Application of porphyra polymer in preparation of medicine for promoting wound healing

Abstract

The present application relates to the field of medicine, and in particular to a new application of Chondrus crispus polysaccharide, and more particularly to the application of Chondrus crispus polysaccharide in a drug for promoting wound healing. Tests show that Chondrus crispus polysaccharide has no toxicity to human umbilical vein endothelial cells (HUVEC) and human skin fibroblasts (HSF), can promote the migration and tube formation of human umbilical vein endothelial cells (HUVEC), and has a significant promoting effect on the healing of the wound of human umbilical vein endothelial cells (HUVEC).

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a novel application of red algae polysaccharide in the preparation of drugs that promote wound healing. Background Technology

[0002] Medical dressings are a type of medical material used to cover damaged wounds. They protect the human body from physical, chemical, and pathogenic microorganisms, and have functions such as heat preservation, moisture retention, and immune protection. Currently, chitosan and alginate are among the glycobased medical dressings that are being researched or applied. Chitosan is used in medical dressings because it is non-toxic and stimulates chondrocyte growth at low concentrations; alginate dressings can promote the regeneration of new wound tissue and improve the speed of wound healing. Therefore, researching non-toxic substances with the potential to promote wound healing is of great significance for finding reasonable measures to promote wound healing.

[0003] Red hairy algae (Bangia fusco-purpure), also known as red hairy vegetable or red cotton algae, is an economically valuable red algae resource endemic to the coastal areas of Fujian, my country, accounting for over 90% of the national total production. In August 2016, the "Putian Red Hairy Vegetable" national geographical indication certification trademark was approved and officially announced, becoming a national geographical indication premium seaweed. Related literature reports that red hairy algae has effects such as lowering blood lipids, lowering blood pressure, nourishing yin and clearing heat, and preventing arteriosclerosis, possessing high edible and medicinal value. However, there are few reports on red hairy algae polysaccharides, and no literature reports on their application in drugs promoting wound healing. Summary of the Invention

[0004] In view of this, the present invention provides a new application of Rhodophyta polysaccharide. The present invention is the first to discover that Rhodophyta polysaccharide can significantly promote the migration and tube formation of human umbilical vein endothelial cells without causing harm to them.

[0005] This invention evaluates the effect of *Rhododendron simsii* polysaccharide on wound healing in human umbilical vein endothelial cells (HUVECs) through experiments. The results show that *Rhododendron simsii* polysaccharide (0–500 μg / mL) has no toxic effect on HUVECs and human skin fibroblasts (HSF). *Rhododendron simsii* polysaccharide concentrations of 100 μg / mL and 500 μg / mL significantly promote wound healing in HUVECs; furthermore, treatment with 100 μg / mL of *Rhododendron simsii* polysaccharide preserves the integrity of the cell tubular structure and promotes tube formation in HUVECs.

[0006] Therefore, this invention provides the application of Rhodophyta polysaccharides in the preparation of drugs that promote wound healing.

[0007] Based on the above experimental results, the red algae polysaccharide provided by this invention has a significant effect on promoting the healing of human umbilical vein endothelial cell (HUVEC) wounds.

[0008] In addition, the application of the *Rhodophyta* polysaccharide proposed in the above embodiments of the present invention may also have the following additional technical features:

[0009] Preferably, this application uses red algae polysaccharide as one or the only active ingredient in the preparation of drugs that promote wound healing.

[0010] Preferably, the red algae polysaccharide promotes the migration of human umbilical vein endothelial cells.

[0011] Preferably, the red algae polysaccharide promotes tube formation in human umbilical vein endothelial cells.

[0012] Preferably, the drug is an ointment, powder, spray film, or dressing.

[0013] Preferably, the polysaccharide from *Rhodophyta rubrum* is not toxic to human umbilical vein endothelial cells and human skin fibroblasts.

[0014] Preferably, the concentration of the red algae polysaccharide is 0–500 μg / mL.

[0015] Preferably, the preparation method of the red algae polysaccharide is as follows:

[0016] Take red algae powder, soak and wash it with methanol, centrifuge, take the precipitate, add distilled water to the precipitate, extract at 90-100℃ for 2h, centrifuge, collect the supernatant, and re-dissolve the residue with distilled water, and extract again at 90-100℃ for 2h. Combine the supernatants obtained from the two extractions, concentrate under reduced pressure to obtain crude polysaccharide solution.

[0017] The crude polysaccharide solution was mixed with Sevag reagent at a ratio of 3:1, shaken, centrifuged, and the supernatant was collected. Anhydrous ethanol was added dropwise to the supernatant until the final concentration reached 75% v / v. The mixture was allowed to stand overnight, centrifuged, and the precipitate was collected. The precipitate was dissolved in water, dialyzed, and freeze-dried to obtain crude polysaccharide from *Rhododendron molle*.

[0018] The crude polysaccharide of *Rhodotorula rubra* was dissolved in ultrapure water to prepare a 5 mg / mL polysaccharide solution. After filtration through a 0.22 μm filter membrane, the polysaccharide solution was separated and purified using an propylene dextran gel S-400. During separation and purification, pure water or 0.1 M NaCl solution was used for elution, with a flow rate controlled at 0.2 mL / min. 3 mL samples were collected per tube using an automated sample collector, and 80 tubes were collected continuously. The phenol-sulfuric acid method was used for monitoring and detection. The eluent from the tube with the best repeatability was collected. After 48 h of ultrapure water dialyzing and freeze-drying, the *Rhodotorula rubra* polysaccharide was obtained.

[0019] Preferably, the Sevag reagent is a mixture of chloroform and n-butanol in a volume ratio of 4:1. Attached Figure Description

[0020] Figure 1 The image shows the purification process of the crude polysaccharide from *Rhizopus rubrum* using an propylene dextran gel S-400.

[0021] Figure 2 These are the experimental results of the cytotoxicity of the red algae polysaccharide of the present invention against HUVEC cells;

[0022] Figure 3 These are the experimental results of the cytotoxicity of the red algae polysaccharide of the present invention against HSF cells;

[0023] Figure 4 These are representative photographs showing how the polysaccharide from *Rhodophyta* promotes HUVEC migration according to the present invention.

[0024] Figure 5 This invention relates to the effect of Rhodophyta polysaccharide on HUVEC migration rate;

[0025] Figure 6 These are the experimental results of the red algae polysaccharide promoting HUVEC tube formation according to the present invention;

[0026] Figure 7 This invention relates to the effect of the red algae polysaccharide on the number of lumens formed in HUVECs. Detailed Implementation

[0027] The technical solution of the present invention is illustrated below through specific examples and accompanying drawings. It should be understood that the one or more method steps mentioned in the present invention do not preclude the existence of other method steps before or after the combined steps, or the insertion of other method steps between these explicitly mentioned steps; it should also be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, unless otherwise stated, the numbering of each method step is merely a convenient tool for identifying each method step, and not for limiting the order of the method steps or defining the scope of the present invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the present invention.

[0028] To better understand the above technical solutions, exemplary embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although exemplary embodiments of the present invention are shown in the drawings, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the invention to those skilled in the art.

[0029] The test materials used in this invention are all commercially available products that can be purchased on the market.

[0030] The present invention will be further illustrated below with reference to the embodiments:

[0031] Example 1: Preparation of Rhodophyta polysaccharides

[0032] Fresh red algae were washed and dried in an oven at 50°C until constant weight, then pulverized into algae powder. The algae powder was repeatedly soaked and washed three times with an appropriate amount of methanol, soaking overnight to partially remove pigments and alcohol-soluble impurities. After soaking, the precipitate was collected by centrifugation at 4000g for 15 min. 1L of distilled water was added to the precipitate, and the mixture was extracted in hot water at 90-100°C for 2 h. After extraction, the mixture was centrifuged at 4000g for 20 min, and the supernatant was collected. The residue was repeatedly extracted in hot water at 90-100°C for 2 h by adding 1L of distilled water, centrifuged, and the supernatants from the two centrifugations were combined and concentrated to 500mL under reduced pressure to obtain a crude polysaccharide solution.

[0033] Sevag reagent was prepared by mixing chloroform and n-butanol at a volume ratio of 4:1. Then, the crude polysaccharide solution was mixed with Sevag reagent at a ratio of 3:1, shaken for 40 min, and centrifuged at 4000g for 10 min to remove the protein precipitate. The supernatant was collected, and this process was repeated three times. Anhydrous ethanol was added dropwise to the supernatant to achieve a final concentration of 75% (v / v), and the mixture was allowed to stand overnight. After overnight standing, the mixture was centrifuged at 5000g for 10 min, and the precipitate was collected. The precipitate was dissolved in an appropriate amount of distilled water, dialyzed through a dialysis membrane with a molecular weight cutoff of 3500 Da for 48 h, and then freeze-dried to obtain crude polysaccharide from *Rhizopus rubrum*.

[0034] Crude polysaccharides from *Rhodotorula rubra* were dissolved in ultrapure water to prepare a 5 mg / mL polysaccharide solution. After filtration through a 0.22 μm filter membrane, 5 mL of the solution was deposited on an S-400 propylene dextran gel (1.6 cm ID × 100 cm). The crude polysaccharides were then separated and purified based on their molecular weight distribution. Elution was performed with pure water or 0.1 M NaCl at a flow rate of 0.2 mL / min. Samples were collected at 3 mL / tube using an automated sample collector, for a total of 80 tubes. The absorbance (OD) at 490 nm was measured using the phenol-sulfuric acid method. 490 Tracking and monitoring were performed, with elution volume as the x-axis and OD... 490 Plot the elution curve on the ordinate, as shown below. Figure 1 As shown. After repeated trials, the eluent from the tube with the best repeat was collected, dialyzed with ultrapure water for 48 hours, and then freeze-dried to obtain the *Rhodophyta* polysaccharide.

[0035] Example 2: Determination of the toxicity of *Rhodophyta rubrum* polysaccharide to human umbilical vein endothelial cells (HUVECs) and human skin fibroblasts (HSFs).

[0036] The toxicity of red algae polysaccharides to human umbilical vein endothelial cells (HUVECs) and human skin fibroblasts (HSFs) was determined using the MTT assay.

[0037] Take preserved HUVECs (or HSF) cells and culture them. HUVECs (or HSF) in the logarithmic growth phase are then cultured at a rate of 3 × 10⁻⁶. 4 Cells were seeded at various concentrations in 96-well cell culture plates and incubated at 37°C with 5% CO2 for 24 h. Cells were then treated with different concentrations of *Rhodophyta rubrum* polysaccharide solution (0–500 μg / mL) for 24 h. After removing the supernatant, 50 μL of MTT solution was added to each well, and incubation continued for 30 min. After removing the supernatant, 100 μL of DMSO was added, and the mixture was shaken for 20 min. The absorbance (OD) at 570 nm was measured using a multifunctional cell imaging system. 570 The cell viability was calculated using the following formula. A safe dosage of *Rhodophyta rubrum* polysaccharide was determined based on a cell viability of ≥90%.

[0038]

[0039] The control group was treated with cell culture medium without the addition of red algae polysaccharide, while the experimental group was treated with culture medium solutions containing different concentrations of red algae polysaccharide (the concentrations set in this embodiment include 0 μg / mL, 31.25 μg / mL, 62.5 μg / mL, 125 μg / mL, 250 μg / mL, and 500 μg / mL).

[0040] The results are as follows Figure 2 and Figure 3 As shown, the cell viability of *Rhododendron molle* after treatment with polysaccharides remained above 90%. Figure 2 As shown in Figure 3, this indicates that the polysaccharide from *Rhodophyta rubrum* is not toxic to HUVECs and HSF at a certain concentration, demonstrating that the raw materials and cells used in the experiment were appropriate.

[0041] Example 3: Effect of Rhodophyta polysaccharide on the migration of human umbilical vein endothelial cells (HUVECs)

[0042] The preserved HUVEC cells were cultured, and human umbilical vein endothelial cells (HUVECs) in the logarithmic growth phase were cultured at a rate of 5 × 10⁻⁶. 5Cells were seeded at a concentration of [insert concentration here] into 6-well cell culture plates and incubated at 37°C, 5% CO2 for 24 h. A monolayer of cells was then prepared using a sterile 200 μL plastic pipette tip, followed by a gentle wash once with PBS to remove the scraped cells. Next, human umbilical vein endothelial cells (HUVECs) were treated with *Rhodophyta rubrum* polysaccharide at concentrations of 0 μg / mL, 20 μg / mL, 100 μg / mL, and 500 μg / mL, respectively. Images were captured at 0 and 24 h using a bright-field microscope (40x magnification). Analysis was performed using ImageJ, and the wound healing rate was calculated using the following formula:

[0043]

[0044] Where T0 is the scratch area at hour 0, T 24 The area of ​​the scratch within 24 hours

[0045] The results are as follows Figure 3 and Figure 4 As shown: An in vitro wound healing model was established using the cell scratch method, and the effect of Rhodophyta polysaccharide on the wound healing ability of HUVECs was determined. Compared with the control group, Rhodophyta polysaccharide at concentrations of 100 μg / mL and 500 μg / mL significantly promoted wound healing in HUVECs.

[0046] Example 4: Effect of Rhodophyta polysaccharide on human umbilical vein endothelial cell (HUVEC) tube formation.

[0047] The Matrigel assay was used to detect cell lumen formation ability. Matrigel was added to pre-chilled 96-well plates at a rate of 40 μL / well and incubated at 37°C for 1 h to allow the matrix gel to solidify. HUVECs containing 0 μg / mL and 100 μg / mL of *Rhodophyta rubrum* polysaccharides were then added at a rate of 3 × 10⁻⁶ cells / well. 4 Cells / wells were seeded onto the solidified matrix gel surface and incubated for 8 hours. Images were taken using a bright-field microscope (40x magnification) and the number of lumens was counted.

[0048] The results are as follows Figure 5 and Figure 6 As shown, compared with the control group, the experimental group treated with 100 μg / mL Rhodophyta polysaccharide had intact tubular structures after 8 h, and the number of tubular structures formed was significantly higher than that of the control group.

[0049] In summary, this invention is the first to propose that *Rhododendron molle* polysaccharides (0-500 μg / mL) have no toxic effects on human umbilical vein endothelial cells and human skin fibroblasts. *Rhododendron molle* polysaccharides at concentrations of 100 μg / mL and 500 μg / mL significantly promoted wound healing in human umbilical vein endothelial cells (HUVECs); furthermore, treatment of HUVECs with 100 μg / mL of *Rhododendron molle* polysaccharides preserved the integrity of the cell tubular structure and promoted tube formation.

[0050] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. The illustrative expressions of the above terms in this specification should not be construed as necessarily referring to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. In addition, those skilled in the art can combine and integrate the different embodiments or examples described in this specification.

[0051] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. The application of red algae polysaccharides in the preparation of drugs that promote wound healing, characterized in that: A drug for promoting wound healing is prepared using red algae polysaccharide as one or the only active ingredient, wherein the red algae polysaccharide promotes the migration of human umbilical vein endothelial cells and promotes the formation of tubes in human umbilical vein endothelial cells. The preparation method of the Rhodophyta polysaccharide is as follows: Take red algae powder, soak and wash it with methanol, centrifuge, take the precipitate, add distilled water to the precipitate, extract at 90~100℃ for 2 h, centrifuge, collect the supernatant, and re-dissolve the residue with distilled water, and extract again at 90~100℃ for 2 h. Combine the supernatants obtained from the two extractions, concentrate under reduced pressure to obtain crude polysaccharide solution. The crude polysaccharide solution was mixed with Sevag reagent at a ratio of 3:1, shaken, centrifuged, and the supernatant was collected. Anhydrous ethanol was added dropwise to the supernatant until the final concentration reached 75% v / v. The mixture was allowed to stand overnight, centrifuged, and the precipitate was collected. The precipitate was dissolved in water, dialyzed, and freeze-dried to obtain crude polysaccharide from *Rhododendron molle*. The crude polysaccharide of *Rhodotorula rubra* was dissolved in ultrapure water to prepare a 5 mg / mL polysaccharide solution. After filtration through a 0.22 μm filter membrane, the polysaccharide solution was separated and purified using an propylene dextran gel S-400. During separation and purification, pure water or 0.1 M NaCl solution was used for elution, with a flow rate controlled at 0.2 mL / min. 3 mL samples were collected per tube using an automated sample collector, and 80 tubes were collected continuously. The phenol-sulfuric acid method was used for monitoring and detection. The eluent from the tube with the best repeatability was collected. After 48 h of ultrapure water dialyzing and freeze-drying, the *Rhodotorula rubra* polysaccharide was obtained.

2. The application as described in claim 1, characterized in that: The drug is an ointment, powder, or spray film.

3. The application as described in claim 2, characterized in that: The polysaccharide from *Rhodophyta rubrum* is not toxic to human umbilical vein endothelial cells or human skin fibroblasts.

4. The application as described in claim 1, characterized in that: The concentration of the polysaccharide from *Rhodophyta rubrum* is 100-500 μg / mL.

5. The application as described in claim 1, characterized in that: The Sevag reagent is a mixture of chloroform and n-butanol in a volume ratio of 4:1.

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