CN115837032B

CN115837032B - Preparation for shaping, beautifying and repairing

Info

Publication number: CN115837032B
Application number: CN202310146610.1A
Authority: CN
Inventors: 任珊珊; 季中蕾; 谭慎兴; 刘媛媛
Original assignee: Affiliated Hospital of Weifang Medical University
Current assignee: Affiliated Hospital of Weifang Medical University
Priority date: 2023-02-22
Filing date: 2023-02-22
Publication date: 2023-05-12
Anticipated expiration: 2043-02-22
Also published as: CN115837032A

Abstract

The invention provides a preparation for shaping, beautifying and repairing, and belongs to the technical field of wound repair. The core active ingredient of the preparation is duckweed polysaccharide prepared by the invention, the duckweed polysaccharide has the function of promoting the repair of skin injury, and the repair of the injury can be accelerated by regulating the proliferation and migration of skin fibroblasts and the expression of collagen and vascular endothelial growth factor. In addition, the duckweed polysaccharide provided by the invention has excellent effect of promoting the expression of collagen genes, so that the effect of repairing scars can be achieved, and the duckweed polysaccharide is more beneficial to wound repair after plastic and cosmetology.

Description

Preparation for shaping, beautifying and repairing

Technical Field

The invention belongs to the technical field of wound repair, and particularly relates to a preparation for shaping, beautifying and repairing.

Background

The cosmetic process often involves debridement suturing, where repair of the wound of the patient after suturing and the patient's appearance have a close impact. Poor wound repair can affect the appearance and confidence of the patient, and in severe cases, the quality of life of the patient.

Skin repair is a very complex biological process that can be generally divided into different phases of inflammatory response, cell proliferation, neovascularization, extracellular matrix synthesis, collagen deposition, re-epithelialization and tissue remodeling. Promotion of skin injury repair by drugs is a major means of clinical skin injury repair treatment, however, although the current clinical drug categories are numerous, the therapeutic effect of most drugs is not ideal. Therefore, the search for new skin injury therapeutic drugs remains an urgent need in the art.

Disclosure of Invention

The invention aims to provide a repairing agent capable of effectively accelerating skin injury, so that the repairing agent is used for rapid wound repair of plastic and cosmetic patients.

To achieve the above object, in a first aspect, the present invention provides an application of duckweed polysaccharide in preparing a wound repair agent, the preparation method of the duckweed polysaccharide comprising the steps of:

(1) Pulverizing dried herba Spirodelae, and sieving with 50 mesh sieve to obtain herba Spirodelae powder;

(2) Weighing the duckweed powder, adding distilled water according to the mass ratio of 1:20, stirring uniformly, and heating at 100 ℃ for extraction for 6 hours;

(3) Filtering with gauze, centrifuging the filtrate in a centrifuge at 8000r/min for 15min to obtain herba Spirodelae crude polysaccharide extractive solution;

(4) Concentrating the duckweed crude polysaccharide extract under reduced pressure to 20% of the original volume to obtain a duckweed crude polysaccharide concentrate;

(5) Adding 4 times of 95% ethanol into the duckweed crude polysaccharide concentrate, precipitating with ethanol, centrifuging at 8000r/min in a centrifuge for 15min to remove supernatant, and freeze-drying the precipitate to obtain duckweed crude polysaccharide;

(6) Preparing the duckweed crude polysaccharide into a 5% duckweed crude polysaccharide solution, removing protein by using a Sevag method, and repeating for 3 times to obtain a duckweed polysaccharide solution;

(7) And (3) passing the duckweed polysaccharide solution through an ultrafiltration membrane with the pore diameter of 30kDa to obtain duckweed polysaccharide with the molecular weight of less than 30 kDa.

Preferably, the repair agent increases proliferation of human skin fibroblasts; the restorative agent enhances migration of human skin fibroblasts.

Preferably, the repair agent increases mRNA expression of Collagen I and Collagen III in human skin fibroblasts.

Preferably, the repair agent increases vascular endothelial growth factor protein expression in human skin fibroblasts.

In a second aspect, the present invention provides the use of duckweed polysaccharide in the preparation of a human skin fibroblast proliferation promoter.

In a third aspect, the present invention provides the use of duckweed polysaccharide in the preparation of a human skin fibroblast migration promoter.

In a fourth aspect, the invention provides the use of duckweed polysaccharide in the preparation of mRNA expression promoters of Collagen I and Collagen III in human skin fibroblasts.

In a fifth aspect, the present invention provides the use of duckweed polysaccharide in the preparation of vascular endothelial growth factor protein expression promoter in human skin fibroblasts.

Preferably, the preparation method of the duckweed polysaccharide comprises the following steps:

(2) Weighing duckweed powder, adding distilled water according to the mass ratio of 1:20, stirring uniformly, and heating and extracting at 100 ℃ for 6 hours;

(6) Preparing 5% duckweed crude polysaccharide solution from duckweed crude polysaccharide, removing protein by using Sevag method, repeating for 3 times to obtain duckweed polysaccharide solution;

(7) The duckweed polysaccharide solution is subjected to an ultrafiltration membrane with the pore diameter of 30kDa, so as to obtain duckweed polysaccharide with the molecular weight of less than 30 kDa;

the promoter is used in proliferation, migration and collagen expression studies of human skin fibroblasts in experimental studies.

The invention has the beneficial effects that:

the duckweed polysaccharide provided by the invention has the function of promoting the repair of skin injury, and can accelerate the repair of injury by regulating the proliferation and migration of human skin fibroblasts and the expression of collagen and vascular endothelial growth factor.

Secondly, the duckweed polysaccharide provided by the invention has excellent effect of promoting the expression of collagen genes, so that the effect of repairing scars can be achieved, and the wound repair after plastic and cosmetology is more facilitated.

Drawings

FIG. 1 shows the results of detection of human skin fibroblast proliferation in control and treatment groups;

FIG. 2 is a graph showing the results of detection of human skin fibroblast migration in control and treatment groups;

FIG. 3 is a statistical result of human skin fibroblast migration for the control and treatment groups;

FIG. 4 shows the relative expression levels of mRNA of Collagen I in the control and treatment groups;

FIG. 5 shows the relative mRNA expression levels of Collagen III in the control and treatment groups;

FIG. 6 shows the protein expression of VEGF-A in control and treatment groups;

FIG. 7 is a graph showing skin repair in mice in the control and treatment groups;

* Represents P < 0.05, P < 0.01, P < 0.001.

Detailed Description

Example 1

In this example, duckweed polysaccharide was prepared according to the following procedure:

1. pulverizing dried herba Spirodelae, sieving with 50 mesh sieve to obtain herba Spirodelae powder;

2. weighing 50g of duckweed powder, adding distilled water according to the mass ratio of 1:20, stirring uniformly, heating to 100 ℃ and leaching for 6 hours;

3. filtering with gauze, centrifuging the filtrate in a centrifuge at 8000r/min for 15min to obtain herba Spirodelae crude polysaccharide extractive solution;

4. concentrating the duckweed crude polysaccharide extract under reduced pressure to 20% of the original volume;

5. adding 4 times of 95% ethanol, precipitating with ethanol for 24 hr, centrifuging at 8000r/min for 15min, removing supernatant, and lyophilizing to obtain herba Spirodelae crude polysaccharide;

6. preparing 5% duckweed crude polysaccharide solution from duckweed crude polysaccharide, removing protein by using Sevag method, repeating for 3 times to obtain duckweed polysaccharide solution;

7. and (3) passing the duckweed polysaccharide solution through a filter membrane with the pore diameter of 30kDa to obtain the duckweed polysaccharide with the molecular weight of less than 30 kDa.

Example 2

In this example, the promoting effect of duckweed polysaccharide on human skin fibroblast proliferation was examined as follows:

1. human skin fibroblasts (HSF cells) were prepared as a Cell suspension and inoculated in 96-well plates, and after the cells were completely adherent, were replaced with medium containing 10mg/mL duckweed polysaccharide (treatment group) or no duckweed polysaccharide (control group);

2. at incubation times 24h,48h,72h, respectively, the absorbance at 450nm was detected by adding 10. Mu.L of CCK-8.

The results are shown in FIG. 1, and it can be seen that the cells of the treated group proliferated faster than the control group. The results demonstrate that duckweed polysaccharide can promote proliferation of human skin fibroblasts.

Example 3

In this example, the promoting effect of duckweed polysaccharide on human skin fibroblast migration was examined as follows:

1. HSF cells were seeded into 6-well plates, and when cells in the Cell well dish were substantially full, the adherent Cell layer was gently slid in the vertical direction of the culture well using a 200 μl gun head, and the detached cells were washed off using PBS;

2. changing to culture medium containing 10mg/mL duckweed polysaccharide (treatment group) or no duckweed polysaccharide (control group), photographing scratch under an inverted microscope, and placing in a cell incubator, wherein each treatment is repeated for 3 times;

3. after incubation for 24h, scratches were photographed under an inverted microscope and mobility calculated.

The results are shown in figures 2 and 3, and it can be seen that the cell mobility is higher in the treated group compared to the control group. The results demonstrate that duckweed polysaccharide can promote migration of human skin fibroblasts.

Example 4

In this example, the effect of duckweed polysaccharide on Collagen I (Collagen I) and Collagen III (Collagen III) gene expression in human skin fibroblasts was examined as follows:

1. inoculating HSF Cell into 6-hole plate, and waiting for Cell growth in Cell hole plate;

2. changing to culture medium containing 10mg/mL duckweed polysaccharide (treatment group) or no duckweed polysaccharide (control group), placing in a cell incubator, and setting 3 replicates for each treatment;

3. after 24 hours of treatment, adding 1mL of TRIZOL into each hole, blowing off cells for multiple times, transferring the mixed solution into a centrifuge tube, and standing for 5 minutes at room temperature;

4. centrifuging to remove precipitate, adding 200 μl of chloroform into the supernatant, mixing, standing at room temperature for 15min, centrifuging, and transferring the supernatant to a new centrifuge tube;

5. adding an equal volume of precooled isopropanol, uniformly mixing, standing on ice for 10min, and centrifuging to remove the supernatant;

6. adding 1mL of 75% ethanol into the precipitate, mixing uniformly by using an oscillator, centrifuging to remove the supernatant, and standing for 10min to obtain RNA precipitate;

7. adding 30 mu L DEPC water to dissolve the precipitate, and determining the purity and concentration of total RNA;

8. reverse transcribing the RNA into cDNA using a TAKARA reverse transcription kit;

9. PCR reactions were performed to detect mRNA expression levels of Collagen I and Collagen III.

The experimental results are shown in FIGS. 4 and 5, and compared with the control group, the mRNA relative expression amount of the Collagen I in the treatment group is 2.047+/-0.049; the relative expression amount of mRNA of the Collagen III in the treatment group is 3.065 +/-0.062, and the duckweed polysaccharide can obviously promote the mRNA expression of the Collagen I and the Collagen III.

Example 5

In this example, the effect of duckweed polysaccharide on vascular endothelial growth factor VEGF-A protein expression in human skin fibroblasts was examined as follows:

3. after 24h of treatment, removing the culture medium, adding 80 mu L of PIPA lysate, fully lysing, scraping cells by using a cell scraper, collecting the cells into a centrifuge tube, placing the cells on ice at 4 ℃ for 30min, and vibrating once every 10 min;

4. centrifuging and sucking the supernatant, measuring the protein concentration by using a Bradford method, adjusting the protein concentration to 2 mug/mu L, and then boiling at 100 ℃ for 5 minutes;

5. preparing SDS-PAGE gel, after preparing, adding 10 mu L protein solution into each hole after assembling the electrophoresis frame, and starting electrophoresis;

6. after the end, the electric rotating clamp is assembled according to a classical sandwich model and is arranged in an electric rotating box, the current is adjusted to be constant current of 250mA, and the film is rotated for 1.5 hours;

7. after the electric conversion is finished, placing the membrane in 5% skimmed milk, and sealing for 1h at room temperature;

incubating vascular endothelial growth factor (VEGF-A) and GAPDH primary antibody at 8.4deg.C, and washing membrane to incubate corresponding secondary antibody after incubation overnight;

9. after the film is washed, development exposure is performed.

The experimental results are shown in FIG. 6, and it can be seen that the VEGF-A protein expression of the treated group is significantly higher than that of the control group, indicating that duckweed polysaccharide can promote the expression of vascular endothelial growth factor in human skin fibroblasts, thereby facilitating angiogenesis of skin.

Example 6

1. 12 Balb/C mice (8-10 weeks, 18-22g body weight, female) were randomized into 2 groups and fasted for 12 hours prior to surgery;

2. before dehairing, 1% pentobarbital sodium is used for intraperitoneal injection anesthesia, after the hair is subtracted by the elbow scissors, 8% sodium sulfide is used for dehairing the back of the mice, and clean water is used for wiping;

3. fixing a mouse on an operation panel, sterilizing by using iodophor, sterilizing by using 75% alcohol, drawing a circle with a trace of 1cm diameter by using a surgical knife, and cutting round skin according to the trace;

4. the treatment group smears 1mL of sterile water containing 10mg/mL duckweed polysaccharide on fresh wound surfaces, the control group smears only 1mL of sterile water, and each group is wrapped by a sterile bandage;

5. photo recordings were made on day 0, day 8, and day 16, respectively.

As shown in fig. 7, it can be seen that the wound healing speed of the treated group was faster and healed better than the control group, indicating that duckweed polysaccharide can help to accelerate wound repair.

In view of the above, it is known that the duckweed polysaccharide extracted by the present invention can achieve better and faster repair of wounds by promoting proliferation, migration, collagen expression and vascular endothelial growth factor expression of human skin fibroblasts, and thus, the duckweed polysaccharide is prepared into a wound repair agent for rapid repair of wounds after plastic and cosmetic treatment.

Claims

1. The application of duckweed polysaccharide in preparing wound repairing agent is characterized in that the preparation method of duckweed polysaccharide comprises the following steps:

2. The use according to claim 1, wherein the repair agent increases proliferation of human skin fibroblasts; the restorative agent enhances migration of human skin fibroblasts.

3. The use according to claim 1, wherein the repair agent increases mRNA expression of Collagen I and Collagen III in human skin fibroblasts.

4. The use according to claim 1, wherein the repair agent increases vascular endothelial growth factor protein expression in human skin fibroblasts.