CN116726041A - Peony pistil polysaccharide and application thereof in wound healing - Google Patents
Peony pistil polysaccharide and application thereof in wound healing Download PDFInfo
- Publication number
- CN116726041A CN116726041A CN202311007388.3A CN202311007388A CN116726041A CN 116726041 A CN116726041 A CN 116726041A CN 202311007388 A CN202311007388 A CN 202311007388A CN 116726041 A CN116726041 A CN 116726041A
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- Prior art keywords
- polysaccharide
- peony pistil
- peony
- pistil
- solution
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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- D—TEXTILES; PAPER
- D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
- D01D—MECHANICAL METHODS OR APPARATUS IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS
- D01D5/00—Formation of filaments, threads, or the like
- D01D5/0007—Electro-spinning
- D01D5/0015—Electro-spinning characterised by the initial state of the material
- D01D5/003—Electro-spinning characterised by the initial state of the material the material being a polymer solution or dispersion
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention provides a peony pistil polysaccharide and application thereof in wound healing, and belongs to the technical field of polysaccharide application. The extracted peony pistil polysaccharide comprises peony pistil polysaccharide 1 and peony pistil polysaccharide 2; molecular weight of peony pistil polysaccharide 113.16 x 10 3 Da; the monosaccharide composition of the peony pistil polysaccharide 1 is 17.45% Ara,9.14% Rha,34.60% Gal,14.58% Glc,10.74% Man,9.33Glc-UA; the molecular weight of the peony pistil polysaccharide 2 is 19.45 x 10 5 Da; the monosaccharide composition of the peony pistil polysaccharide 2 was 18.55% Ara,9.91% Rha,34.06% Gal,13.01% Glc,9.60% Man,9.19Glc-UA. The peony pistil polysaccharide extracted by the invention can effectively promote wound healing.
Description
Technical Field
The invention belongs to the technical field of polysaccharide application, and particularly relates to a peony pistil polysaccharide and application thereof in wound healing.
Background
Peony, also known as Paeonia ostii, is commonly referred to as Paeonia lactiflora, which is derived from five generations at the earliest, has a long history and is known as "King in flowers". The root, bark, flower, seed and other parts of peony have various nutritional components and utilization values, and have the effects of regulating qi and blood, maintaining liver, relieving cough and the like. The peony pistil has rich nutrition, high contents of carbohydrate, protein, total sugar, mineral elements, vitamins and total flavone, and good and reasonable composition of fatty acid and amino acid, can regulate intestines and stomach, dredge and activate blood, and has various physiological activity functions of reducing blood fat, reducing blood sugar, preventing cardiovascular and cerebrovascular diseases and the like. However, the research on the stamen of peony is mainly focused on the aspects of flavonoid activity and the like, and the research and application on the polysaccharide of the stamen of peony are less.
Wound damage refers to damage or destruction of skin, tissue or organ of a part of the body by external forces or other factors. The severity of the wound injury may vary depending on the size, depth of the wound and the location where it is located. Common wound types include bruises, cuts, lacerations, punctures, pressure wounds, and the like. Common hazards of wound damage include the following: increased risk of infection, resulting in bleeding and hemorrhage, resulting in tissue damage and dysfunction, and severe wound damage can also have mental and psychological impact on the patient. Therefore, how to efficiently achieve rapid healing of wounds is a problem that needs to be solved at present.
Electrospinning is a technique for producing nanoscale continuous fibers by jet spinning a polymer solution or melt under the action of a strong electric field. The nano-scale fiber membrane prepared by the process has the advantages of large specific surface area, high porosity, large active ingredient loading capacity and the like, so that how to enable the electrostatic spinning to exert better effect in wound healing is a problem which needs to be solved at present.
Disclosure of Invention
The invention aims to provide a peony pistil polysaccharide and application thereof in wound healing, thereby providing a novel therapeutic drug for wound healing.
In order to achieve the above purpose, the present invention provides the following technical solutions:
firstly, the invention provides application of peony pistil polysaccharide in preparing a medicament for promoting wound healing, wherein the peony pistil polysaccharide comprises peony pistil polysaccharide 1 and peony pistil polysaccharide 2;
the molecular weight of the peony pistil polysaccharide 1 is 13.16 x 10 3 Da；
The monosaccharide composition of the peony pistil polysaccharide 1 is 17.45% Ara,9.14% Rha,34.60% Gal,14.58% Glc,10.74% Man,9.33Glc-UA;
the molecular weight of the peony pistil polysaccharide 2 is 19.45 x 10 5 Da；
The monosaccharide composition of the peony pistil polysaccharide 2 is 18.55% Ara,9.91% Rha,34.06% Gal,13.01% Glc,9.60% Man,9.19Glc-UA.
Preferably, the medicament reduces oxidative damage caused by DPPH radicals, OH radicals and ABTS +. Radicals.
Preferably, the agent reduces the inflammatory response caused by increased levels of NO, TNF- α and IL-6.
Preferably, the drug promotes migration of skin fibroblasts.
Preferably, the preparation method of the peony pistil polysaccharide comprises the following steps:
(1) Reflux-extracting the stamen of Paeonia suffruticosa with 95% ethanol for 3 times to remove oil;
(2) Adding 20 times of distilled water in water bath at 80deg.C for 3 hr, removing supernatant, repeating the operation for 2 times again, and mixing the 3 supernatants to obtain flos moutan polysaccharide extractive solution;
(3) Concentrating the peony pistil polysaccharide into 20% of the original volume by rotary evaporation, adding 4 times of ethanol solution, standing at 4 ℃ for filtering, centrifuging, and taking precipitate to obtain peony pistil crude polysaccharide;
(4) Preparing the peony pistil crude polysaccharide into a peony pistil crude polysaccharide solution with the concentration of 0.01g/mL by using distilled water, and mixing the peony pistil crude polysaccharide solution with chloroform: n-butanol=4:1 volume ratio to configure Sevag reagent;
(5) Mixing the peony pistil crude polysaccharide solution and the Sevag reagent according to the volume ratio of 5:1, and repeating the operation for 8 times to remove protein;
(6) The crude polysaccharide solution of the peony pistil with protein removed and AB-8 macroporous resin are mixed according to the following ratio of 1:4, adding the mixture into a constant temperature oscillator for oscillating for 16 hours to remove the pigment of the polysaccharide;
(7) Filtering with 10kDa filter membrane, removing filtrate, and passing the retentate through 100kDa filter membrane to obtain filtrate which is 10-100kDa peony pistil polysaccharide solution 1 and retentate which is more than 100kDa peony pistil polysaccharide solution 2;
(8) And (3) freeze-drying the peony pistil polysaccharide solution 1 and the peony pistil polysaccharide solution 2 to obtain the peony pistil polysaccharide 1 and the peony pistil polysaccharide 2.
Secondly, the invention provides application of the peony pistil polysaccharide in preparing a skin fibroblast migration promoter, wherein the peony pistil polysaccharide comprises peony pistil polysaccharide 1 and peony pistil polysaccharide 2;
the molecular weight of the peony pistil polysaccharide 1 is 13.16 x 10 3 Da；
The monosaccharide composition of the peony pistil polysaccharide 1 is 17.45% Ara,9.14% Rha,34.60% Gal,14.58% Glc,10.74% Man,9.33Glc-UA;
the molecular weight of the peony pistil polysaccharide 2 is 19.45 x 10 5 Da；
The monosaccharide composition of the peony pistil polysaccharide 2 is 18.55% Ara,9.91% Rha,34.06% Gal,13.01% Glc,9.60% Man,9.19Glc-UA.
Secondly, the invention provides application of the peony pistil polysaccharide in preparing a dressing for promoting wound healing, which is characterized in that the core active ingredient of the dressing is the peony pistil polysaccharide;
the peony pistil polysaccharide comprises peony pistil polysaccharide 1 and peony pistil polysaccharide 2;
the molecular weight of the peony pistil polysaccharide 1 is 13.16 x 10 3 Da；
The monosaccharide composition of the peony pistil polysaccharide 1 is 17.45% Ara,9.14% Rha,34.60% Gal,14.58% Glc,10.74% Man,9.33Glc-UA;
the molecular weight of the peony pistil polysaccharide 2 is 19.45 x 10 5 Da；
The monosaccharide composition of the peony pistil polysaccharide 2 is 18.55% Ara,9.91% Rha,34.06% Gal,13.01% Glc,9.60% Man,9.19Glc-UA.
Secondly, the invention provides a composite nanofiber membrane for wound healing, wherein the core active ingredients of the composite nanofiber membrane are chitosan and peony pistil polysaccharide 1;
the molecular weight of the peony pistil polysaccharide 1 is 13.16 x 103Da;
the monosaccharide composition of the peony pistil polysaccharide 1 is 17.45% Ara,9.14% Rha,34.60% Gal,14.58% Glc,10.74% Man,9.33Glc-UA.
Preferably, the preparation method of the composite nanofiber membrane comprises the following steps:
(1) Respectively dissolving 25% gelatin and 9-10% chitosan in 70% acetic acid to obtain gelatin solution and chitosan solution;
(2) Mixing gelatin solution and chitosan solution according to the volume ratio of 4:1 to obtain mixed solution A;
(3) Adding 1% of peony pistil polysaccharide into the mixed solution A to obtain a mixed solution B;
(4) And carrying out electrostatic spinning to obtain the composite nanofiber membrane.
Preferably, the composite nanofiber membrane enhances wound healing rate, inhibits wound inflammatory response, and promotes wound neovascular number.
The invention has the beneficial effects that:
the peony pistil polysaccharide with different molecular weights is obtained through ultrafiltration, and the peony pistil polysaccharide has good antioxidant capacity through free radical scavenging experiments; in vitro cell experiments show that the peony pistil polysaccharide can inhibit the expression of inflammatory factors TNF-alpha and IL-6 and can accelerate the migration speed of cells; in vivo animal experiments show that the peony pistil polysaccharide composite nanofiber membrane can be cooperated with chitosan to remarkably accelerate wound healing, promote granulation tissue and capillary angiogenesis around the wound, and reduce the levels of inflammatory factors NO, TNF-alpha and IL-6 in skin cells at the wound healing position. Thus, the peony pistil polysaccharide prepared by the invention can be used for preparing more effective wound healing medicines or dressings singly or in combination with chitosan.
Drawings
FIG. 1 is a molecular weight map of the extracted peony pistil polysaccharide of the present invention;
FIG. 2 is a graph showing the results of monosaccharide composition of the extracted peony pistil polysaccharide of the present invention;
FIG. 3 is an infrared absorption spectrum of the extracted peony pistil polysaccharide of the present invention;
FIG. 4 is a SEM result of the extracted peony pistil polysaccharide of the present invention;
FIG. 5 is a graph showing the results of the antioxidant capacity of the extracted peony pistil polysaccharide of the present invention;
FIG. 6 is a graph showing the effect of the extracted peony pistil polysaccharide on NO, TNF-alpha and IL-6 cytokine secretion;
FIG. 7 is a graph showing the effect of the peony pistil polysaccharide of the present invention on cell scratch healing;
FIG. 8 is an SEM of the peony pistil polysaccharide composite nanofiber membrane prepared according to the present invention;
FIG. 9 is a graph showing the effect of the peony pistil polysaccharide composite nanofiber membrane prepared by the invention on wound healing of mice;
FIG. 10 is a graph showing the effect of the complex nanofibrous membrane of peony pistil polysaccharide on NO, TNF-alpha and IL-6 cytokine secretion at the wound healing site of mice;
FIG. 11 is a graph showing the effect of the peony pistil polysaccharide composite nanofiber membrane prepared by the invention on skin tissues at the wound healing site of mice.
Detailed Description
EXAMPLE 1 preparation of peony pistil polysaccharide
(1) Reflux-extracting 500g of peony pistil with 3L of 95% ethanol for 3 times to remove oil;
(2) Adding 10L distilled water in water bath at 80deg.C for 3 hr, removing supernatant, repeating the operation for 2 times again, and mixing the 3 supernatants to obtain flos moutan polysaccharide extractive solution;
(3) Concentrating the peony pistil polysaccharide into 20% of the original volume by rotary evaporation, adding 4 times of ethanol solution, standing at 4 ℃ for filtering, centrifuging and taking precipitate to obtain the peony pistil crude polysaccharide;
(4) The crude polysaccharide of the peony pistil is prepared into 500mL of crude polysaccharide solution of the peony pistil with the concentration of 0.01g/mL by using distilled water, and chloroform is used for preparing the crude polysaccharide solution: n-butanol=4:1 volume ratio to configure Sevag reagent;
(5) Mixing the peony pistil crude polysaccharide solution and the Sevag reagent according to the volume ratio of 5:1, and repeating the operation for 8 times to remove protein;
(6) The crude polysaccharide solution of the peony pistil with protein removed and AB-8 macroporous resin are mixed according to the following ratio of 1:4, adding the mixture into a constant temperature oscillator for oscillating for 16 hours to remove the pigment of the polysaccharide;
(7) Filtering with 10kDa filter membrane, removing filtrate, and passing the retentate through 100kDa filter membrane to obtain filtrate which is the peony pistil polysaccharide solution 1 with molecular weight of 10-100kDa and retentate which is the peony pistil polysaccharide solution 2 with molecular weight of more than 100 kDa.
(8) And (3) freeze-drying the peony pistil polysaccharide solution 1 and the peony pistil polysaccharide solution 2 to obtain the peony pistil polysaccharide 1 and the peony pistil polysaccharide 2.
EXAMPLE 2 high Performance gel permeation chromatography determination of polysaccharide molecular weight and purity
The molecular weight and purity of the peony pistil polysaccharide were measured by high performance gel permeation chromatography, and the results are shown in figure 1.
As can be seen from FIG. 1, both the peony pistil polysaccharides (PN 1 and PN 2) of two molecular weights have a single symmetrical peak, indicating that the component is pure polysaccharide and has a uniform molecular weight. Obtaining a correction curve by taking the molecular weight of dextran as a control, and calculating to obtain the molecular weight of PN1 of 13.16 x 10 3 Da, PN2 molecular weight 19.45 x 10 5 Da, withThe ultrafiltration results were consistent.
EXAMPLE 3 detection of polysaccharide composition by ion chromatography
Fucose, murine Li Tang, arabinose, galactose, glucose, xylose, mannose, fructose, galacturonic acid glucose, glucuronic acid and mannuronic acid were used as monosaccharide standards.
(1) Sample 5 mg was weighed and 1mL of 2M trifluoroacetic acid (TFA, 3 mol/L) was added and heated at 121℃for 3 hours;
(2) Then, it was dried under a nitrogen stream. Subsequently, 50mL of water was added for vortex mixing;
(3) After filtration, the supernatants were analyzed using HPIC equipped with Dionex ™ CarboPac ™ PA20 (150 x 3.0mm,10 μm) liquid chromatography column and electrochemical detector;
(4) 0.1M NaOH, 0.1M NaOH and 0.2M NaAc were used as mobile phases, the flow rate was set to 0.5 mL/min, the sample injection amount was 5uL, and the column temperature was 30 ℃;
(5) The monosaccharide standard solution is treated by the same method, and the molar ratio is calculated according to the molar mass of the monosaccharide;
(6) Monosaccharide composition of the peony-pistil polysaccharide samples of different molecular weights was analyzed by HPIC, the composition was determined using IC and by matching the retention time in the chromatographic profile with the retention time of the monosaccharide standard, and the results are shown in table 1 and fig. 2.
TABLE 1 monosaccharide composition of peony pistil polysaccharides of different molecular weights
| Group | Ara | Rha | Gal | Glc | Man | Glc-UA |
| PNS1 | 17.45 | 9.14 | 34.60 | 14.58 | 10.74 | 9.33 |
| PNS2 | 18.55 | 9.91 | 34.06 | 13.01 | 9.60 | 9.19 |
As can be seen from Table 1 and FIG. 2, the peony pistil polysaccharide consists of arabinose, rhamnose, galactose, glucose, mannose and glucuronic acid, the monosaccharide composition ratio of the two polysaccharides is not greatly different, wherein the highest molar ratio is galactose, and the molar ratio reaches 34%.
Example 4 determination of organic functional groups of polysaccharides by Fourier transform Infrared Spectroscopy
The organic functional group of the peony pistil polysaccharide was measured by fourier transform infrared spectroscopy, and the results are shown in fig. 3.
As can be seen from FIG. 3, the FT-IR spectrum shows 500-4000cm -1 The absorption peak between 3393 and 3393 cm -1 A broad and strong absorption peak appears nearby, indicating the presence of-OH; at 2935 cm -1 Weak absorption peak appears at the position, which is caused by C-H asymmetric stretching vibrationThe above 2 absorption peaks are characteristic absorption peaks of the saccharide compound. At 1620 cm -1 Sum 1403 cm -1 The nearby characteristic absorption peaks are respectively caused by C=O asymmetry and C-H angle-changing stretching vibration, which shows that the polysaccharide contains uronic acid and is an acidic polysaccharide, and the result is consistent with the result of the content of uronic acid measured before; at 1651 cm -1 And 1555 cm -1 The absence of N-H absorption peaks in the vicinity indicates that the polysaccharide protein content is lower and the purification is better. At 1112cm -1 The vicinity may be caused by a corner-changing vibration peak of C-O-H, O-H, and it is presumed to be pyranose. At 840 cm -1 The weak absorption peak indicates that the polysaccharide contains a small amount of alpha-type glycosidic bonds. The above results show that the peony pistil polysaccharide may be an acid polysaccharide of alpha-configuration pyranose, and has a typical polysaccharide structure, which indicates that the extracts obtained by the experimental method are all polysaccharide substances.
Example 5 detection of polysaccharide morphology
The morphology of the peony pistil polysaccharide was observed by a scanning electron microscope, and the result is shown in fig. 4.
As can be seen from fig. 4, PN1 was an irregular block structure at 600 x, and bonded convex particles were observed under a 6000 x mirror. PN2 is a filament-like and small-fragment structure at 600X, with irregular clusters of clusters, and more holes are observed at 6000X-fold mirrors, possibly due to the mutual repulsion of intermolecular attractive forces.
Example 6 detection of antioxidant Property of the peony pistil polysaccharide extracted by the invention
Oxidative damage can lead to oxidative stress and death of cells, and has negative effect on wound healing of patients, so that the invention firstly detects the antioxidant capacity of the peony pistil polysaccharide.
(1) Antioxidant capacity of the peony pistil polysaccharide was determined by DPPH free radical scavenging assay,. OH free radical scavenging assay and ABTS +. Free radical scavenging assay:
DPPH radical scavenging experiment:
(1) Diluting polysaccharide samples with different concentrations by distilled water, wherein the sample group takes ascorbic acid (VC) as a positive control and absolute ethyl alcohol as a blank control;
(2) The absorbance was measured at 517 nm by mixing 0.1 mM DPPH solution with the same volume of the sample solution of the curdlan in a 96-well plate.
The calculation formula of the DPPH free radical scavenging activity is DPPH free radical scavenging rate (%) = [1- (A) 1 -A 0 )]/A 2 ×100%, A 0 Absorbance without sample, A 1 Absorbance of the added sample A 2 -absorbance of distilled water;
ABTS +. Free radical scavenging experiments:
(1) 7.4 mmol/l ABTS and 2.6 mmol/l K 2 S 2 O 8 Mixing at volume of 1:1, storing at room temperature in dark place for 12 h, diluting with absolute ethanol at 734-nm for 40-50 times, and measuring absorbance value of 0.7+ -0.02;
(2) Mixing the peony pistil polysaccharide samples with different concentrations (0, 0.625, 1.25, 2.5, 5 and 10 mg/ml) with the ABTS working solution according to the volume of 1:4, shaking uniformly for 10 s, and standing in the dark for 6 min:
(3) The absorbance of the samples was measured on 96-well plates at 734 nm with VC and distilled water as positive and blank controls, respectively. The calculation formula is ABTS clearance (%) = (1-A) 1 /A 0 ) × 100%；A 0 Absorbance without sample, A 1 -absorbance of the added sample;
OH radical scavenging experiments:
(1) 1 mmol/l ferrous sulfate solution (FeSO) was added to the peony pistil polysaccharide samples 4 ) 9 mmol/l hydrogen peroxide solution (H) 2 O 2 ) And 3 mmol/l salicylic acid-ethanol solution, respectively at different concentrations (0, 0.625, 1.25, 2.5, 5, 10 mg/ml);
(2) Then reacting in a constant temperature water bath at 37 ℃ for 30 min, cooling to room temperature, and measuring absorbance at 510 and nm;
(3) VC and distilled water are used as positive control and blank control respectively, and the clearance calculation formula is that OH clearance (%) = [1- (A) 1 -A 0 )]/A 2 ×100%；A 0 Absorbance without sample, A 1 Absorbance of the added sample A 2 Absorbance of distilled water.
The results of the above experiments are shown in FIG. 5.
As can be seen from FIG. 5, the DPPH radical scavengers,. OH scavengers and ABTS+. Scavengers of PN1 and PN2 all increase with increasing mass concentration, although less than the equivalent concentration of VC, but all have good antioxidant activity, wherein the IC50 of the PN 1. OH, ABTS+ & DPPH radicals are 3.69, 3.75, 0.45 μg/mL respectively, and are slightly lower than the PN2 IC50, indicating that PN1 has antioxidant activity slightly better than PN2, which may be related to molecular weight, number of active groups and glycosidic linkages in the polysaccharide, and possibly monosaccharide composition (glucuronic acid, galactose and glucose).
EXAMPLE 7 detection of the Effect of the extracted peony pistil polysaccharide of the present invention on the level of inflammatory factor in lipopolysaccharide-induced cells
Excessive or prolonged inflammatory responses may negatively impact wound healing. Excessive inflammation can lead to pain, swelling, and tissue damage, and can delay the wound healing process. Long-standing inflammation may also lead to fibrosis, scar tissue generation, or affect neovascularization. Thus, the effect of the peony pistil polysaccharide on inflammatory response will be examined in the following.
(1) RAW264.7 cells with good growth state are inoculated into 24-well plates to make the cell density of each well be 4 multiplied by 10 5 Culturing in an incubator at 37 ℃ with CO2 concentration of 5% for 24h, adding the peony pistil polysaccharide solution with different concentrations into a sample group, continuously culturing for 4 hours, adding 1 mug/mL of LPS, and adding the same dose of LPS into a model group; normal groups of cells did not apply any treatment. After further culturing 18. 18 h, the cell supernatants of each group were collected and assayed for NO and IL-6, TNF- α concentrations by ELISA.
Example 8 detection of the Effect of the peony pistil polysaccharide extracted according to the present invention on fibroblast L929 cell migration
(1) L929 cells with good growth state were inoculated into a labeled 6-well plate with a density of 1×10 5 Individual cells/wells;
(2) Scribing the well plate with a sterile ruler and 200 μl tips of micropipettes along the diameter of the well plate when the cells account for 80% of the well plate;
(3) Gently washing the separated cells with PBS solution for 3 times, adding 5ml of serum-free culture medium into blank group, adding culture medium containing 1% of fetal bovine serum in equal amount into control group, and adding culture medium containing 1% of fetal bovine serum +250ug/ml of peony pistil polysaccharide into drug group;
(4) After 24h incubation, each scratch area was observed under an inverted microscope and the cell scratch healing rate was calculated using Image J software, the results are shown in fig. 7.
As can be seen from fig. 7, the healing rate of cell scratches was significantly increased after both PN1 and PN2 actions of 12 h and 24 hours, and both PN1 and PN2 significantly stimulated closure of cell scratches, as compared to the control group.
Example 9 preparation of composite nanofiber membranes
(1) Dissolving 25% (w/v) gelatin and 10% (w/v) chitosan in 70% acetic acid respectively, mixing gelatin and chitosan at a ratio of 4:1 (v/v), and directly preparing into smooth fiber film by electrostatic spinning to obtain GL/CS of a control group;
(2) 25% (w/v) gelatin and 9% (w/v) chitosan are respectively dissolved in 70% acetic acid, gelatin and chitosan are mixed in a ratio of 4:1 (v/v), 1% (w/v) of peony pistil polysaccharide PN1 with better antioxidant and anti-inflammatory effects is added for mixing, and smooth fiber films are directly manufactured through electrostatic spinning, so that high-dose groups of GL/CS/PNH are obtained;
(3) 25% (w/v) gelatin and 9.5% (w/v) chitosan are respectively dissolved in 70% acetic acid, gelatin and chitosan are mixed in a ratio of 4:1 (v/v), 0.5% (w/v) PN1 is added for mixing, and smooth fiber films are directly manufactured through electrostatic spinning, so that GL/CS/PNH of a low dose group is obtained.
An SEM of the polysaccharide composite nanofiber membrane is shown in fig. 8. It can be seen that the fibrous membranes of different proportions exhibit different fibrous states. The diameter of the fibrous membrane after the addition of the peony pistil polysaccharide is increased from 60 nm to about 80 nm. Compared with the low concentration, the high-concentration peony pistil polysaccharide composite fiber film has good continuity, no yarn breakage phenomenon and no obvious nodule.
Example 10 detection of the effects of fibrous Membrane on wound healing in ICR mice
(1) 24 SPF grade ICR mice are randomly divided into 4 groups, 6 groups each, each half of which has a weight of 22+/-2 g, and the groups comprise a normal group and a control group, the back hair is shaved after anesthesia and disinfected, 1cm diameter wounds are cut out by a puncher, a blank group is not treated, the control group covers GL/CS films, and an experimental group covers GL/CS/PNH and GL/CS/PNL films respectively;
(2) Photographs of wounds were observed on days 0, 3, 7, and 12, and wound healing rates were calculated using Image J software, and the results are shown in fig. 9.
As shown in fig. 9, the control and experimental mice had faster wound healing rates compared to the blank group, with significantly increased wound healing rates at interventions 3, 7 and 12 d; compared with a control group, the wound healing speed of mice in the experimental group is higher, the wound healing rate is obviously increased during the intervention of 3 rd, 7 th and 12d, which indicates that the electrostatic spinning membrane can accelerate the wound healing, and the synergistic effect of chitosan and peony pistil polysaccharide enables the wound healing to be faster.
EXAMPLE 11 Effect of polysaccharide composite nanofiber membrane on inflammation at wound healing sites
(1) The mice treated in example 10 were blood collected from the orbital sockets into sterile tubes at days 3, 7, 12, centrifuged at 3000r/min and 4 ℃ for 10min to extract supernatant serum and stored at-80 ℃.
(2) The amounts of NO and IL-6, TNF- α in serum were measured using an enzyme-linked immunosorbent assay (ELISA) kit from Shanghai enzyme-linked biotechnology Co., ltd, according to the manufacturer's instructions, and the results are shown in FIG. 10.
As can be seen from FIG. 10, the mice treated with the fiber membranes effectively reduced the levels of NO and IL-6, TNF- α in the serum. The fibrous membrane with the addition of the peony-pistil polysaccharide inhibited the release of NO and IL-6, TNF-alpha more effectively than the control. The result shows that the polysaccharide composite nanofiber membrane can inhibit the inflammatory reaction of wounds, and the synergistic effect of chitosan and the peony pistil polysaccharide can remarkably inhibit the inflammatory reaction.
Example 12 pathological observations of the effects of polysaccharide composite nanofiber membranes on wound healing tissue
(1) Mice were anesthetized at 7 and 12d post-intervention, wounds and their peripheral tissues were harvested, and HE stained for wound histopathological changes, the results of which are shown in fig. 11.
As shown in fig. 11, all groups developed epithelial hyperplasia with concomitant inflammatory infiltrate and wound contraction. However, the fibrous membrane treated wounds healed more heally histologically, the inflammatory infiltrate was significantly reduced, the epidermis and dermis became more mature, and the wound had a greater number of newly formed blood vessels than the blank group. Compared with the control group, the synergistic effect of chitosan and the peony pistil polysaccharide obviously reduces inflammatory infiltration, the epidermis and the dermis become more mature, and the number of newly formed blood vessels of the wound is more.
Claims (10)
1. The application of the peony pistil polysaccharide in preparing the medicine for promoting the wound healing is characterized in that the peony pistil polysaccharide comprises peony pistil polysaccharide 1 and peony pistil polysaccharide 2;
the molecular weight of the peony pistil polysaccharide 1 is 13.16 x 10 3 Da；
The monosaccharide composition of the peony pistil polysaccharide 1 is 17.45% Ara,9.14% Rha,34.60% Gal,14.58% Glc,10.74% Man,9.33Glc-UA;
the molecular weight of the peony pistil polysaccharide 2 is 19.45 x 10 5 Da；
The monosaccharide composition of the peony pistil polysaccharide 2 is 18.55% Ara,9.91% Rha,34.06% Gal,13.01% Glc,9.60% Man,9.19Glc-UA.
2. The use according to claim 1, wherein the medicament reduces oxidative damage caused by DPPH radicals, OH radicals and ABTS +. Radicals.
3. The use according to claim 1, wherein the medicament reduces inflammatory responses caused by increased levels of NO, TNF- α and IL-6.
4. The use according to claim 1, wherein the medicament promotes migration of skin fibroblasts.
5. The use according to claim 1, wherein the process for the preparation of the peony pistil polysaccharide comprises the steps of:
(1) Reflux-extracting the stamen of Paeonia suffruticosa with 95% ethanol for 3 times to remove oil;
(2) Adding 20 times of distilled water in water bath at 80deg.C for 3 hr, removing supernatant, repeating the operation for 2 times again, and mixing the 3 supernatants to obtain flos moutan polysaccharide extractive solution;
(3) Concentrating the peony pistil polysaccharide into 20% of the original volume by rotary evaporation, adding 4 times of ethanol solution, standing at 4 ℃ for filtering, centrifuging, and taking precipitate to obtain peony pistil crude polysaccharide;
(4) Preparing the peony pistil crude polysaccharide into a peony pistil crude polysaccharide solution with the concentration of 0.01g/mL by using distilled water, and mixing the peony pistil crude polysaccharide solution with chloroform: n-butanol=4:1 volume ratio to configure Sevag reagent;
(5) Mixing the peony pistil crude polysaccharide solution and the Sevag reagent according to the volume ratio of 5:1, and repeating the operation for 8 times to remove protein;
(6) The crude polysaccharide solution of the peony pistil with protein removed and AB-8 macroporous resin are mixed according to the following ratio of 1:4, adding the mixture into a constant temperature oscillator for oscillating for 16 hours to remove the pigment of the polysaccharide;
(7) Filtering with 10kDa filter membrane, removing filtrate, and passing the retentate through 100kDa filter membrane to obtain filtrate which is 10-100kDa peony pistil polysaccharide solution 1 and retentate which is more than 100kDa peony pistil polysaccharide solution 2;
(8) And (3) freeze-drying the peony pistil polysaccharide solution 1 and the peony pistil polysaccharide solution 2 to obtain the peony pistil polysaccharide 1 and the peony pistil polysaccharide 2.
6. The application of the peony pistil polysaccharide in the preparation of the skin fibroblast migration promoter is characterized in that the peony pistil polysaccharide comprises peony pistil polysaccharide 1 and peony pistil polysaccharide 2;
the molecular weight of the peony pistil polysaccharide 1 is 13.16 x 10 3 Da；
The monosaccharide composition of the peony pistil polysaccharide 1 is 17.45% Ara,9.14% Rha,34.60% Gal,14.58% Glc,10.74% Man,9.33Glc-UA;
the molecular weight of the peony pistil polysaccharide 2 is 19.45 x 10 5 Da；
The monosaccharide composition of the peony pistil polysaccharide 2 is 18.55% Ara,9.91% Rha,34.06% Gal,13.01% Glc,9.60% Man,9.19Glc-UA.
7. The application of the peony pistil polysaccharide in preparing the dressing for promoting the wound healing is characterized in that the core active ingredient of the dressing is the peony pistil polysaccharide;
the peony pistil polysaccharide comprises peony pistil polysaccharide 1 and peony pistil polysaccharide 2;
the molecular weight of the peony pistil polysaccharide 1 is 13.16 x 10 3 Da；
The monosaccharide composition of the peony pistil polysaccharide 1 is 17.45% Ara,9.14% Rha,34.60% Gal,14.58% Glc,10.74% Man,9.33Glc-UA;
the molecular weight of the peony pistil polysaccharide 2 is 19.45 x 105Da;
the monosaccharide composition of the peony pistil polysaccharide 2 is 18.55% Ara,9.91% Rha,34.06% Gal,13.01% Glc,9.60% Man,9.19Glc-UA.
8. The composite nanofiber membrane for wound healing is characterized in that the core active ingredients of the composite nanofiber membrane are chitosan and peony pistil polysaccharide 1;
the molecular weight of the peony pistil polysaccharide 1 is 13.16 x 10 3 Da；
The monosaccharide composition of the peony pistil polysaccharide 1 is 17.45% Ara,9.14% Rha,34.60% Gal,14.58% Glc,10.74% Man,9.33Glc-UA.
9. The composite nanofiber membrane of claim 8, wherein the method of preparing the composite nanofiber membrane comprises the steps of:
(1) Respectively dissolving 25% gelatin and 9-10% chitosan in 70% acetic acid to obtain gelatin solution and chitosan solution;
(2) Mixing gelatin solution and chitosan solution according to the volume ratio of 4:1 to obtain mixed solution A;
(3) Adding 1% of peony pistil polysaccharide into the mixed solution A to obtain a mixed solution B;
(4) And carrying out electrostatic spinning to obtain the composite nanofiber membrane.
10. The composite nanofiber membrane of claim 9, wherein the composite nanofiber membrane increases wound healing rate, inhibits wound inflammatory response, and promotes wound neovascular number.
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