CN117122720A - Preparation method of sponge containing bletilla striata polysaccharide - Google Patents
Preparation method of sponge containing bletilla striata polysaccharide Download PDFInfo
- Publication number
- CN117122720A CN117122720A CN202310948533.1A CN202310948533A CN117122720A CN 117122720 A CN117122720 A CN 117122720A CN 202310948533 A CN202310948533 A CN 202310948533A CN 117122720 A CN117122720 A CN 117122720A
- Authority
- CN
- China
- Prior art keywords
- polysaccharide
- sponge
- bsp
- bletilla
- chitosan
- 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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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/22—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing macromolecular materials
- A61L15/225—Mixtures of macromolecular compounds
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- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
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- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
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- A61L2300/232—Monosaccharides, disaccharides, polysaccharides, lipopolysaccharides
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Abstract
The invention discloses a preparation method of a sponge containing bletilla striata polysaccharide, and belongs to the technical field of medical materials. The method provided by the invention comprises the following steps: providing a crosslinking reaction solution containing bletilla striata polysaccharide, chitosan and a crosslinking agent, and separating solid matters in the crosslinking reaction solution after the crosslinking reaction is finished to obtain a crosslinking product of the bletilla striata polysaccharide and the chitosan; dispersing the crosslinked product in a swelling solvent to obtain a swelling solution, and removing the swelling solvent after the crosslinked product is swelled to obtain the sponge containing bletilla polysaccharide. The bletilla striata polysaccharide and the chitosan are crosslinked through the crosslinking agent so as to optimize the physical properties of the sponge, such as liquid absorption property, tensile stress, compressive stress and the like. And the bletilla polysaccharide is matched with the chitosan, so that the effects of stopping bleeding, inhibiting bacteria, resisting inflammation, promoting cell proliferation and the like of the sponge containing the bletilla polysaccharide are improved, and the repairing rate of the sponge containing the bletilla polysaccharide in treating wound surfaces is improved.
Description
Technical Field
The invention relates to the technical field of medical materials, in particular to a preparation method of a sponge containing bletilla striata polysaccharide.
Background
Trauma is a common human injury in life and clinic. Skin acts as the first line of defense for the human body and, when damaged, undergoes healing processes such as hemostasis, inflammation, proliferation and remodeling. However, if the wound is improperly treated during the healing process, there is a risk of infection, and complications such as fever, pain and the like are induced, and even necrosis of tissues and organs, amputation, shock and death are caused. Analysis of medical insurance beneficiaries in 2018 finds that about 820 thousands of wounds with infection or non-infection exist, more than 60 tens of thousands of wounds are serious sequelae and even death caused by untimely or incorrect treatment of wounds each year, and the treatment of wounds is a necessary basic medical guarantee for human beings, so that safe, simple and effective treatment of wounds is a key of treatment. Wound dressing is an important tool for wound treatment, however, the traditional dressing has the problems of single efficacy, poor antibacterial and anti-infection effects, easy adhesion to tissues, secondary injury and the like. Therefore, some novel polymer dressings, such as microspheres, gels, and polymer sponges, have been developed, and the novel dressings have become a research hotspot in recent years.
Rhizoma Bletillae (Bletilla striata (Thunb.) Reichb.f.) is dry tuber of rhizoma Bletillae of Orchidaceae, and has astringent, hemostatic, repercussive, and granulation promoting effects. The rhizoma bletillae is rich in polysaccharide components, is a natural polysaccharide, mainly comprises beta-glucose, alpha-mannose and beta-mannose, can reach the content of more than 25% in the rhizoma bletillae, and has good biocompatibility, biological activity and safety. The bletilla striata polysaccharide not only can be used as a drug carrier, but also can be used as a drug effect active ingredient, and can be singly used or used in combination with other drugs to play a role, so that the bletilla striata polysaccharide is an ideal biological macromolecule material. Is commonly used for preparing wound dressing, vascular embolism material, drug targeting carrier, polymer micelle and the like. The research and development of polysaccharide in rhizoma bletilla can overcome the defects of unstable quality of medicinal materials, difficult control of dosage, unsatisfactory single medicine effect, long and tedious medicine decoction process, inconvenient carrying and emergency use and the like when rhizoma bletillae is used as a traditional Chinese medicine. In order to better exert the treatment characteristics of the rhizoma bletillae such as astringing to stop bleeding, detumescence and promoting granulation, the novel function and the novel use of the rhizoma bletillae as a raw material of a high-molecular sponge material are developed, the high-quality and high-efficiency development of the rhizoma bletillae industry is promoted, and a novel thought is provided for the modern development of the traditional Chinese medicine rhizoma bletillae.
The patent with the application number of CN202110702861.4 discloses a compound sponge dressing of bletilla striata and coptis chinensis and a preparation method thereof, wherein the compound sponge dressing of bletilla striata and coptis chinensis comprises bletilla striata polysaccharide and coptis chinensis total alkaloids, and the bletilla striata polysaccharide and the coptis chinensis total alkaloids form a porous lamellar sponge structure. The bletilla striata-coptis chinensis compound sponge dressing can be prepared by a one-step freeze drying method, and has the advantages of high porosity, good water absorption performance, good moisture retention, excellent elasticity and medicine slow release effect, and the preparation process is simple and the production cost is low.
The sponge auxiliary material obtained by the patent has the advantages of high porosity, good water absorption performance and the like, but still has the following problems: 1. the bletilla striata polysaccharide and the coptis total alkaloids form a porous lamellar sponge structure in a two-dimensional form, so that the water absorption and the elasticity of the sponge are easily reduced;
2. the bletilla polysaccharide and the coptis total alkaloids are only mixed by simple physics, and the mixture of the bletilla polysaccharide and the coptis total alkaloids is obtained, wherein the bletilla polysaccharide and the coptis total alkaloids are independent and do not form new substances through chemical reaction, so that the functions of the bletilla polysaccharide and the coptis total alkaloids are exerted independently even if the bletilla polysaccharide and the coptis total alkaloids are combined, and the functions of the bletilla polysaccharide and the coptis total alkaloids are not increased remarkably.
Disclosure of Invention
The invention aims to solve at least one problem in the background art, and provides a preparation method of sponge containing bletilla striata polysaccharide.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a preparation method of sponge containing rhizoma bletilla polysaccharide comprises providing crosslinking reaction solution containing rhizoma bletilla polysaccharide, chitosan and crosslinking agent, and separating solid substances in the crosslinking reaction solution to obtain crosslinking product of rhizoma bletilla polysaccharide and chitosan after crosslinking reaction is completed;
dispersing the crosslinked product in a swelling solvent to obtain a swelling solution, and removing the swelling solvent after the crosslinked product is swelled to obtain the sponge containing bletilla polysaccharide.
According to one embodiment of the invention, in the crosslinking reaction solution, the mass concentration ratio of the bletilla striata polysaccharide to the chitosan is 1-3:1-3.
According to one embodiment of the present invention, in the crosslinking reaction solution, the concentration of the bletilla striata polysaccharide is 10mg/mL to 30mg/mL, and the concentration of the chitosan is 10mg/mL to 30mg/mL.
According to one embodiment of the invention, the step of preparing the crosslinking reaction solution comprises the steps of reacting bletilla striata polysaccharide, chitosan and a dissolution solvent to obtain a first reaction solution, and adding the crosslinking agent into the first reaction solution to react to obtain the crosslinking reaction solution.
According to one embodiment of the invention, the duration of the crosslinking reaction is 15min to 20min.
According to one embodiment of the invention, the concentration of the crosslinked product in the swelling solution is 7 mg/mL-80 mg/mL
According to one embodiment of the invention, the method of removing the swelling solvent comprises freeze drying.
The invention has the following beneficial effects:
1. the invention is based on the bletilla polysaccharide and chitosan to carry out free radical chemical crosslinking under the action of the crosslinking agent, so as to obtain the high molecular sponge material with good biocompatibility and good antibacterial and anti-infective properties, and the high molecular sponge material is applied to wound repair in life and clinic. By setting the conditions for preparing the bletilla polysaccharide sponge, the bletilla polysaccharide sponge has high-density pores, good liquid absorption capacity, stable mechanical properties, certain antibacterial property and biological activity, can promote benign healing of wound surfaces, avoid complications and rejection caused by traditional materials, further reduce pain of patients and increase use compliance of the patients.
2. When the bletilla polysaccharide sponge is in the wound blood seepage period, the bletilla polysaccharide in the bletilla polysaccharide sponge can absorb wound blood, aggregate platelets and accelerate coagulation while absorbing wound seepage in time. In the wound repair period, the bletilla striata polysaccharide can exert the bacteriostasis and reduce the wound infection risk. In addition, the good biological activity of the polysaccharide of bletilla striata allows it to regulate cytokines (e.g. coagulation factors, platelet-derived growth factors, transforming growth factors, etc.) and inflammatory cells (e.g. leukocytes, platelets, fibroblasts, etc.) and a variety of coagulation-related enzymes (prothrombin, thrombin, plasmin). Therefore, the bletilla polysaccharide sponge can accelerate hemostasis and promote wound healing through the mechanism.
Drawings
FIG. 1 shows a schematic flow chart of the polysaccharide and chitosan obtained by crosslinking the polysaccharide and chitosan under oxalyl chloride;
FIG. 2 shows an SEM image of an understanding of the present invention and a polysaccharide sponge;
FIG. 3a shows a tensile strain curve of an understanding of the present invention and a polysaccharide sponge;
FIG. 3b shows a graph of compressive strain of a polysaccharide sponge as understood by the present invention;
FIG. 4 shows SEM images of different polysaccharide to chitosan ratios, with different polysaccharide sponges, as understood by the present invention;
FIG. 5 shows a plot of porosity versus wicking ratio for different amounts of bletilla striata polysaccharide to chitosan ratios of the present invention;
FIG. 6a shows a graph of tensile strain for different amounts of bletilla striata polysaccharide to chitosan according to the invention;
FIG. 6b shows a graph of compressive strain for different amounts of bletilla striata polysaccharide to chitosan in accordance with the present invention;
FIG. 7 shows SEM images of different lyophilized volumes of bletilla polysaccharide sponges of the present invention;
FIG. 8 shows a line graph of porosity and imbibition ratio of different lyophilization volumes of bletilla polysaccharide sponges of the invention;
FIG. 9a shows a graph of tensile strain of different lyophilization volumes of bletilla polysaccharide sponges of the present invention;
FIG. 9b shows a graph of compressive strain of different lyophilization volumes of bletilla polysaccharide sponges of the present invention;
FIG. 10 shows SEM images of bletilla striata polysaccharide sponges prepared according to the invention at different oxalyl chloride concentrations;
FIG. 11 shows a line graph of the bletilla polysaccharide sponges of the invention prepared at different oxalyl chloride concentrations with different porosities and imbibition ratios;
FIG. 12a shows graphs of different tensile strains of bletilla polysaccharide sponges prepared with different oxalyl chloride concentrations according to the invention;
FIG. 12b shows graphs of different compressive strains of bletilla polysaccharide sponges prepared with different oxalyl chloride concentrations according to the invention;
FIG. 13 shows SEM images of bletilla striata polysaccharide sponges of the present invention showing different reaction sequences of polysaccharide, chitosan, DMF, oxalyl chloride;
FIG. 14 shows a line graph of the present invention showing that bletilla polysaccharide sponges have different porosities and imbibition ratios for different reaction sequences of polysaccharide, chitosan, DMF, oxalyl chloride;
FIG. 15a shows a graph of different tensile strains of bletilla striata polysaccharide sponges according to the present invention for different reaction sequences of polysaccharide, chitosan, DMF, oxalyl chloride;
FIG. 15b shows a graph of the present invention showing that bletilla striata polysaccharide sponges have different compressive strains for different reaction sequences of polysaccharide, chitosan, DMF, oxalyl chloride;
FIG. 16 shows FTIR spectra of BSP, CS-BSP-CK, CS-BSP-1, CS-BSP-2 in the present invention;
FIG. 17a shows hydrogen spectra of BSP, CS-BSP-CK, CS-BSP-1, CS-BSP-2 in the present invention;
FIG. 17b shows a hydrogen spectrum of the delta 8.3 magnification of FIG. 17a in accordance with the present invention;
FIG. 18 shows X-ray diffraction patterns of BSP, CS-BSP-CK, CS-BSP-1, CS-BSP-2 in accordance with the present invention;
FIG. 19a shows a line graph of OD values of CS-BSP-1 according to the present invention;
FIG. 19b shows a line graph of OD values of CS-BSP-2 according to the present invention;
FIG. 20 is a line graph showing weight change in 21 days after molding mice with CK, CS-BSP-1 and CS-BSP-2, respectively, in accordance with the present invention;
FIG. 21 shows a diagram of a bacteriostatic media according to the invention obtained by a paper diffusion method;
FIG. 22 is a bar chart showing the status of the zone of inhibition obtained by the co-cultivation method of the paper sheet of the present invention;
FIG. 23 shows a histogram of blood loss after hemostasis using CK, CS-BSP-1 and CS-BSP-2, respectively, in accordance with the present invention;
FIG. 24 is a diagram showing bleeding after hemostasis using CK, CS-BSP-1, CS-BSP-2, respectively, in accordance with the present invention;
FIG. 25 shows a graph of cell mortality in the present invention;
FIG. 26 is a view showing the healing of a wound in the present invention;
fig. 27 shows a simulated view of the wound healing area in the present invention;
FIG. 28 shows a graph of wound healing rate in the present invention;
FIG. 29 is a graph showing the recovery of inflammatory cell infiltration of the wound surface according to the present invention;
FIG. 30 is a view showing recovery of collagen in the present invention;
FIG. 31a is a bar graph showing the reduction of TNF-alpha by polysaccharide sponges as understood by the present invention;
FIG. 31b is a bar graph showing that the present invention recognizes and polysaccharide sponges reduce IL-6 inflammatory factors;
FIG. 31c is a bar graph showing the effect of polysaccharide sponges on VEGF content as understood by the present invention;
FIG. 32 shows a schematic diagram of a test procedure for a mouse wound model of the present invention.
Detailed Description
The present invention will be described in further detail in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the particular embodiments described herein are illustrative only and are not intended to limit the invention, i.e., the embodiments described are merely some, but not all, of the embodiments of the invention.
The embodiment provides a preparation method of a sponge containing bletilla striata polysaccharide, and provides a crosslinking reaction solution containing the bletilla striata polysaccharide, chitosan and a crosslinking agent, and after the crosslinking reaction is completed, solid substances in the crosslinking reaction solution are separated to obtain a crosslinking product of the bletilla striata polysaccharide and the chitosan;
dispersing the crosslinked product in a swelling solvent to obtain a swelling solution, and removing the swelling solvent after the crosslinked product is swelled to obtain the sponge containing bletilla polysaccharide.
In the embodiment, chitosan is used as a natural polymer material with rich sources and low manufacturing cost, and has good performances of moisture preservation, adsorption, biocompatibility, antibiosis, hemostasis and the like. The bletilla polysaccharide and the chitosan are matched for use, so that the sponge containing the bletilla polysaccharide in the embodiment simultaneously contains the treatment effects of the bletilla polysaccharide and the chitosan, and the treatment effects of the sponge containing the bletilla polysaccharide such as hemostasis, antibiosis, cell growth promotion, wound healing promotion and the like are further optimized.
The porosity of the sponge was obtained by setting the mass ratio of bletilla polysaccharide to chitosan material, as shown in figure 5. By setting the proportion of the bletilla polysaccharide to the chitosan, the porosity is 17.5% -27%, and when the component amount of the bletilla polysaccharide is higher than that of the chitosan, the porosity is lower than 23.5%; when the component amount of bletilla polysaccharide is lower than that of chitosan, the porosity is higher than 20.5%; when the component amount of bletilla polysaccharide is equal to that of chitosan, the porosity is lower than 18.5%.
In some embodiments, oxalyl chloride is used as a cross-linking agent, and when the bletilla polysaccharide, chitosan, and oxalyl chloride are mixed, one end of the oxalyl chloride radical attacks the-OH in the chemical structure of the bletilla polysaccharide, so that the oxalyl chloride is connected with the chemical structure of the bletilla polysaccharide to form a chemical structure, and the other end of the oxalyl chloride radical attacks the-NH in the chemical structure of the chitosan 2 Thus, the chemical structure of bletilla striata polysaccharide is synthesized into a chemical substance through the chemical structure of oxalyl chloride and chitosan, as shown in figure 1. Since in the chemical structure of bletilla polysaccharide, there are a plurality of structural sites of-OH, and in the chemical structure of chitosan, there are a plurality of structural sites of-NH 2 So that when one end of the oxalyl chloride is free to be connected with the-OH of one of the sites, the other end of the oxalyl chloride is free to be connected with the-NH of one of the sites 2 Is connected, so that the chemical structure of the bletilla polysaccharide and the chemical structure of the chitosan have multiple connection modes, namely, in the same reactionIn the product, different combination modes of the chemical structure of the bletilla polysaccharide and the chemical structure of the chitosan exist, so that the bletilla polysaccharide is connected with the chitosan through oxalyl chloride to form a film-shaped structure, the films are mutually interwoven to form a three-dimensional hole structure, and an obtained powder sponge SEM image is displayed as a network interpenetrating structure, as shown in figure 2.
In some embodiments, the crosslinker is an acyl halide containing a diacyl structure (e.g., malonyl, oxalyl, methylmalonyl, ethylmalonyl, butylmalonyl, succinyl, 2-methylsuccinyl, 2-dimethylsuccinyl, 2-ethyl-2-methyl-succinyl, 2, 3-dimethylsuccinyl, glutaryl, 2-methylglutaryl, 3-methylglutaryl, 2-dimethylglutaryl, 2, 3-dimethylglutaryl, 3-dimethylglutaryl, adipoyl, pimeloyl, maleyl, fumaryl), wherein the halogen element in the acyl halide is chlorine or bromine, more preferably chlorine.
Further, the acid halide may be malonyl chloride, oxalyl chloride, methylchloromalonyl chloride, oxalyl chloride malonyl chloride, ding Lvbing diacid chloride, succinyl chloride, 2-methylchlorosuccinyl chloride, 2-dimethylchlorosuccinyl chloride, 2-ethylchloro-2-methylchlorosuccinyl chloride, 2, 3-dimethylchlorosuccinyl chloride, glutaryl chloride, 2-methylchloroglutaryl chloride, 3-methylchloroglutaryl chloride, 2-dimethylchloroglutaryl chloride, 2, 3-dimethylchloroglutaryl chloride, 3-dimethylchloroglutaryl chloride, adipoyl chloride, pimeloyl chloride, maleic chloride, fumaroyl chloride.
In some embodiments, the molecular structure of the cross-linking agent has two active sites, one for attachment of the bletilla polysaccharide molecular structure and the other for attachment of the chitosan molecular structure.
In some embodiments, chitosan may be replaced with other polymeric polysaccharides.
According to one embodiment of the invention, in the crosslinking reaction solution, the mass concentration ratio of the bletilla striata polysaccharide to the chitosan is 1-3:1-3.
According to one embodiment of the present invention, in the crosslinking reaction solution, the concentration of the bletilla striata polysaccharide is 10mg/mL to 30mg/mL, and the concentration of the chitosan is 10mg/mL to 30mg/mL.
According to one embodiment of the invention, the step of preparing the crosslinking reaction solution comprises the steps of reacting bletilla striata polysaccharide, chitosan and a dissolution solvent to obtain a first reaction solution, and adding the crosslinking agent into the first reaction solution to react to obtain the crosslinking reaction solution.
According to one embodiment of the invention, the duration of the crosslinking reaction is 15min to 20min.
According to one embodiment of the invention, the concentration of the crosslinked product in the swelling liquid is 7 mg/mL-80 mg/mL.
According to one embodiment of the invention, the method of removing the swelling solvent comprises freeze drying.
Preparation of bletilla striata polysaccharide sponge: harvesting rhizoma bletilla, cleaning, cutting off fibrous roots and overground parts, drying at 65deg.C for 48 hr, pulverizing into powder, and sieving with 60 mesh sieve.
Weighing rhizoma bletilla powder (according to mass ratio of rhizoma bletilla to ultrapure water=1:12), adding ultrapure water, leaching at 90 ℃ for 24 hours, centrifuging, taking supernatant, adding absolute ethanol to a final concentration of 80%, centrifuging, collecting precipitate, freeze-drying, and crushing to obtain rhizoma bletilla polysaccharide freeze-dried powder.
The preparation method of the bletilla striata polysaccharide sponge comprises the following steps:
precisely weighing 100mg of bletilla polysaccharide freeze-dried powder and 150mg of chitosan, dissolving in 10mL of DMF, continuously stirring, adding 0.1mL of oxalyl chloride, continuously reacting for 15min, centrifuging, discarding supernatant to obtain a product, adding 20mL of distilled water to swell uniformly, and freeze-drying at-60-80 ℃ for 12-18 h to obtain CS-BSP-1.
Precisely weighing 100mg of bletilla polysaccharide freeze-dried powder and 30mg of chitosan, dissolving in 10mL of DMF, continuously stirring, adding 0.2mL of oxalyl chloride, continuously reacting for 15min, centrifuging, discarding supernatant to obtain a product, adding 25mL of distilled water to swell uniformly, and freeze-drying at-60-80 ℃ for 12-18 h to obtain CS-BSP-2.
Among them, DMF may also be other solvents including but not limited to DMSO, pyridine, furans, etc.
Wherein BSP represents bletilla polysaccharide and CS represents chitosan.
The pH value of the water can be alkaline, neutral or acidic, and when the pH value is alkaline, the prepared bletilla polysaccharide sponge has better looseness. When the sponge is acidic, the prepared bletilla polysaccharide sponge has good viscosity. And the neutral water is used for preparing the bletilla striata polysaccharide sponge with good comprehensiveness.
As shown in fig. 1, a schematic flow chart of the bletilla polysaccharide sponge obtained by crosslinking bletilla polysaccharide and chitosan with oxalyl chloride is shown.
Test examples
1. Test preparation
The chitosan has the viscosity of 100-200 mPas, the deacetylation degree of more than or equal to 98 percent and is available from Shanghai Ala Biochemical technology Co., ltd; DMF, anhuizhen technologies inc; oxalyl chloride, shanghai microphone Lin Biochemical technology Co., ltd; PBS, siemens technologies Co., ltd; chloral hydrate, shanghai Michelia Biochemical technology Co., ltd; normal saline, wuhansai wile biotechnology limited; 4% paraformaldehyde, blue Ke Jie technologies; 4% sodium citrate anticoagulant, italian biotechnology Limited company; a number 0.5 tube manufactured by the Pinctada martensii Biotechnology Co., ltd; nutrient agar, produced by Beijing Obocin Biotechnology Limited liability company; LB broth, produced by Qingdao high technology Industrial Yuan Haibo Biotechnology Co., ltd; ELISA kit (ELISA, IL-6, TNF-alpha, VEGF), whan Seville Biotechnology Co., ltd; mouse embryo cells (NIH-3T 3), wohplaunorace life technologies Co., ltd; coli (Yersinia enterocoLitica, lot 20220420), staphylococcus aureus (StaphyLococcus aureus, lot 20220420), pseudomonas aeruginosa (p. Aeromonas, lot 20220420) were all purchased from the Shanghai collection biotechnology center.
2. Characterization of bletilla polysaccharide sponges
2.1 scanning electron microscope, taking trace bletilla polysaccharide sponge to directly adhere to conductive adhesive, and spraying metal for 45s by using an Oxford Quorum SC7620 sputter film plating instrument, wherein the metal spraying is 10mA; and then a scanning electron microscope (SEM, TESCAN MIRA LMS) is used for shooting the morphology of the bletilla polysaccharide sponge, the accelerating voltage is 15kV during shooting, and the detector is an SE2 secondary electron detector.
By scanning electron microscopy of the bletilla polysaccharide sponges CS-BSP-1 and CS-BSP-2, as shown in figure 2, in SEM images of CS-BSP-1 and CS-BSP-2, the sponges have membranous layered structures and rich holes, and with some fibrous structures, the surfaces of the films are smooth and have high concavo-convex, the films extend in the horizontal direction and the vertical direction, the films extending in the horizontal direction are interwoven with each other, and the films extending in the vertical direction are interwoven with each other to form holes; or the films extending along the horizontal direction are interwoven with the films extending along the vertical direction to form holes; the film is provided with fibrous films which are mutually interwoven to form holes; or the fibrous thin film is interwoven with the thin film extending along the horizontal direction to form holes, or the fibrous thin film is interwoven with the thin film extending along the vertical direction to form holes; the holes are extended along the horizontal direction and also extended along the vertical direction, the films extended along different directions are interwoven with the fibrous films, and the fibrous films are interwoven with each other to form a three-dimensional hole-shaped structure. From the figure, it can be seen that the size of the film structure is 10 μm to 70 μm, the thickness of the film is 0.1 μm to 2 μm, and the diameter of the hole is 1 μm to 90 μm. The three-dimensional porous structure has a crucial influence on the physical properties of bletilla polysaccharide sponges.
In addition, CS-BSP-1 and CS-BSP-2 were subjected to tensile strain and compressive strain tests.
As shown in FIG. 3a, the sponge still has good recovery property when the tensile force is continuously applied to the CS-BSP-1 sponge, the tensile strain rate of the sponge is close to 13% when the tensile force applied to the CS-BSP-1 sponge reaches approximately 0.07MPa, then the tensile strain rate of the sponge is higher than 10% when the tensile force is reduced to approximately 0.03MPa, and the tensile strain rate of the sponge is still higher than 10% when the tensile force is reduced. When the tensile force is continuously applied to the CS-BSP-2 sponge to be close to 0.04Mpa, the tensile strain rate of the sponge exceeds 13 percent, and then the tensile strain rate of the sponge reaches 17.5 percent in the process of reducing the tensile force. The bletilla striata polysaccharide sponge has good recovery capacity after stretching.
As shown in FIG. 3b, the compression strain rate of the bletilla polysaccharide sponges exceeded 80% when the compression force was continuously applied to CS-BSP-1 and CS-BSP-2 approaching 0.3 MPa. The bletilla striata polysaccharide sponge has good recovery capacity after compression.
From the results, the physical properties of the bletilla polysaccharide sponge are improved to a certain extent by crosslinking the bletilla polysaccharide and the chitosan through oxalyl chloride. This has relevance to the fact that bletilla polysaccharide sponges have three-dimensional pore-like structures, and the size, thickness, pore diameter, etc. of the films.
2.1.1 different ratios of Bletillae polysaccharide to chitosan with different SEM images of Bletillae polysaccharide sponges
TABLE 1 different bletilla polysaccharide and chitosan content ratios
As shown in fig. 4, when the ratio of bletilla polysaccharide to chitosan is 1:1, the SEM image of bletilla polysaccharide sponge shows a distinct layered structure, and the arrangement of the membranous structure and the porous structure is more compact. As the chitosan content increases, the porosity of the sponge increases. As the number of BSPs increases, the sponge-like and porous structures of the bletilla polysaccharide decrease, gradually forming lumps. As the number of BSPs increases, the phenomenon of forming lumps becomes more pronounced. Therefore, the proportion of the bletilla polysaccharide to the chitosan is properly controlled, the membranous structure and the porous structure of the bletilla polysaccharide sponge can be adjusted, the quantity of the chitosan is properly higher than that of the bletilla polysaccharide, and the bletilla polysaccharide sponge can generate better loose and porous structures.
2.1.1.1 different ratios of bletilla striata polysaccharide to chitosan have different porosities and imbibition ratios
As shown in fig. 5, when the amount of chitosan is gradually increased compared to the amount of bletilla polysaccharide, the porosity and the liquid absorption ratio of the bletilla polysaccharide sponge are correspondingly increased. Ratio when the ratio of bletilla polysaccharide to chitosan was 1:2, 1:3, the bletilla polysaccharide sponge exhibited better porosity and liquid absorption. When the quantity of the bletilla polysaccharide is gradually increased compared with that of the chitosan, the porosity and the liquid absorption ratio of the bletilla polysaccharide sponge are reduced instead. The chitosan is higher than the bletilla polysaccharide, so that the bletilla polysaccharide sponge has better porosity and liquid absorption. From this, it is known that chitosan plays a critical role in the porosity and the liquid absorption ratio of bletilla polysaccharide sponges.
It can also be derived from fig. 5 that the porosity and the liquid absorption have a substantially positive correlation.
2.1.1.2 different ratios of bletilla striata polysaccharide to chitosan have different tensile and compressive strain curves
As shown in fig. 6b, the strain rate of the sponge after compression of BL1-BL5 reaches 80%, and when the applied pressure is continuously increased to the sponge of BL1-BL5, the strain rate of the sponge is also increased accordingly, and then when the pressure is decreased, the strain rate of the sponge is also increased. Among the quantitative ratios of BL1 to BL5, BL1 exhibits the best strain capacity compared to other BL.
As shown in FIG. 6b, when a tensile force of not more than 0.13MPa is applied only to the sponge, the strain rate can reach 12%. When the tensile force is reduced, the strain rate exceeds 17%.
2.1.2 different lyophilized volumes of bletilla polysaccharide sponges with different SEM images
TABLE 2 different lyophilization volumes
As shown in fig. 7, the porosity of the bletilla polysaccharide sponge showed a biphasic response to the lyophilized volume of the bletilla polysaccharide sponge, the pore structure was first increased, and the three-dimensional pore structure was more pronounced, followed by a decrease in the pore structure, which was also gradually decreased. These observations indicate that too small or too large a lyophilization volume is not the best choice for achieving the ideal pore structure of the bletilla polysaccharide sponge. The freeze-dried volume of the bletilla polysaccharide sponge has a crucial effect on the pore structure formation of the bletilla polysaccharide sponge.
2.1.2.1 different lyophilized volumes of bletilla polysaccharide sponges have different porosities and imbibition ratios
As shown in FIG. 8, when V2 is 10mL, the bletilla polysaccharide sponge has better porosity and liquid absorption, and as the volume increases, the porosity and liquid absorption of the bletilla polysaccharide sponge decreases. From this, it was found that the lyophilization volume had an effect on the porosity and liquid absorption of the bletilla polysaccharide sponge.
2.1.2.2 different freeze-dried volumes of bletilla polysaccharide sponges have different tensile and compressive strain curves
As shown in fig. 9a and 9b, different lyophilization volumes of sponges have different tensile and compressive strain curves. From this, it was found that the freeze-dried volume had an effect on the deformation of the sponge.
2.1.3 different oxalyl chloride concentrations of bletilla polysaccharide sponges with different SEM images
TABLE 3 oxalyl chloride at different concentrations
As shown in fig. 10, the bletilla polysaccharide sponge of C1 is an SEM image without oxalyl chloride, and it can be seen that the bletilla polysaccharide sponge has a dense lamellar structure, and the lamellar structure is similar to the arrangement, and no obvious pore structure is seen. With the increase of oxalyl chloride concentration, the morphology and structure of the bletilla polysaccharide sponge changed. The bletilla polysaccharide sponge with low concentration of oxalyl chloride has a block structure which is scattered and is complicated to interweave, and interweaving is firm. As oxalyl chloride concentration increases, the sponge gradually converts into a membranous structure, resulting in a looser, more organized internal structure, and eventually a three-dimensional porous structure. As oxalyl chloride concentration further increases, the sponge retains its porous structure, but the membranous structure becomes less pronounced, resulting in tighter internal structures, increased thickness of the membrane, affecting the strength of the bletilla polysaccharide sponge. It is known that the space morphology of the bletilla polysaccharide sponge after the addition of oxalyl chloride is different from that of the bletilla polysaccharide sponge without the addition of oxalyl chloride, and the bletilla polysaccharide sponge with the addition of oxalyl chloride is interwoven through the membrane to form a three-dimensional porous structure, but the bletilla polysaccharide sponge with the addition of oxalyl chloride does not have the characteristic of the three-dimensional porous structure. From this, oxalyl chloride is a key factor in the three-dimensional pore structure of bletilla polysaccharide sponges.
2.1.3.1 Bletillae polysaccharide sponges with different oxalyl chloride concentrations have different porosities and imbibition ratios
As shown in fig. 11, the bletilla polysaccharide sponge without oxalyl chloride added exhibited lower porosity and liquid absorption, and the bletilla polysaccharide sponge with low concentration of oxalyl chloride also had lower liquid absorption but higher porosity. From this, oxalyl chloride is a key factor affecting the porosity of bletilla polysaccharide sponges; oxalyl chloride concentration is a key factor affecting the liquid absorption ratio of bletilla polysaccharide sponges. Whereas too high a concentration of oxalyl chloride results in a decrease in porosity and liquid absorption of the bletilla polysaccharide sponge. Thus, the proper oxalyl chloride concentration can improve the porosity and liquid absorption of the bletilla polysaccharide sponge.
2.1.3.2 Bletillae polysaccharide sponges with different oxalyl chloride concentrations have different tensile and compressive strain curves
As can be seen in fig. 12a and 12b, sponges of different oxalyl chloride concentrations have different tensile and compressive strain curves. From this, it was found that the oxalyl chloride concentration had an effect on the deformation of the sponge.
2.1.4 different feed sequences of bletilla polysaccharide sponges with different SEM pictures
TABLE 4 different charging sequences
As shown in FIG. 13, S1 is a sponge of bletilla polysaccharide having a three-dimensional, film-like porous structure with a relatively loose film structure and a relatively regular arrangement of pores and films. The sponge prepared by the other three reactants still has a certain porous structure, but the arrangement of the films is tighter, and the concave-convex degree is reduced; therefore, the condition of S1 is more favorable for the formation of the sponge structure of bletilla polysaccharide.
2.1.4.1 the rhizoma bletilla polysaccharide sponges with different charging sequences have different porosities and imbibition ratios
As shown in FIG. 14, the porosity and liquid absorption of the S1 bletilla polysaccharide sponge is higher than that of the S2-S4 bletilla polysaccharide sponges. Thus, the order of addition affects the porosity and the liquid absorption ratio of the resulting bletilla polysaccharide sponge.
2.1.2.2 the bletilla polysaccharide sponges with different charging sequences have different tensile strain curves and compressive strain curves
As shown in fig. 15a and 15b, sponges of different order of addition have different tensile strain curves and compressive strain curves. It follows that different feeding sequences have an influence on the deformation of the sponge.
2.2FTIR spectral characterization
By KBr tabletting technique, the tablet is compressed in the middle infrared (400 cm) -1 ～4000cm -1 ) FTIR spectroscopic analysis was performed in the range. 10mg of the sample was homogeneously mixed with 300mg of KBr. A sample sphere of diameter 7 mm was pressed at a pressure of 2 tons for 3 minutes. An infrared spectrum of the sample was obtained in the mid-infrared range using a Thermo Scientific Nicolet 6700 spectrophotometer. For each sample, 32 scans were recorded, with a resolution of 4cm -1 。
As shown in FIG. 16, at 3436cm -1 The absorption peak of the physical mixing (CS-BSP-CK) sponge of bletilla polysaccharide (BSP), BSP and Chitosan (CS) is most obvious, which shows that the hydroxyl of BSP in CS-BSP-CK is not consumed at the moment, and the hydroxyl of BSP is consumed by adding oxalyl chloride as a crosslinking agent, and simultaneously, the absorption of CS-BSP-1 and CS-BSP-2 is reduced at the position and moves to the vicinity of 3350cm < -1 >, and the absorption is presumably caused by the influence of hydrogen bonds on amide bonds formed after the reaction of oxalyl chloride and CS. At 1524cm -1 Here, the sponges of BSP and CS-BSP-CK are gentle, and the C=O stretching vibration peaks of secondary amide appear in CS-BSP-1 and CS-BSP-2, and at the same time, 1418cm -1 Here, C-N stretching peaks of the primary amide appear, all of which confirm the presence of the amide bond.
2.3 characterization of nuclear magnetic resonance hydrogen spectrum
Using D 2 O is used as a solvent, and the lyophilized BSP, CS-BSP-CK, CS-BSP-1, CS-BSP-2 are exchanged with deuteriumThree times. Then measured by NMR spectrometer (Bruker AVANCE III HD MHz) 1 H NMR spectrum.
As shown in FIGS. 17a and 17b, the hydrogen of CS-BSP-CK and BSP is mainly concentrated in the high field region, which is hydrogen on hydrocarbon groups, and there is almost no hydrogen signal in the low field region. While CS-BSP-1 and CS-BSP-2 have hydrogen signals near the low field region delta 8.3, and the chemical shift value of the amide bond is generally near delta 5-8.5, the hydrogen signals near delta 8.3 can be judged as the hydrogen signals in the amide bond generated by the reaction. The above results all indicate that a sponge of CS-crosslinked BSP was successfully prepared.
2.4X-ray diffraction characterization
The BSP and BSP sponges were characterized by X-ray diffraction (XRD) using a p-analytical X' Pert powder diffractometer at a voltage of 40kV and a current of 40 mA. XRD diffraction spectrum is in the range of 10-70 deg. and scanning speed is 2 deg. min -1 The step size is 0.02 °.
As shown in fig. 18, BSP and CK have a broad peak around 2θ=20°, and the peak of CS-BSP-CK is strong and sharp corresponding to the crystalline region, whereas CS-BSP-1, CS-BSP-2 have a weak peak around 2θ=23°, indicating a decrease in crystallinity. The lower the crystallinity of the composite, the better the miscibility of the material components in the composite sponge. The bletilla striata polysaccharide and the chitosan have good miscibility, and the bletilla striata polysaccharide is crosslinked with the chitosan through oxalyl chloride, so that the miscibility is increased.
3. In vitro test of bletilla polysaccharide sponges
3.1 cell growth promoting Performance test
After 1mL of 0.2% pancreatin was used to digest 3min for logarithmic growth phase cells using mouse fibroblasts (NIH-3T 3), the digestion was stopped by adding an equal amount of DMEM medium, centrifuging at 1000rpm for 4min, removing the supernatant, adding fresh medium, gently beating the cells to be suspended in the medium.
Inoculating NIH-3T3 cells with density of 104/mL into 96-well culture dish, each well having volume of 100 μl, setting one circle around the well plate as blank group, adding PBS solution, adding only culture solution into negative control group without adding cells, and adding CO at 37deg.C and 5% 2 Incubation was carried out for 24h to allow cell attachment.
The culture medium was replaced with the prepared CS-BSP-1 and CS-BSP-2 extracts (100. Mu.L), and after co-culturing for 24 hours, 48 hours and 72 hours, the original culture medium was aspirated, 100. Mu.L of fresh culture medium was added to each well, CCK-8 (10. Mu.L) was added to each well and culturing was continued at 37℃for 4 hours. The absorbance at 450nm was measured by microplate reader.
As a result of toxicity analysis of CS-BSP-1 and CS-BSP-2 by CCK-8 method, as shown in FIGS. 19a and 19b, the OD values of CS-BSP-1 and CS-BSP-2 were higher than that of DMEM group from 0.04g to 0.16g in 72h, showing that CS-BSP-1 and CS-BSP-2 were not only non-cytotoxic but also had some growth promoting effect on cells, especially evident at 0.04g, and after 72h, the OD value of CS-BSP-1 was 1.95 times that of DMEM, and the OD value of CS-BSP-2 was 1.71 times that of DMEM, with significant difference (P < 0.0001). It is presumed that at low concentrations, white polysaccharide and sponge have a strong promoting effect on cell growth and proliferation.
By analyzing the weight of the mice after molding, whether the mice are toxic or not can be estimated after the bletilla polysaccharide sponge is treated, the weight change of the mice in the treatment process is shown as a figure 20, and the weight of each group of mice is basically and stably increased within 21 days without statistical difference among groups, which proves that the bletilla polysaccharide sponge has no obvious toxicity to the mice.
3.2 antibacterial Property test of bletilla polysaccharide sponge
The antibacterial activity of the polysaccharide sponges of the present invention was measured by the filter paper agar diffusion method and the co-culture method.
10mgCS-BSP-1 and CS-BSP-2 sponge samples are weighed and immersed in 1mL PBS for 24 hours to prepare sample lixivium. Respectively soaking sterile filter paper sheets (with the diameter of 6 mm) in a sample leaching solution for 30min, absorbing 200 mu L of 3 bacterial suspensions (such as escherichia coli, staphylococcus aureus and pseudomonas aeruginosa) with the concentration of about 1 multiplied by 108cfu/mL, uniformly coating on a nutrient agar plate, placing the soaked sterile filter paper sheets on the agar plate at equal intervals, taking PBS as a negative control, then inversely incubating the plate for 24h at the temperature of 37 ℃, and recording the diameter of a bacteriostasis ring.
mu.L of bacterial suspension diluted to 1X 108CFU/mL with PBS was added to a 96-well plate. Next, 180 μl of sample leaching solution and PBS solution (CK) were added to each well. The 96-well plate is placed in a 37 ℃ incubator for 4 hours, then taken out, the solution in the well plate is blown uniformly by a pipette, then bacterial suspension in the well plate is sucked, 20 mu L of each well is sucked, then 1mLPBS solution is used for dilution, 60 mu L of the diluted bacterial suspension is dripped on an agar plate, and finally all agar plates are placed in the 37 ℃ incubator for 24 hours after being uniformly coated by a completely sterilized triangular glass rod. Bacterial growth was observed.
Bacteria are a major cause of many infections in humans, as they are often found in the mucous membranes or skin of humans, leading to inflammation and exacerbation of wounds. Therefore, the CS-BSP-1 and CS-BSP-2 were tested for their resistance to E.coli, staphylococcus aureus and Pseudomonas aeruginosa by both the paper diffusion method and co-culture method.
As shown in FIG. 21, the sheet diffusion method showed that CS-BSP-1 and CS-BSP-2 had a distinct zone of inhibition, whereas CK did not have a zone of inhibition due to lack of antimicrobial.
As shown in FIG. 22, the co-culture method shows that after 24 hours of culture, CS-BSP-1 and CS-BSP-2 have obvious antibacterial effects, and CS-BSP-1 has stronger resistance to staphylococcus aureus and pseudomonas aeruginosa, and has better inhibition effects on escherichia coli, CS-BSP-1 and CS-BSP-2.
3.3 Bletillae polysaccharide sponge hemostatic Performance test
The tail-breaking model test of mice, the test mice are SPF-grade healthy male ICR mice, and the weight is about 18 g. The mice are housed in cages at room temperature, water and food can be freely obtained, 5 mice in each cage, and the total number of the mice is 3. The water and wood chip pads were changed 2 times a week, fed daily, and entered the test 2-3 weeks after laboratory feeding.
The anesthetized mice were quantitatively intraperitoneally injected with 4% chloral hydrate and fixed on a surgical cork plate, and the tail length of the mice was cut off 2/3 with surgical scissors. Pre-weighed filter paper is placed under the wound to accurately estimate the blood loss, the tail of the mouse is suspended for 5s after cutting to ensure the normal blood loss, a hemostatic sponge is applied to the wound to stop bleeding, the hemostatic time and the weight of the filter paper (blood loss) are recorded, each test is repeated three times, and a tail-breaking model which is not treated by the sponge is used as a Control (CK).
As shown in FIGS. 23 and 24, the blood loss in the model of the tail-breaking of the CS-BSP-1 treated mice was significantly reduced, and the hemostatic time was the shortest, the blood loss was (17.80.+ -. 0.95 mg), the hemostatic time was (277.33.+ -. 5.51 s), and the blood loss in the model of the tail-breaking of the mice without any treatment (CK) was (30.83.+ -. 0.85 mg), the hemostatic time was (428.67.+ -. 8.02) (P < 0.05), and CK was nearly 2-fold higher than CS-BSP-1 treatment, both in blood loss and hemostatic time, as expressed by CK > CS-BSP-2 > CS-BSP-1. As shown in FIG. 25, the BCI is a clinical common blood coagulation judgment index, the lower the BCI is, the better the blood coagulation effect is, which shows that the better the hemostatic effect is, the hemostatic index is expressed as CS-BSP-CK > CS-BSP-2 > CS-BSP-1, and the hemostatic coefficient of CS-BSP-CK is obviously higher than CS-BSP-1 and CS-BSP-2. As can be seen from FIGS. 23-25, CS-BSP-1 has excellent hemostatic effect, which is closely related to the ratio of chitosan to bletilla gum in CS-BSP-1, and in addition, CS-BSP-1 has good tissue adhesion and mechanical strength, providing a physical barrier for the wound to accelerate the clotting rate of the wound.
4. In vivo test of bletilla polysaccharide sponges
4.1 mouse wound model test
4.1.1 wound model establishment
The wound healing capacity of bletilla polysaccharide sponges was evaluated using a full thickness skin defect model. The test material and the mice in the earlier stage were fed in the same manner as 2.5.3, 5 mice in each cage, and a total of 12 cages were randomly divided into 3 groups of 20 mice each. 60 mice were anesthetized using intraperitoneal injection of 4% chloral hydrate. After the mice were anesthetized, the back hair of the mice was shaved off by a shaver, and a full-thickness skin defect wound having a diameter of 10mm was formed in the back region of the mice, and molding was completed.
4.1.2 mice weight change and wound healing Condition
After the molding was completed, the mice were randomly divided into 3 groups of 20 mice each, and the wounds of the mice were treated with CS-BSP-1 and CS-BSP-2, respectively, and medical gauze treatment was used as a negative Control (CK), and the mice were taken out of the dressing every 7d to record the degree of wound healing during the treatment starting period, and at the same time, the weight change of the mice was recorded every 3d and the dressing was replaced, and the test was continued for 28d as shown in FIGS. 20, 26 and 32.
In order to study the influence of the compound sponge on wound tissue regeneration, the wound healing area and the wound healing rate in the mouse body are measured. The wound healing rate was calculated using Image J software, and from the simulated figures of the wound healing rate and the healing area, as shown in fig. 27 and 28, the CS-BSP-1 and CS-BSP-2 had no significant difference in healing effect, but significant difference from CK.
On day 7 of treatment, CS-BSP-1 and CS-BSP-2 substantially scab, CS-BSP-1 wound area was significantly reduced, but CK had not yet scab completely, inflammation was the heaviest, and exudates were present. This is because BSP itself can enhance wound healing efficiency by inducing proliferation and migration of vascular endothelial cells. And the composite CS can adjust the mechanical force, water absorption, antibacterial property and other capacities of the sponge prepared by the BSP, and further improve the healing degree of the wound surface.
On treatment day 14, the wound surfaces of CS-BSP-1 and CS-BSP-2 are obviously reduced, and the healing degree is higher. This is due to the porous structure and excellent wicking ability of the bletilla polysaccharide sponge, which removes any excess exudates, creates a suitable fluid environment, and promotes wound healing rate. In addition, the sponge based on the composite CS has good antibacterial property, promotes cell growth performance, avoids secondary injury caused by external bacterial infection, and promotes wound surface to heal rapidly.
On treatment day 21, the CS-BSP-1 and CS-BSP-2 wounds healed completely, the mice had healthy hair growth, and CK was also visible as an unrepeated wound, indicating that CS-BSP-1 and CS-BSP-2 could heal the wounds faster.
4.2H & E staining and Masson staining
Immediately after the mice are sacrificed, the wound skin is excised, 4% chloral hydrate is used for intraperitoneal injection to anesthetize the mice, and wound tissues are cut off along the wound surface of about 2-3mm, so that the materials are obtained. The wound tissue is immersed in 4% paraformaldehyde for fixation, and the steps of flushing, dewatering, transparentizing, waxing, embedding, repairing, slicing and spreading are sequentially carried out, and then H & E dyeing and Masson dyeing are carried out.
To get a better understanding of how the bletilla polysaccharide sponge promoted wound healing, mice from the 4.1 mouse wound model test described above were sacrificed at different time intervals and the state of regenerating skin tissue was analyzed by H & E staining and Masson staining. H & E staining and Masson staining can identify inflammatory cell infiltration and collagen recovery of the wound.
As shown in fig. 29, in 7d, a large number of inflammatory cells including lymphocytes, plasma cells and macrophages can be infiltrated and aggregated under the wound surface mirror of the CK group, the hair follicle structure is less, the cell arrangement is more disordered, and the new blood vessels are not obviously increased; the wound surfaces of the CS-BSP-1 and the CS-BSP-2 are accompanied by infiltration of scattered inflammatory cells while granulation tissues are generated, and the CS-BSP-1 has more newly generated capillaries, so that the dermis of a treatment group is thinned, and skin appendages are increased; all 3 groups showed epithelial shedding and no epithelial regeneration was seen.
After 14d, CK granulation tissue is still mainly inflammatory cells, collagen and fibroblasts are less common; obvious epidermis regeneration occurs in the wounds of CS-BSP-1 and CS-BSP-2, collagen in the dermis layer is orderly arranged, granulation tissues are obviously increased, and in addition, compared with the wounds of CS-BSP-1 and CS-BSP-2, skin appendages of CS-BSP-1 such as hair follicles, sweat glands and the like are more proliferated.
After 21d, each group has obvious epidermic regeneration, CS-BSP-1 and CS-BSP-2 epidermic tissues tend to be complete, the keratinized layer is more obvious, the accessory glands are increased, the connective tissue recombination is good, and a large number of new blood vessels appear; CK still has massive inflammatory cell infiltration, few accessory glands in dermis, few fibroblasts in granulation tissue, disordered and discontinuous collagen arrangement, incomplete connective tissue recombination in dermis, and massive gaps.
After 28d, the epidermis of the negative group tended to be intact, the keratinized layer was evident, all groups of wounds had healed, there was apparent appearance of skin appendages, collagen was abundant and neat, similar to normal skin, and the healing process had been substantially completed.
Collagen deposition is an important phenomenon for wound recovery, and as shown in FIG. 30, CS-BSP-1, CS-BSP-2 and CK all showed small amounts of collagen production after 7d of treatment.
By 14d, collagen deposition was increased for each group relative to 7d, but the CK arrangement was sparse, the collagen deposition amounts of CS-BSP-1 and CS-BSP-2 were slightly larger than CK, and the skin appendages such as hair follicles, sweat glands, etc. of CS-BSP-1 were more proliferated.
After 21d, the collagen deposition of the wound dermis layers of the CS-BSP-1 and the CS-BSP-2 is still more than that of CK, the arrangement is more compact and ordered, the skin appendages at the wound of the CS-BSP-2 are more obviously diffused, and the protein deposition is also increased.
By 28 days, all groups of wound collagen deposition became dense and ordered, appearance of wound skin appendages, apparent wound healing and apparent collagen deposition were similar to normal skin.
In conclusion, CS-BSP-1 and CS-BSP-2 can promote wound healing by regulating the inflammation level of the wound, promoting collagen deposition and epithelial cell regeneration, and the promoting effect of CS-BSP-1 is better and more obvious.
4.3 enzyme-linked immunosorbent assay (ELISA)
At the beginning of the treatment, one cage of mice was sacrificed every 7d for each treatment, and mouse whole blood was collected, i.e., 7d, 14d, 21d, 28d. The collected whole blood was subjected to 6000 rpm Zhong Lixin min at room temperature, serum was collected in a frozen tube, labeled and stored at 80℃under zero. The ELISA kit is used for determining the content of inflammatory factors IL-6, TNF-alpha and VEGF in each serum sample, and specific test steps are referred to the kit instruction.
Symptomatic cells are the basis of wound healing, especially macrophages and neutrophils. However, a large amount of data suggests that the prolongation and overexpression of various inflammatory cells eventually prevent the healing process. In the inflammatory phase, important inflammatory factors tumor necrosis factor-alpha (TNF-alpha) and interleukin 6 (IL-6) are selected in the experiment.
As a result, as shown in FIGS. 31a, 31b and 31c, the IL-6 content of CK was 31.35% higher than CS-BSP-1 and 19.41% higher than CS-BSP-2 on day 7 of the treatment; the TNF-alpha content of CK is 40.18% higher than CS-BSP-1 and 33.49% higher than CS-BSP-2.
On day 14, the IL-6 content of CK was 37.02% higher than CS-BSP-1 and 24.32% higher than CS-BSP-2; the TNF-alpha content of CK is 62.08% higher than CS-BSP-1 and 43.28% higher than CS-BSP-2.
From the treatment day 7 to the treatment day 21, the TNF-alpha and IL-6 in CS-BSP-1 and CS-BSP-2 are obviously lower than CK, so that the polysaccharide sponge can reduce the over-expression of inflammatory factors such as TNF-alpha, IL-6 and the like, the wound inflammation period is faster, and the wound healing speed of mice is accelerated. In the wound healing process, angiogenesis is important in tissue formation induced by various growth factors such as Vascular Endothelial Growth Factor (VEGF).
And after CS-BSP-1 and CS-BSP-2 treatment, the VEGF content of CK was 76.69%,65.95% and 87.77% of CS-BSP-1, respectively, from day 7 to day 21; the content of experimental groups is 84.65%,79.56% and 94.78 of CS-BSP-2, which are all obviously higher than CK, so that it is clear that polysaccharide sponge can improve VEGF expression, thereby inducing the formation of wound tissue and leading the wound to heal rapidly.
5. Statistical analysis
Statistical analysis was performed using social science statistical program 22.0 software (SPSS, IBM, USA). Data are expressed as mean ± Standard Deviation (SD) of continuous variables, mean analyzed by independent sample t-test and one-way anova. A value of p <0.05 is considered statistically significant.
6. Conclusion(s)
The bletilla striata polysaccharide is used as a natural high molecular polysaccharide extracted from rare and rare traditional Chinese medicine bletilla striata, and a cross-linking technology is used for successfully preparing the sponge with the network interpenetrating structure. It not only has obvious hemostatic effect, but also can be used as a high-quality medicine carrier. Through the crosslinking of oxalyl chloride and chitosan, not only the mechanical property and microstructure of the bletilla polysaccharide sponge can be better regulated, but also the biological activities of hemostasis, antibiosis and the like of the bletilla polysaccharide sponge can be enhanced. By comparing CS-BSP-1 with CS-BSP-2, the CS-BSP-1 has better hemostatic, antibacterial, cell growth promoting, wound healing promoting effects, etc., which may be the reason for higher content of bletilla striata polysaccharide in CS-BSP-1. The cck-8 experimental results of CS-BSP-1 and CS-BSP-2 sponges show that the sponges have remarkable promotion effect on NIH-3T3 cells, and are also one of the advantages of natural polysaccharide materials.
In sum, the bletilla striata polysaccharide sponge not only has better biocompatibility and good hemostatic, antibacterial and anti-inflammatory capabilities, but also has the effects of promoting cell proliferation and accelerating wound healing, and is beneficial to preventing wound infection in the treatment process. And can be used for treating wound caused by different factors, such as incised wound, scald, etc.
And when the requirements for using different bletilla polysaccharide sponges are different, the bletilla polysaccharide sponges with different physical properties and therapeutic effects are obtained by adjusting the quantity ratio of the bletilla polysaccharide to the chitosan, the concentration of oxalyl chloride, the sequence of feeding reaction and the freeze-drying volume of the bletilla polysaccharide sponges, so that the bletilla polysaccharide sponges are wide in application prospect and have the characteristic of targeted use.
The above examples merely illustrate specific embodiments of the application, which are described in more detail and are not to be construed as limiting the scope of the application. It should be noted that it is possible for a person skilled in the art to make several variants and modifications without departing from the technical idea of the application, which fall within the scope of protection of the application.
Claims (7)
1. A preparation method of a sponge containing rhizoma bletillae polysaccharide is characterized in that,
providing a crosslinking reaction solution containing bletilla striata polysaccharide, chitosan and a crosslinking agent, and separating solid matters in the crosslinking reaction solution after the crosslinking reaction is finished to obtain a crosslinking product of the bletilla striata polysaccharide and the chitosan;
dispersing the crosslinked product in a swelling solvent to obtain a swelling solution, and removing the swelling solvent after the crosslinked product is swelled to obtain the sponge containing bletilla polysaccharide.
2. The method for preparing a sponge containing bletilla striata polysaccharide according to claim 1, wherein the mass concentration ratio of the bletilla striata polysaccharide to the chitosan in the crosslinking reaction solution is 1-3:1-3.
3. The method for preparing a sponge containing bletilla striata polysaccharide according to claim 2, wherein the concentration of the bletilla striata polysaccharide in the crosslinking reaction solution is 10-30 mg/mL, and the concentration of the chitosan is 10-30 mg/mL.
4. The method for preparing a sponge containing bletilla striata polysaccharide according to claim 1, wherein the step of preparing a crosslinking reaction solution comprises the steps of reacting bletilla striata polysaccharide, chitosan and a dissolution solvent to obtain a first reaction solution, and adding the crosslinking agent into the first reaction solution to react to obtain the crosslinking reaction solution.
5. A method of preparing a sponge comprising bletilla striata polysaccharide as claimed in claim 1, wherein the cross-linking reaction is carried out for a period of time of from 15 minutes to 20 minutes.
6. The method for preparing a sponge containing bletilla striata polysaccharide according to claim 1, wherein the concentration of the cross-linked product in the swelling solution is 7mg/mL to 80mg/mL.
7. A method of preparing a sponge comprising bletilla striata polysaccharide as claimed in claim 1, wherein the method of removing swelling solvent comprises freeze drying.
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