouth, stopping bleeding, removing putrefaction, promoting granulation, relieving swelling and pain, sterilizing and inhibiting bacteria and the like, and improve the healing efficiency of wound repair. The hydrogel is used as a good drug slow-release carrier and a wound dressing, and animal and plant extracts are loaded on the hydrogel, so that the hydrogel is a great development trend of the wound dressing in the future. Studies have reported the therapeutic potential of hydrogels consisting of extracts of corynia brachycarpa as wound healing promoters in vivo models.
Wound repair phase 2 begins with the release of proteolytic enzymes and proinflammatory factors from invading immune cells into the wound area, producing an inflammatory immune response, which produces reactive oxygen species that promote the wound repair process, but excessively high levels of ROS can prevent wound healing by increasing oxidative stress and lipid peroxidation even causing severe cell damage.
The wound dressing can cover and protect wounds and promote wound healing of trauma, burns, diabetic feet and postoperative incisions. The ideal wound dressing can provide a moist environment, prevent secondary infection, clear excess tissue exudate, promote tissue regeneration near the wound surface, improve the wound healing quality and reduce the generation of scars. Compared with the artificially synthesized polymer hydrogel, the natural biological polymer hydrogel (such as polysaccharide hydrogel, polypeptide hydrogel and the like) has the advantages of good biocompatibility, degradability, biospecificity, no immune and toxic reaction, controllable degradation time, no toxicity and immunogenicity of degradation products, capability of discharging final metabolic products out of a body and the like. Since the preparation of DOPA (DOPA) modified hydrogels inspired by mussel in 2002 was reported, modification of hydrogels with DOPA building blocks has received much attention. The key structural unit of DOPA is a catechol structure, and the DOPA can form strong covalent bonds and non-covalent bonds with various inorganic/organic/metal surfaces, so that the adhesiveness and the mechanical property of the hydrogel are greatly improved. Meanwhile, a large number of researches show that the catechol-functionalized hydrogel tissue has excellent adhesiveness and strong antioxidant activity.
Research shows that the codonopsis pilosula contains various chemical components such as polysaccharides, phytosterols, terpenes, alkaloids and phenylpropanoid glycosides, wherein the polysaccharides have the largest proportion, and are main active components of the codonopsis pilosula with various effects of enhancing the immunity of the organism, resisting aging, reducing blood sugar and the like. Studies have shown that Codonopsis Pilosula Polysaccharide (CPP) has the effect of scavenging superoxide anion free radicals, has a certain antioxidant activity and has a protective effect on partial injury of organisms. At present, the research on the biological activity of codonopsis pilosula polysaccharide is mostly limited in the aspect of regulating the function of an immune system, which hinders the development of related new medicine resources of the medicinal material codonopsis pilosula to a certain extent, so that the added value of the medicinal material codonopsis pilosula is not high, and the development of codonopsis pilosula planting and related industries is restricted. The biological activity and physicochemical properties of natural polysaccharides are closely related to their structure. In order to better develop the efficacy of polysaccharides and to make them more widely used, researchers often modify or modify polysaccharide molecules. The modification of polysaccharide can improve the bioactivity and absorption efficiency of polysaccharide and endow polysaccharide with new effect. Meanwhile, the polysaccharide modification technology can also provide a new utilization approach for polysaccharides which cannot exert the activity or be put into industrial production due to the structural property. However, many modification methods have disadvantages such as high modification cost, harsh modification conditions, hazardous modification processes, modification by-products that hinder the modification or cause contamination, etc., which greatly limits the large-scale application of polysaccharide modification techniques.
Notoginseng radix is the dried root and rhizome of Panax notoginseng belonging to Araliaceae, and has effects of removing blood stasis, stopping bleeding, and relieving swelling and pain. The Notoginseng radix extract mainly contains Notoginseng radix total saponin, dencichine, flavone, amino acids, saccharides and microelements. The extraction method comprises water decoction and ethanol precipitation, ethanol reflux and percolation. A large number of researches show that the extraction mode has certain influence on the activity of the medicine, but the influence of the preparation process of the pseudo-ginseng on the biological activity is not reported at present.
Disclosure of Invention
In order to solve the technical problems, effectively improve the extraction efficiency of the active ingredients of the pseudo-ginseng and meet the requirement of hospitals on the source diversity of hydrogel dressings, the invention provides the hydrogel wound dressing loaded with the pseudo-ginseng extract. Firstly, the applicant carries out acetylation modification on the codonopsis pilosula polysaccharide, and carries out blending, grafting and crosslinking on the modified codonopsis pilosula polysaccharide and dopamine to obtain the hydrogel with better adhesiveness and higher antioxidant bioactivity, and further tests show that the antibacterial performance of the hydrogel is greatly improved compared with that of the hydrogel without the dopamine. Then, the applicant artificially develops a characteristic hydrogel with new functions, pseudo-ginseng extract is loaded to increase the hemostatic effect of the hydrogel, and experiments show that the pseudo-ginseng extract extracted by an enzymatic hydrolysis method has higher biological activity, and the wound dressing prepared by compounding the pseudo-ginseng extract with the hydrogel has good hemostatic effect, so that the wound dressing is applied to wound repair and treatment, and not only can promote the rapid coagulation and closure of wounds, but also can further increase the anti-inflammatory and healing promotion effects, and can accelerate the speed and quality of wound healing.
In order to achieve the above object, the present invention adopts the following technical solutions:
a wound dressing with good hemostatic performance comprises Notoginseng radix extract as raw material.
Preferably, the panax notoginseng extract is an enzymolyzed panax notoginseng alcohol extract.
Preferably, the enzyme used for enzymolysis is one or more of cellulase, xylanase and amylase.
Preferably, the enzyme used for the enzymatic hydrolysis is cellulase or amylase.
Preferably, the enzymolysis conditions are as follows: performing enzymolysis on 100U/g Notoginseng radix ethanol extract and 20U/g amylase of Notoginseng radix ethanol extract at pH4.5 and 50 deg.C for 2 hr.
Preferably, the raw material components of the wound dressing further comprise acetylated codonopsis pilosula polysaccharide, dopamine and a cross-linking agent.
Preferably, the acetylation modifier adopted by the acetylated codonopsis pilosula polysaccharide is one of glacial acetic acid or acetic anhydride. The acetyl can change the orientation and the horizontal sequence of polysaccharide molecules, thereby changing the physical properties of the polysaccharide, and the introduction of the acetyl can change the stretching of the molecules, and finally can lead to the exposure of carboxyl groups of the polysaccharide and increase the solubility in water.
Preferably, the cross-linking agent is one or more of genipin and citric acid. Hydrogels can be formed by physical or chemical methods, the physically cross-linked hydrogels tend to have better biocompatibility but poorer mechanical properties, the chemically cross-linked hydrogels are more stable, but the introduction of chemical cross-linking agents tends to result in poorer cell compatibility and thus limit the applications thereof. Genipin and citric acid belong to natural biological cross-linking agents, can be cross-linked with amino-containing polymers to form biological materials, and are far less toxic than glutaraldehyde and other chemical cross-linking agents.
Further, the applicant provides a method for preparing said wound dressing, characterized in that it comprises the following steps:
1) preparing acetylated codonopsis pilosula polysaccharide: fully dissolving codonopsis pilosula polysaccharide with deionized water, adding an acetylation modifying reagent, controlling the system pH to 8.0-10.0 with NaOH solution, stirring and reacting for 1-2h, adjusting the pH to be neutral with HCl solution, dialyzing for 36-48h after the reaction is stopped, concentrating the dialyzate in a rotary evaporator under reduced pressure, adding absolute ethyl alcohol, precipitating with ethanol at 4 ℃ for 24-48h, and freeze-drying to obtain acetylated codonopsis pilosula polysaccharide;
2) adding the acetylated codonopsis pilosula polysaccharide obtained in the step 1) into deionized water, heating and stirring until the acetylated codonopsis pilosula polysaccharide is dissolved to obtain an acetylated codonopsis pilosula polysaccharide aqueous solution, adding a cross-linking agent aqueous solution, mixing, stirring uniformly at room temperature, standing, adding a dopamine aqueous solution, heating and stirring uniformly, adding a pseudo-ginseng extract, and standing to obtain a finished product.
Preferably, the mass concentration of the acetylated codonopsis pilosula polysaccharide aqueous solution is 5-10 wt%; the mass concentration of the dopamine aqueous solution is 1-8 wt%; the mass concentration of the cross-linking agent aqueous solution is 1-2 wt%; the mass concentration of the pseudo-ginseng extract is 0.5-1.5 wt%.
The beneficial effects of adopting the technical scheme are that: firstly, the raw material components adopted by the hydrogel preparation are natural substances existing in nature, are non-toxic and non-irritant to skin and bodies, and have good biodegradability. The codonopsis pilosula polysaccharide is subjected to acetylation modification by a polysaccharide modification technology, so that the capacity of removing free radicals of the codonopsis pilosula polysaccharide is improved, the codonopsis pilosula polysaccharide has good mechanical properties and biological tissue adhesion after being combined with dopamine for mixed grafting and crosslinking, and meanwhile, a natural crosslinking agent is adopted in the reaction, so that the codonopsis pilosula polysaccharide has the advantage of good biocompatibility compared with a common chemical crosslinking agent, namely glutaraldehyde. In addition, the wound repair process is often accompanied by bacterial infection, so that the antibacterial performance of the hydrogel is tested, and the result shows that the antibacterial performance of the hydrogel is greatly improved. Secondly, in order to enhance the efficacy of the hydrogel and improve the healing efficiency of wound repair, the pseudo-ginseng extract is prepared by an enzymatic hydrolysis method, and the wound dressing is prepared by loading the pseudo-ginseng extract so as to exert the hemostatic function of pseudo-ginseng active ingredients. The wound dressing prepared by the invention has good oxidation resistance, certain biological tissue adhesion, excellent antibacterial property and hemostatic property, and has wide prospects in acute wound treatment and skin wound treatment.
Detailed Description
The technical solutions in the embodiments of the present invention will be reviewed and completely described below with reference to the embodiments of the present invention, so as to further explain the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. Given the embodiments of the present invention, all other embodiments that can be obtained by a person of ordinary skill in the art without any inventive step are within the scope of the present invention.
Main experimental reagents and materials:
pseudo-ginseng was purchased from Yunnan province, and codonopsis pilosula polysaccharide, dopamine, genipin, citric acid and glutaraldehyde were purchased from commercially available products. Coli and s.aureus were purchased from the national collections of microorganisms.
EXAMPLE 1 enzymatic treatment of Panax notoginseng extract
The alcohol extract of notoginseng: taking 25g of pseudo-ginseng, crushing, sieving with a 200-mesh sieve, mixing with 80% ethanol in a proportion of 1: extracting at 50 deg.C for 2 hr, filtering, evaporating filtrate, and drying to obtain ethanol extract.
TABLE 1
EXAMPLE 2 formulation and preparation of hydrogels
TABLE 2
Note: "-" means no such component; dressing 1-4 are sequentially added with notoginseng alcohol extract enzymolysis product 1-4 respectively.
The preparation method of the dressing comprises the following steps: 1) preparing acetylated codonopsis pilosula polysaccharide: fully dissolving codonopsis pilosula polysaccharide with deionized water, adding an acetylation modifying reagent, controlling the system pH to 8.0-10.0 with NaOH solution, stirring and reacting for 1-2h, adjusting the pH to be neutral with HCl solution, dialyzing for 36-48h after the reaction is stopped, concentrating the dialyzate in a rotary evaporator under reduced pressure, adding absolute ethyl alcohol, precipitating with ethanol at 4 ℃ for 24-48h, and freeze-drying to obtain the acetylated codonopsis pilosula polysaccharide. 2) Adding the acetylated codonopsis pilosula polysaccharide obtained in the step 1) into deionized water, heating and stirring until the acetylated codonopsis pilosula polysaccharide is dissolved to obtain an acetylated codonopsis pilosula polysaccharide aqueous solution, adding a cross-linking agent aqueous solution, mixing, stirring uniformly at room temperature, standing, adding a dopamine aqueous solution, heating and stirring, adding a pseudo-ginseng extract, and standing to obtain a finished product.
The remaining hydrogels in the examples were prepared in substantially identical process steps except for formulation components and amounts.
Example 3 in vitro antioxidant Activity assay of hydrogels
Measurement of DPPH radical scavenging ability: dissolving the hydrogel sample with 0.05M/L acetic acid solution, respectively taking 2.0mL of sample solution, adding 2.5mL of 0.1mmol/L DPPH 95% ethanol solution, mixing well, shaking at room temperature, and reacting completely. The reaction was kept in the dark for 30min, and the absorbance at 517nm was measured. Each sample was replicated three times. Using distilled water as a blank control to replace hydrogel sample liquid, and calculating the clearance according to the following formula: DPPH radical scavenging ratio/% (A) 2 -A 1 )/A 2 X 100. In the formula, A 1 Is the absorbance of the experimental group; a. the 2 Absorbance in blank. The results are shown in Table 1. From the data in table 3, it can be found that the hydrogel prepared from acetylated codonopsis pilosula polysaccharide has higher DPPH free radical scavenging rate compared with unmodified codonopsis pilosula polysaccharide, which indicates that the acetylated modified codonopsis pilosula polysaccharide can improve the biological antioxidant activity of the acetylated codonopsis pilosula polysaccharide to a certain extent, and the antioxidant activity of the hydrogel prepared from natural cross-linking agent is higher than that of the hydrogel prepared from glutaraldehyde as cross-linking agent, and the DPPH free radical scavenging rate reaches more than 82%.
TABLE 3
Example 4 characterization of the adhesive Properties of the hydrogels
The bioadhesion was tested using an AG-Xplus Universal Material testing machine. A sheet-like bio-adhesive hydrogel was cut into a specimen having a length of 20mm, a width of 10mm and a thickness of 1mm, and the specimen was contacted with the skin of the back of a human hand under a pressure of 100g for 1min to adhere, and then the non-adhered part of the hydrogel was sandwiched and stretched upward to peel it from the epidermis of the human body, thereby obtaining a peel stress-strain curve of bio-adhesive of the hydrogel, and the average stress at the time of peeling was taken as the adhesive strength of the hydrogel. From the experimental data in table 4, it can be found that the hydrogel prepared by using dopamine has higher viscosity than the hydrogel without dopamine, which indicates that the biological tissue adhesiveness of the gel can be improved to some extent by blending, grafting and crosslinking of dopamine.
TABLE 4
Example 5 bacteriostatic Activity testing of hydrogels
Preparing a bacterial liquid of escherichia coli and staphylococcus aureus: respectively recovering and subculturing Escherichia coli and Staphylococcus aureus with LB liquid culture medium, uniformly dispersing bacteria in centrifuge tube filled with PBS buffer solution with inoculating loop, and placing in shaking table for constant temperature shaking culture to obtain OD 600 A bacterial suspension of 0.5. Laboratory articles such as glass instruments, culture media, PBS buffer solution and the like used in the experiment are sterilized by an autoclave, and hydrogel samples are sterilized by ultraviolet disinfection.
The bacteria inhibiting method comprises the following steps: cylindrical hydrogel samples 9mm in diameter and 1mm in height were placed in solid media coated with a suspension of Staphylococcus aureus and Escherichia coli bacteria, respectively. All samples were incubated in a constant temperature biochemical incubator at 37 ℃ for 24h, and finally the zone of inhibition was measured. The inhibition (PI) criteria for each sample are as follows: PI ═ di/dh × 100%; wherein di and dh denote the bacteriostatic zone diameter (mm) and the hydrogel diameter, respectively.
TABLE 5
The experimental data in Table 5 show that the antibacterial performance of the hydrogel disclosed by the invention is greatly improved after the acetylated codonopsis pilosula polysaccharide and the dopamine are blended and crosslinked, the inhibition rate of staphylococcus aureus reaches 162-fold 166%, the inhibition rate of escherichia coli reaches 173-fold 178%, and the inhibition rate is not much higher than that of the hydrogel without dopamine, and the data show that the antibacterial performance of the hydrogel can be improved to a certain extent by adopting a natural crosslinking agent, and the inhibition rate is higher when genipin and citric acid are mixed for use.
EXAMPLE 6 hemostatic Properties of dressings
30 mice, with weight of 25 +/-2 g and half of each male and female, were randomly grouped, fresh blood was drawn, and sodium citrate anticoagulant (volume ratio of blood to anticoagulant 9: 1) was immediately added for future use. After 0.2mL of fresh blood is dripped on the surfaces of 4 dressing samples, 0.2mol/L CaCl is immediately dripped 2 The coagulation process was started with 0.04mL of the solution, 25mL of deionized water was added after 5min, followed by shaking at 37 ℃ for 5min, and then the solution was taken out and its absorbance value (wavelength 415nm) was measured by UV spectrophotometer. The absorbance value after 0.25mL of blood diluted to 25mL with deionized water was assumed to be 100 as a blank. In vitro coagulation index (BCI) 100% × sample absorbance value/blank absorbance value. Generally, a smaller value of BCI indicates a better hemostatic effect of the hemostatic material. As can be seen from the experimental data in table 6, compared with the pseudo-ginseng alcohol extract composite hydrogel material, the in vitro blood coagulation index of the pseudo-ginseng extract composite hydrogel material extracted by enzymolysis is greatly reduced, which indicates that the pseudo-ginseng extract after enzymolysis has higher bioactivity, wherein the in vitro blood coagulation index of the dressing 4 is smaller by only 42.37%, and indicates that the wound dressing obtained by loading the pseudo-ginseng extract extracted by using cellulase and amylase as the composite enzymolysis preparation with hydrogel has excellent hemostatic effect.
TABLE 6
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention, including any reference to the above-mentioned embodiments. Various modifications to these embodiments will be readily apparent to those skilled in the art. The general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
1. A wound dressing with good hemostatic performance is characterized in that raw material components of the wound dressing comprise pseudo-ginseng extract.
2. The wound dressing of claim 1, wherein the notoginseng extract is an enzymatically hydrolyzed notoginseng alcohol extract.
3. The wound dressing of claim 1, wherein the enzyme used for enzymatic hydrolysis is one or more of cellulase, xylanase and amylase in combination.
4. A wound dressing according to claim 3, wherein the enzymes used for enzymatic hydrolysis are cellulases and amylases.
5. A wound dressing according to claim 4, wherein the enzymatic conditions are: performing enzymolysis on 100U/g Notoginseng radix ethanol extract and 20U/g amylase of Notoginseng radix ethanol extract at pH4.5 and 50 deg.C for 2 hr.
6. The wound dressing of claim 1, wherein the raw material components of the wound dressing further comprise acetylated codonopsis pilosula polysaccharide, dopamine and a cross-linking agent.
7. A wound dressing according to claim 6, wherein the acetylation modifier adopted for acetylating Codonopsis pilosula polysaccharide is one of glacial acetic acid or acetic anhydride.
8. The wound dressing of claim 6, wherein the cross-linking agent is one or more combinations of genipin and citric acid.
9. A method of manufacturing a wound dressing according to any of claims 1 to 8, comprising the steps of:
1) preparing acetylated codonopsis pilosula polysaccharide: fully dissolving codonopsis pilosula polysaccharide with deionized water, adding an acetylation modifying reagent, controlling the system pH to 8.0-10.0 with NaOH solution, stirring and reacting for 1-2h, adjusting the pH to be neutral with HCl solution, dialyzing for 36-48h after the reaction is stopped, concentrating the dialyzate in a rotary evaporator under reduced pressure, adding absolute ethyl alcohol, precipitating with ethanol at 4 ℃ for 24-48h, and freeze-drying to obtain acetylated codonopsis pilosula polysaccharide;
2) adding the acetylated codonopsis pilosula polysaccharide obtained in the step 1) into deionized water, heating and stirring until the acetylated codonopsis pilosula polysaccharide is dissolved to obtain an acetylated codonopsis pilosula polysaccharide aqueous solution, adding a cross-linking agent aqueous solution, mixing, stirring uniformly at room temperature, standing, adding a dopamine aqueous solution, heating and stirring uniformly, adding a pseudo-ginseng extract, and standing to obtain a finished product.
10. The preparation method according to claim 9, wherein the mass concentration of the acetylated codonopsis pilosula polysaccharide water solution is 5-10 wt%; the mass concentration of the dopamine aqueous solution is 1-8 wt%; the mass concentration of the cross-linking agent aqueous solution is 1-2 wt%; the mass concentration of the pseudo-ginseng extract is 0.5wt% -1.5 wt%.
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