A biological polysaccharide dressing for promoting wound repair and a preparation method thereof

Abstract

The application relates to a biological polysaccharide dressing for promoting wound repair and a preparation method, which comprises the following components in percentage by mass volume: 2.5-3.0% of sodium carboxymethyl cellulose, 0.6-0.9% of sodium chloride, 0.2-0.3% of anhydrous sodium acetate, 0.3-0.5% of sodium gluconate, 0.02-0.03% of magnesium chloride, 0.02-0.04% of potassium chloride, 0.14-0.18% of extract of Radix Dendrobii, 0.08-0.12% of extract of Marchantia, 0.04-0.08% of extract of Gekko, 0.03-0.05% of inactivated Lactobacillus, and the balance of purified water. The dressing can promote the migration of keratinocytes, accelerate re-epithelialization of a wound, and relieve scar itching through the synergistic effect of the polysaccharide matrix and multiple components, and is suitable for postoperative non-chronic wound care.

Description

Technical Field

[0001] This invention belongs to the field of extracorporeal wound rehabilitation dressing technology, and specifically relates to a biological polysaccharide dressing that promotes wound repair and its preparation method. Background Technology

[0002] Sodium carboxymethyl cellulose, as the core matrix component of biological polysaccharide rehabilitation dressings, can form a continuous, moist gel protective layer on the wound surface, promoting moist wound healing through physical barriers and water retention.

[0003] The quality of postoperative wound healing directly affects the patient's recovery experience and long-term aesthetic results. In clinical practice, two closely related issues are often overlooked: First, delayed postoperative wound healing, characterized by slow epidermal re-epithelialization and initial dermal collagen deposition rates, leads to prolonged wound openness, increasing the potential risk of infection and disrupting the subsequent collagen remodeling rhythm, thus increasing the probability of abnormal scar formation; second, scar formation can cause persistent local itching, the mechanism of which involves the abnormal aggregation of mast cells within the scar tissue. Summary of the Invention

[0004] This invention provides a bio-polysaccharide dressing that promotes wound repair and its preparation method, which can help improve the speed of postoperative wound healing and relieve intractable itching caused by scarring.

[0005] Technical solution:

[0006] A bio-polysaccharide dressing for promoting wound healing comprises, by mass and volume percentage: 2.5%–3.0% sodium carboxymethyl cellulose, 0.6%–0.9% sodium chloride, 0.2%–0.3% anhydrous sodium acetate, 0.3%–0.5% sodium gluconate, 0.02%–0.03% magnesium chloride, 0.02%–0.04% potassium chloride, 0.14%–0.18% *Gnaphalium affine* extract, 0.08%–0.12% *Lysimachia christinae* extract, 0.04%–0.08% *Gnaphalium affine* extract, 0.03%–0.05% inactivated *Lactobacillus* cells, with the balance being purified water; the degree of substitution of sodium carboxymethyl cellulose is 0.7–0.9, and its 2% aqueous solution has a dynamic viscosity of 2500–3000 mPa·s at 25°C;

[0007] In the above formulation, sodium carboxymethyl cellulose serves as a thickening matrix, working with sodium chloride, potassium chloride, and magnesium chloride to simulate the extracellular fluid ion environment. Anhydrous sodium acetate and sodium gluconate form a pH buffer pair, ensuring the stability of the dressing during moist heat sterilization and storage. The three plant extracts and inactivated lactobacillus cells act as active ingredients, synergistically promoting epithelialization, relieving itching, enhancing the penetration of active ingredients, and regulating the wound microecology, thereby achieving the expected effects of accelerating postoperative wound healing and reducing persistent itching after scar formation.

[0008] The inactivated Lactobacillus cells were prepared using the following method:

[0009] A1. Inoculate the Lactobacillus strain into MRS liquid medium, incubate at 37°C for 18–24 h, collect the bacterial cells by centrifugation, and wash 2–3 times with sterile phosphate buffer.

[0010] A2. After washing, the bacterial cells are resuspended in water for injection and inactivated by moist heat at 121°C for 15-20 min. The inactivated bacterial cells are collected by centrifugation and washed 2-3 times with purified water.

[0011] A3. Freeze-dry the washed and inactivated bacterial cells, grind them at low temperature, and pass them through a 200-mesh sieve to obtain the final product.

[0012] This process thoroughly destroys bacterial activity through moist heat inactivation, while preserving active structures such as lipoteichoic acid and peptidoglycan in the cell wall, enabling them to exert more effective immunomodulatory effects in subsequent dressings. Multiple washes effectively remove culture medium components and intracellular substances released after inactivation, ensuring the safety and efficacy of the product.

[0013] The extract of *Houttuynia cordata* was prepared by the following process: *Houttuynia cordata* roots and stems were pulverized into a coarse powder of 15-20 mesh, and extracted 2-3 times with 8-10 times the amount of 70% ethanol under reflux for 90-120 minutes each time. The extracts were combined and concentrated under reduced pressure to a clear extract with a relative density of 1.10-1.15 at 60℃. This extract was loaded onto a D101 macroporous resin column and eluted sequentially with 3-5 column volumes of purified water, 3-5 column volumes of 30% ethanol, and 4-6 column volumes of 70% ethanol. The 70% ethanol eluent was collected, concentrated under reduced pressure, and then freeze-dried to obtain the final product. 70% ethanol can effectively dissolve steroidal saponins, and the gradient elution of the macroporous resin removes water-soluble and moderately polar impurities while enriching *Houttuynia cordata* glycoside A and removing other possible impurities.

[0014] The extract of *Lysimachia nummularia* is prepared by the following process: the whole herb of *Lysimachia nummularia* is crushed into a coarse powder of 15-20 mesh, 10-12 times the amount of 70% ethanol is added, and ultrasonic extraction is performed 2-3 times at a power of 300-400W, each time for 30-40 minutes. The extracts are combined and concentrated under reduced pressure to a clear extract with a relative density of 1.05-1.10 at 60℃. The extract is then freeze-dried to obtain the final product. Ultrasonic extraction utilizes cavitation effect and mechanical vibration to promote the rapid dissolution of terpenoid components such as serpentine. The low operating temperature avoids the degradation of active ingredients that may be caused by traditional heat reflux.

[0015] The extract of *Smilax china* was prepared by the following process: The whole herb of *Smilax china* was crushed into a coarse powder of 15-20 mesh. It was then cold-extracted 2-3 times with 8-10 times the volume of 0.05 mol / L sodium hydroxide aqueous solution for 10-12 hours each time. The extracts were combined, and hydrochloric acid was added under stirring to adjust the pH to 4.0-4.5. The mixture was allowed to stand for 8-12 hours, and the precipitate was collected. The precipitate was washed with purified water until the eluent was neutral, and then freeze-dried to obtain the final product. The enrichment of the extract was achieved by utilizing the property of *Smilax china* acid components to form salts in alkaline solutions and then precipitate free after acidification. The cold-extraction conditions protected the structure of the amphiphilic saponins, and washing with water after acid precipitation removed residual acids, alkalis, and salts.

[0016] A method for preparing a biological polysaccharide dressing, characterized by comprising the following steps:

[0017] S1. The freeze-dried powders of *Houttuynia cordata* extract, *Gnaphalium affine* extract, and *Gnaphalium affine* extract are passed through a 200-mesh sieve and stored in the dark and refrigerated for later use.

[0018] S2. Take 60% of the formula amount of water for injection, heat it to 40-50℃, and slowly add sodium carboxymethyl cellulose while stirring at 100-120r / min. Continue stirring for 1.5-2h until it is completely swollen and forms a transparent and uniform viscous solution.

[0019] S3. Cool the adhesive solution to 25-30℃, and add sodium chloride, anhydrous sodium acetate, sodium gluconate, magnesium chloride and potassium chloride in sequence according to the formula, and stir until completely dissolved;

[0020] S4. Adjust the pH of the system to 6.5–7.5 using 0.1 mol / L sodium hydroxide solution or 0.1 mol / L acetic acid solution;

[0021] S5. After pre-dispersing the three extracts and the formulated amount of inactivated lactobacillus cells prepared in step S1 with 6 to 8 times the total weight of the extracts in water for injection, slowly add them to the above gel solution under light protection and stirring at 60 to 80 r / min. Add the remaining water for injection to the total volume and continue stirring for 20 to 30 minutes until the mixture is uniform.

[0022] S6. Fill the well-mixed adhesive solution into a medical-grade brown light-proof container and seal it. Sterilize it with moist heat at 115℃~121℃ for 25~30 minutes. Cool it using a gradient cooling method: first cool it to 80℃ at a rate of 1℃ / min, and then let it cool naturally to room temperature.

[0023] The above method involves first fully swelling sodium carboxymethyl cellulose to construct a gel network, then sequentially introducing ionic components and active ingredients to achieve uniform dispersion; light-protected operation can protect photosensitive substances, and adjusting the pH value to near neutral can ensure compatibility with the physiological environment of the wound; moist heat sterilization can guarantee the sterility of the product, and gradient cooling can prevent the dressing from developing internal stress or phase separation due to sudden cooling, making the finished product stable and uniform.

[0024] Compared with the prior art, the beneficial effects of the present invention are:

[0025] 1. The combination of *Houttuynia cordata* extract and sodium carboxymethyl cellulose matrix can accelerate the directional migration and terminal differentiation of keratinocytes, significantly speeding up the re-epithelialization process of the wound and shortening the window time for the postoperative wound to enter the stable scar-forming period, thereby reducing the risk of infection and the probability of abnormal scar formation.

[0026] 2. The introduction of snake worm extract transforms the dressing from a simple physical barrier and moisturizing agent into one capable of intervening in pruritus-related biological pathways. Animal studies show that regular use of this product effectively inhibits scratching behavior in the scar area, and no rebound of pruritus occurs after discontinuation, indicating that this product has a long-lasting inhibitory effect on scar-related pruritus.

[0027] 3. The amphiphilic saponins contained in the snake vine extract can gently and reversibly improve the permeability of the stratum corneum, allowing the active ingredients in the hairy vine extract and snake vine extract to reach the basal layer of the epidermis and the superficial dermis more effectively, significantly improving the bioavailability of the active ingredients and avoiding the technical defects of poor absorption caused by simply adding active ingredients.

[0028] 4. Inactivated Lactobacillus cells function as "post-biotics." Their cell wall peptidoglycan and lipoteichoic acid, among other structural components, can competitively occupy wound adhesion sites, helping to inhibit the colonization of pathogenic bacteria such as Staphylococcus aureus. At the same time, they gently activate local immune responses, providing a more favorable microenvironment for wound healing without introducing the risk of live bacteria.

[0029] 5. Sodium carboxymethyl cellulose, within a specific degree of substitution and viscosity range, forms a viscous fluid that combines the fluidity of irrigation with the coverage of the wound surface after application. It effectively removes exudate and contaminants from the wound through irrigation while simultaneously forming a continuously moisturizing hydration layer on the wound surface. A single application simplifies postoperative wound management by completing both irrigation and care procedures. The buffer system composed of anhydrous sodium acetate and sodium gluconate maintains the pH stability of the product during moist heat sterilization and storage, while the weak coordination between magnesium chloride and steroidal saponins from the *Houttuynia cordata* extract helps protect the glycosidic bond structure of the active ingredients. Detailed Implementation

[0030] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.

[0031] Example 1

[0032] A bio-polysaccharide dressing for promoting wound repair comprises, by mass and volume percentage: 2.5% sodium carboxymethyl cellulose, 0.6% sodium chloride, 0.2% anhydrous sodium acetate, 0.3% sodium gluconate, 0.02% magnesium chloride, 0.02% potassium chloride, 0.14% *Gnaphalium affine* extract, 0.08% *Gnaphalium affine* extract, 0.04% *Gnaphalium affine* extract, 0.03% inactivated *Lactobacillus* cells, with the balance being purified water; the degree of substitution of sodium carboxymethyl cellulose is 0.7, and its 2% aqueous solution has a dynamic viscosity of 2500 mPa·s at 25°C.

[0033] The inactivated Lactobacillus cells were prepared using the following method:

[0034] A1. Inoculate the Lactobacillus strain into MRS liquid medium, incubate at 37°C for 18 hours, collect the bacterial cells by centrifugation, and wash twice with sterile phosphate buffer.

[0035] A2. After washing, the bacterial cells were resuspended in water for injection and inactivated by moist heat at 121°C for 15 min. The inactivated bacterial cells were collected by centrifugation and washed twice with purified water.

[0036] A3. Freeze-dry the washed and inactivated bacterial cells, grind them at low temperature, and pass them through a 200-mesh sieve to obtain the final product.

[0037] The extract of *Houttuynia cordata* was prepared by the following process: the roots and stems of *Houttuynia cordata* were taken, crushed into 15-mesh coarse powder, and extracted twice by reflux with 8 times the amount of 70% ethanol, each time for 90 min. The extracts were combined and concentrated under reduced pressure to a clear extract with a relative density of 1.10 at 60℃. The clear extract was loaded onto a D101 macroporous resin column and eluted sequentially with 3 column volumes of purified water, 3 column volumes of 30% ethanol, and 4 column volumes of 70% ethanol. The 70% ethanol eluent was collected, concentrated under reduced pressure, and then freeze-dried to obtain the final product.

[0038] The extract of *Lysimachia nummularia* was prepared by the following process: the whole herb of *Lysimachia nummularia* was taken, crushed into 15-mesh coarse powder, 10 times the amount of 70% ethanol was added, and ultrasonic extraction was performed twice at 300W power for 30 minutes each time. The extracts were combined and concentrated under reduced pressure to a clear extract with a relative density of 1.05 at 60℃. The extract was then freeze-dried to obtain the final product.

[0039] Snakeberry extract is prepared by the following process: Take the whole herb of snakeberry, crush it into 15-mesh coarse powder, add 8 times the amount of 0.05mol / L sodium hydroxide aqueous solution for cold soaking and extraction twice, 10h each time, combine the extracts, add hydrochloric acid under stirring to adjust the pH to 4.0, let stand for 8h, collect the precipitate, wash with purified water until the effluent is neutral, freeze dry to obtain the extract.

[0040] A method for preparing a biological polysaccharide dressing, characterized by comprising the following steps:

[0041] S1. The freeze-dried powders of *Houttuynia cordata* extract, *Gnaphalium affine* extract, and *Gnaphalium affine* extract are passed through a 200-mesh sieve and stored in the dark and refrigerated for later use.

[0042] S2. Take 60% of the formula amount of water for injection, heat it to 40℃, and slowly add sodium carboxymethyl cellulose while stirring at 100r / min. Continue stirring for 1.5h until it is completely swollen to form a transparent and uniform viscous solution.

[0043] S3. Cool the adhesive solution to 25°C, and add sodium chloride, anhydrous sodium acetate, sodium gluconate, magnesium chloride and potassium chloride in sequence according to the formula, and stir until completely dissolved;

[0044] S4. Adjust the pH of the system to 6.5 using 0.1 mol / L sodium hydroxide solution or 0.1 mol / L acetic acid solution;

[0045] S5. After pre-dispersing the three extracts and the formulated amount of inactivated lactobacillus cells prepared in step S1 with 6 times the total weight of the extracts in water for injection, slowly add them to the above gel solution under light protection and stirring at 60 r / min. Add the remaining water for injection to the total volume and continue stirring for 20 min until the mixture is uniform.

[0046] S6. Fill the well-mixed adhesive solution into a medical-grade brown light-proof container and seal it. Sterilize it at 115℃ for 25 minutes using moist heat. Cool it using a gradient cooling method: first cool it to 80℃ at a rate of 1℃ / min, and then let it cool naturally to room temperature.

[0047] Example 2

[0048] A bio-polysaccharide dressing for promoting wound healing comprises, by mass and volume percentage: 3.0% sodium carboxymethyl cellulose, 0.9% sodium chloride, 0.3% anhydrous sodium acetate, 0.5% sodium gluconate, 0.03% magnesium chloride, 0.04% potassium chloride, 0.18% *Gnaphalium affine* extract, 0.12% *Gnaphalium affine* extract, 0.08% *Gnaphalium affine* extract, 0.05% inactivated *Lactobacillus* cells, with the balance being purified water; the degree of substitution of sodium carboxymethyl cellulose is 0.9, and its 2% aqueous solution has a dynamic viscosity of 3000 mPa·s at 25°C.

[0049] The inactivated Lactobacillus cells were prepared using the following method:

[0050] A1. Inoculate the Lactobacillus strain into MRS liquid medium, incubate at 37°C for 24 hours, collect the bacterial cells by centrifugation, and wash 3 times with sterile phosphate buffer.

[0051] A2. After washing, the bacterial cells were resuspended in water for injection and inactivated by moist heat at 121°C for 20 min. The inactivated bacterial cells were collected by centrifugation and washed three times with purified water.

[0052] A3. Freeze-dry the washed and inactivated bacterial cells, grind them at low temperature, and pass them through a 200-mesh sieve to obtain the final product.

[0053] The extract of *Houttuynia cordata* was prepared by the following process: the roots and stems of *Houttuynia cordata* were taken, crushed into 20-mesh coarse powder, and extracted three times by reflux with 10 times the amount of 70% ethanol, each time for 120 min. The extracts were combined and concentrated under reduced pressure to a clear extract with a relative density of 1.15 at 60℃. The clear extract was loaded onto a D101 macroporous resin column and eluted sequentially with 5 column volumes of purified water, 5 column volumes of 30% ethanol, and 6 column volumes of 70% ethanol. The 70% ethanol eluent was collected, concentrated under reduced pressure, and then freeze-dried to obtain the final product.

[0054] The extract of *Lysimachia nummularia* was prepared by the following process: the whole herb of *Lysimachia nummularia* was crushed into a coarse powder of 20 mesh, 12 times the amount of 70% ethanol was added, and the extract was ultrasonically extracted three times at 400W power for 40 minutes each time. The extracts were combined and concentrated under reduced pressure to a clear extract with a relative density of 1.10 at 60℃. The extract was then freeze-dried to obtain the final product.

[0055] Snakeberry extract is prepared by the following process: Take the whole herb of snakeberry, crush it into 20-mesh coarse powder, add 10 times the amount of 0.05mol / L sodium hydroxide aqueous solution for cold soaking and extraction 3 times, 12h each time, combine the extracts, add hydrochloric acid under stirring to adjust the pH to 4.5, let stand for 12h, collect the precipitate, wash with purified water until the effluent is neutral, freeze dry to obtain the extract.

[0056] A method for preparing a biological polysaccharide dressing, characterized by comprising the following steps:

[0057] S1. The freeze-dried powders of *Houttuynia cordata* extract, *Gnaphalium affine* extract, and *Gnaphalium affine* extract are passed through a 200-mesh sieve and stored in the dark and refrigerated for later use.

[0058] S2. Take 60% of the formula amount of water for injection, heat it to 50℃, and slowly add sodium carboxymethyl cellulose while stirring at 120r / min. Continue stirring for 2 hours until it is completely swollen to form a transparent and uniform viscous solution.

[0059] S3. Cool the adhesive solution to 30°C, and add sodium chloride, anhydrous sodium acetate, sodium gluconate, magnesium chloride and potassium chloride in sequence according to the formula, and stir until completely dissolved;

[0060] S4. Adjust the pH of the system to 7.5 using 0.1 mol / L sodium hydroxide solution or 0.1 mol / L acetic acid solution;

[0061] S5. After pre-dispersing the three extracts and the formulated amount of inactivated lactobacillus cells prepared in step S1 with 8 times the total weight of the extracts in water for injection, slowly add them to the above gel solution under light protection and stirring at 80 r / min. Add the remaining water for injection to the total volume and continue stirring for 30 min until the mixture is uniform.

[0062] S6. Fill the well-mixed adhesive solution into a medical-grade brown light-proof container and seal it. Sterilize it at 121°C for 30 minutes using moist heat. Cool it using a gradient cooling method: first cool it to 80°C at a rate of 1°C / min, and then let it cool naturally to room temperature.

[0063] Example 3

[0064] A bio-polysaccharide dressing for promoting wound repair comprises, by mass and volume percentage: 2.7% sodium carboxymethyl cellulose, 0.7% sodium chloride, 0.25% anhydrous sodium acetate, 0.4% sodium gluconate, 0.025% magnesium chloride, 0.03% potassium chloride, 0.16% *Gnaphalium affine* extract, 0.1% *Lysimachia christinae* extract, 0.06% *Gnaphalium affine* extract, 0.04% inactivated *Lactobacillus* cells, with the balance being purified water; the degree of substitution of sodium carboxymethyl cellulose is 0.8, and its 2% aqueous solution has a dynamic viscosity of 2700 mPa·s at 25°C.

[0065] The inactivated Lactobacillus cells were prepared using the following method:

[0066] A1. Inoculate the Lactobacillus strain into MRS liquid medium, incubate at 37°C for 20 h, collect the bacterial cells by centrifugation, and wash twice with sterile phosphate buffer.

[0067] A2. After washing, the bacterial cells were resuspended in water for injection and inactivated by moist heat at 121°C for 18 min. The inactivated bacterial cells were collected by centrifugation and washed three times with purified water.

[0068] A3. Freeze-dry the washed and inactivated bacterial cells, grind them at low temperature, and pass them through a 200-mesh sieve to obtain the final product.

[0069] The extract of *Houttuynia cordata* was prepared by the following process: the roots and stems of *Houttuynia cordata* were taken, crushed into 17-mesh coarse powder, and extracted three times by reflux with 9 times the amount of 70% ethanol, each time for 100 min. The extracts were combined and concentrated under reduced pressure to a clear extract with a relative density of 1.13 at 60℃. The clear extract was loaded onto a D101 macroporous resin column and eluted sequentially with 4 column volumes of purified water, 4 column volumes of 30% ethanol, and 5 column volumes of 70% ethanol. The 70% ethanol eluent was collected, concentrated under reduced pressure, and then freeze-dried to obtain the final product.

[0070] The extract of *Lysimachia nummularia* was prepared by the following process: the whole herb of *Lysimachia nummularia* was crushed into 18-mesh coarse powder, 11 times the amount of 70% ethanol was added, and the extract was ultrasonically extracted three times at 350W power for 35 minutes each time. The extracts were combined and concentrated under reduced pressure to a clear extract with a relative density of 1.05 at 60℃. The extract was then freeze-dried to obtain the final product.

[0071] Snakeberry extract is prepared by the following process: the whole herb of snakeberry is crushed into 18-mesh coarse powder, and 9 times the amount of 0.05mol / L sodium hydroxide aqueous solution is added for cold soaking and extraction 3 times, 11h each time. The extracts are combined, and hydrochloric acid is added under stirring to adjust the pH to 4.0. After standing for 10h, the precipitate is collected, washed with purified water until the effluent is neutral, and then freeze-dried to obtain the extract.

[0072] A method for preparing a biological polysaccharide dressing, characterized by comprising the following steps:

[0073] S1. The freeze-dried powders of *Houttuynia cordata* extract, *Gnaphalium affine* extract, and *Gnaphalium affine* extract are passed through a 200-mesh sieve and stored in the dark and refrigerated for later use.

[0074] S2. Take 60% of the formula amount of water for injection, heat it to 45℃, and slowly add sodium carboxymethyl cellulose while stirring at 110r / min. Continue stirring for 1.8h until it is completely swollen to form a transparent and uniform viscous solution.

[0075] S3. Cool the adhesive solution to 27°C, and add sodium chloride, anhydrous sodium acetate, sodium gluconate, magnesium chloride and potassium chloride in sequence according to the formula, and stir until completely dissolved;

[0076] S4. Adjust the pH of the system to 7.0 using 0.1 mol / L sodium hydroxide solution or 0.1 mol / L acetic acid solution;

[0077] S5. After pre-dispersing the three extracts and the formulated amount of inactivated lactobacillus cells prepared in step S1 with 7 times the total weight of the extracts in water for injection, slowly add them to the above gel solution under light protection and stirring at 70 r / min. Add the remaining water for injection to the total volume and continue stirring for 25 min until the mixture is uniform.

[0078] S6. Fill the well-mixed adhesive solution into a medical-grade brown light-proof container and seal it. Sterilize it at 120℃ for 30 minutes using moist heat. Cool it using a gradient cooling method: first cool it to 80℃ at a rate of 1℃ / min, and then let it cool naturally to room temperature.

[0079] Comparative Example 1

[0080] The difference from Example 3 is that 0.16% of the extract of *Houttuynia cordata*, 0.1% of the extract of *Gnaphalium affine*, 0.06% of the extract of *Gnaphalium affine*, and 0.04% of the inactivated *Lactobacillus* cells, as well as their related preparation processes or steps, are removed. Otherwise, the same as Example 3 is used.

[0081] Comparative Example 2

[0082] The difference from Example 3 is that 0.06% of the snake stalk extract and 0.04% of the inactivated lactobacillus cells were removed, along with their related preparation processes or steps; otherwise, they are the same as in Example 3.

[0083] Comparative Example 3

[0084] The difference from Example 3 is that 0.16% of the extract of *Houttuynia cordata* and its related preparation process or steps are removed, while the rest is the same as Example 3.

[0085] Comparative Example 4

[0086] The difference from Example 3 is that 0.1% of the snake-root extract and its related preparation process or steps are removed, while the rest is the same as Example 3.

[0087] Comparative Example 5

[0088] The difference from Example 3 is that 0.04% of inactivated Lactobacillus cells and its related preparation process or steps are removed; otherwise, it is the same as Example 3.

[0089] I. In vitro cell scratch assay

[0090] 1. Detection Method

[0091] Take mouse keratinocyte cell line PAM212, and use 5×10 5 Each well was seeded with a sample of the dressing in 6-well plates and cultured in DMEM medium containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator until confluence reached 90% or higher. The dressing was then vertically scratched using a 200 μL sterile pipette tip, washed with PBS, and then treated with a 10-fold dilution of serum-free DMEM extract (preparation method: add serum-free DMEM to the dressing sample at a 1:10 ratio, extract at 4°C for 2 h, centrifuge at 12000 r / min for 20 min, and sterilize the supernatant through a 0.22 μm filter). A blank control group was included, with an equal volume of serum-free DMEM added. Three replicates were performed for each group. Images were taken at the same location under an inverted microscope at 0 h and 24 h. The scratch width was measured using ImageJ software, and the healing rate was calculated as: Healing rate (%) = (0 h width - 24 h width) / 0 h width × 100%.

[0092] 2. Results Data

[0093] Blank control group 35.2 Comparative Example 1 36.1 Comparative Example 2 59.5 Comparative Example 3 38.4 Comparative Example 4 65.2 Comparative Example 5 69.3 Example 1 77.7 Example 2 78.5 Example 3 78.9

[0094] 3. Analysis

[0095] Comparative Example 1 showed a healing rate close to that of the blank control group, indicating that the basic component had no significant epithelialization-promoting activity. Comparative Example 3, which did not contain *Houttuynia cordata* extract, had a healing rate close to that of Comparative Example 1, confirming that *Houttuynia cordata* extract is the core component for promoting epithelialization. Comparative Examples 2, 4, and 5 all contained *Houttuynia cordata* extract, and their healing rates were significantly higher than those of Comparative Examples 1 and 3, indicating that the epithelialization-promoting ability was significantly enhanced upon the addition of *Houttuynia cordata* extract.

[0096] However, there were significant differences between Comparative Examples 2, 4, and 5 and the Example Group. Comparative Example 2, lacking both *Gnaphalium affine* extract and inactivated bacterial cells, had the lowest healing rate; Comparative Example 4, lacking *Gnaphalium affine* extract, had a slightly higher healing rate than Comparative Example 2; Comparative Example 5, lacking inactivated bacterial cells, had the highest healing rate among the three comparative examples containing *Gnaphalium affine*, but it was still significantly lower than the Example Group. This indicates that *Gnaphalium affine* extract, *Gnaphalium affine* extract, and inactivated bacterial cells all contribute to epithelialization to varying degrees, and the absence of any one component weakens the effect. The healing rates of the three Examples were all higher than those of the comparative examples, demonstrating the optimal synergistic effect when all four active ingredients are present.

[0097] II. Mast cell degranulation inhibition experiment

[0098] 1. Detection Method

[0099] Mast cells were purified from peritoneal lavage fluid of BALB / c mice by Percoll density gradient centrifugation, and their viability was assessed as >95% by trypan blue staining. The cell suspension was then adjusted to a density of 1×10⁻⁶ cells / mL using Tyrode buffer. 6 Cells / mL were seeded into 96-well plates. Each group's dressing extract (prepared using the same method as the scratch assay) pretreated with Tyrode buffer was added and pre-incubated for 15 min. The mixture was then stimulated with C48 / 80 for 30 min, and the reaction was terminated on ice. The supernatant was collected by centrifugation. Detection was performed using the β-aminohexosidase release assay: the supernatant was mixed with the substrate p-Nitrophenyl-N-acetyl...

[0100] -β-D-glucosaminidase was incubated at 37℃ for 1 h, terminated with glycine-NaOH buffer, and absorbance was measured at 405 nm. The model control group was treated with an equal volume of Tyrode buffer instead of the dressing dilution and stimulated with C48 / 80. A blank control group (without C48 / 80 stimulation) and a maximum release group (cells were lysed with 1% Triton X-100) were also included. Calculations were performed using the following formula:

[0101] Degranulation rate (%) = (Absorbance of experimental group - Absorbance of blank control group) / (Absorbance of maximum release group - Absorbance of blank control group) × 100%;

[0102] Degranulation inhibition rate (%) = [1 - Degranulation rate of experimental group / Degranulation rate of model control group] × 100%.

[0103] 2. Results Data

[0104] Model control group 73.8 — Comparative Example 1 71.2 3.5 Comparative Example 2 39.9 45.9 Comparative Example 3 38.4 48.0 Comparative Example 4 69.5 5.8 Comparative Example 5 36.9 50.0 Example 1 27.8 62.3 Example 2 28.8 61.0 Example 3 28.2 61.8

[0105] 3. Analysis

[0106] Comparative Examples 1 and 4 did not contain *Gnaphalium affine* extract, and their degranulation inhibition rates were extremely low. This indicates that the matrix or other components such as *Gnaphalium affine* extract, *Gnaphalium affine* extract, and inactivated bacteria alone cannot effectively inhibit mast cell degranulation, suggesting that *Gnaphalium affine* extract is an important component for achieving this function.

[0107] Among the groups containing *Gnaphalium affine* extract, the data showed that Comparative Example 2 had the weakest inhibition rate, Comparative Example 3 was in the middle, and Comparative Example 5 was better than the former two but still significantly lower than the Example group. This indicates that while *Gnaphalium affine* extract provides the core antipruritic activity, relying on only one or two auxiliary components cannot achieve the best effect. Only when *Gnaphalium affine* extract, *Gnaphalium affine* extract, and inactivated bacteria work together with *Gnaphalium affine* extract can the maximum efficacy in inhibiting mast cell degranulation be fully realized. The synergistic effect among the four active components is indispensable.

[0108] III. Animal Model Experiment of Postoperative Scar Itching

[0109] 1. Detection Method

[0110] Fifty-four male SPF-grade BALB / c mice, aged 6-8 weeks, were used. A full-thickness skin defect with a diameter of 0.8 cm was created on the back near the neck, and the wound was not sutured (the mice could scratch the wound normally with their hind limbs). On postoperative day 7, the mice were randomly divided into 9 groups (model control group, comparative examples 1-5, and examples 1-3), with 6 mice in each group. Starting from the day of grouping, the corresponding sample (1 mL / wound) was rinsed daily and allowed to air dry. The model control group was rinsed with physiological saline. The medication was administered continuously for 21 days (from postoperative day 7 to day 28). Behavioral video recordings were performed for 24 hours on postoperative days 14, 21, and 28. The number of scratches on the back wound by each mouse was counted (one scratch was defined as a single mouse continuously scratching its back until it puts its paw down). The average number of scratches at each time point was taken as the scratch count for that group. Medication was discontinued on postoperative day 28, and observation continued until day 42. The scratch count was recorded again on days 35 and 42 using the same method.

[0111] 2. Results Data

[0112] Model control group 76.0 101.9 129.0 140.1 150.9 Comparative Example 1 73.6 97.1 123.1 134.7 145.8 Comparative Example 2 48.0 50.7 55.2 53.3 52.4 Comparative Example 3 45.0 48.2 52.6 50.8 49.2 Comparative Example 4 71.1 92.8 118.2 130.0 141.7 Comparative Example 5 36.9 39.4 43.7 41.0 38.6 Example 1 26.9 25.7 28.1 25.1 22.8 Example 2 28.1 27.0 29.8 26.1 23.9 Example 3 24.6 23.7 26.3 23.0 20.6

[0113] 3. Analysis

[0114] 3.1 The number of scratches in the model control group, Comparative Example 1, and Comparative Example 4 all increased continuously over time. These three groups did not contain *Corydalis esculenta* extract, indicating that without intervention during scar formation, itching progressively worsens. Comparative Examples 2, 3, and 5, as well as the Example group, all contained *Corydalis esculenta* extract, and the number of scratches was effectively inhibited, confirming that *Corydalis esculenta* extract is the core component for inhibiting scar-related pruritus.

[0115] 3.2 Comparative Example 2, lacking both *Smilax china* extract and inactivated bacteria, exhibited the weakest antipruritic effect. Comparative Example 3, lacking *Smilax china* extract, showed an effect similar to Comparative Example 2, indicating that the absence of *Smilax china* extract resulted in delayed wound healing, insufficient protection of nerve endings, and incomplete itch relief. Comparative Example 5, lacking inactivated bacteria, showed an antipruritic effect superior to Comparative Examples 2 and 3, but still significantly weaker than the Example 5 group, indicating a significant reduction in the long-term itch-suppressing ability after the absence of inactivated bacteria. The number of scratches at each time point in all three Examples was far lower than in the Comparative Examples, demonstrating the synergistic effect among the four active ingredients.

[0116] 3.3 During the observation period after drug withdrawal, the number of scratches in the model control group, Comparative Example 1, and Comparative Example 4 continued to increase. No significant rebound in the number of scratches was observed in any of the groups containing *Gnaphalium affine* extract after drug withdrawal. However, the number of scratches in the Example group remained consistently lower than in the comparative examples after drug withdrawal, indicating that the Example group also maintained a sustained antipruritic effect after drug withdrawal.

[0117] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A bio-polysaccharide dressing that promotes wound healing, characterized in that, By mass-volume percentage, it includes 2.5%–3.0% sodium carboxymethyl cellulose, 0.6%–0.9% sodium chloride, 0.2%–0.3% anhydrous sodium acetate, 0.3%–0.5% sodium gluconate, 0.02%–0.03% magnesium chloride, 0.02%–0.04% potassium chloride, 0.14%–0.18% *Gnaphalium affine* extract, 0.08%–0.12% *Gnaphalium affine* extract, 0.04%–0.08% *Gnaphalium affine* extract, 0.03%–0.05% inactivated Lactobacillus cells, and the balance being purified water.

2. The bio-polysaccharide dressing for promoting wound repair according to claim 1, characterized in that, The inactivated Lactobacillus cells were prepared by the following method: A1. Inoculate the Lactobacillus strain into MRS liquid medium, incubate at 37°C for 18–24 h, collect the bacterial cells by centrifugation, and wash 2–3 times with sterile phosphate buffer. A2. After washing, the bacterial cells are resuspended in water for injection and inactivated by moist heat at 121°C for 15-20 min. The inactivated bacterial cells are collected by centrifugation and washed 2-3 times with purified water. A3. Freeze-dry the washed and inactivated bacterial cells, grind them at low temperature, and pass them through a 200-mesh sieve to obtain the final product.

3. The bio-polysaccharide dressing for promoting wound repair according to claim 1, characterized in that, The extract of *Houttuynia cordata* is prepared by the following process: *Houttuynia cordata* roots and stems are pulverized into 15-20 mesh coarse powder, and extracted 2-3 times with 8-10 times the amount of 70% ethanol under reflux for 90-120 minutes each time. The extracts are combined and concentrated under reduced pressure to a clear extract with a relative density of 1.10-1.15 at 60°C. The extract is then loaded onto a D101 macroporous resin column and eluted sequentially with 3-5 column volumes of purified water, 3-5 column volumes of 30% ethanol, and 4-6 column volumes of 70% ethanol. The 70% ethanol eluent is collected, concentrated under reduced pressure, and then freeze-dried to obtain the final product.

4. The bio-polysaccharide dressing for promoting wound repair according to claim 1, characterized in that, The snake-root extract is prepared by the following process: take the whole snake-root herb, crush it into a coarse powder of 15-20 mesh, add 10-12 times the amount of 70% ethanol, and extract it by ultrasonic extraction 2-3 times at a power of 300-400W, each time for 30-40 minutes. Combine the extracts, concentrate them under reduced pressure to a clear extract with a relative density of 1.05-1.10 at 60℃, and freeze-dry to obtain the final product.

5. The bio-polysaccharide dressing for promoting wound repair according to claim 1, characterized in that, The extract of *Smilax china* is prepared by the following process: take the whole herb of *Smilax china*, crush it into coarse powder of 15-20 mesh, add 8-10 times the amount of 0.05mol / L sodium hydroxide aqueous solution for cold soaking and extraction 2-3 times, each time for 10-12 hours, combine the extracts, add hydrochloric acid under stirring to adjust the pH to 4.0-4.5, let stand for 8-12 hours, collect the precipitate, wash with purified water until the effluent is neutral, freeze dry to obtain the extract.

6. The bio-polysaccharide dressing for promoting wound repair according to claim 1, characterized in that, The degree of substitution of the sodium carboxymethyl cellulose is 0.7 to 0.9, and the dynamic viscosity of its 2% aqueous solution at 25°C is 2500 to 3000 mPa·s.

7. A method for preparing the biopolysaccharide dressing according to any one of claims 1 to 6, characterized in that, Includes the following steps: S1. The freeze-dried powders of *Houttuynia cordata* extract, *Gnaphalium affine* extract, and *Gnaphalium affine* extract are passed through a 200-mesh sieve and stored in the dark and refrigerated for later use. S2. Take 60% of the formula amount of water for injection, heat it to 40-50℃, and slowly add sodium carboxymethyl cellulose while stirring at 100-120r / min. Continue stirring for 1.5-2h until it is completely swollen and forms a transparent and uniform viscous solution. S3. Cool the adhesive solution to 25-30℃, and add sodium chloride, anhydrous sodium acetate, sodium gluconate, magnesium chloride and potassium chloride in sequence according to the formula, and stir until completely dissolved; S4. Adjust the pH of the system to 6.5–7.5 using 0.1 mol / L sodium hydroxide solution or 0.1 mol / L acetic acid solution; S5. After pre-dispersing the three extracts prepared in step S1 and the inactivated lactobacillus cells in the prescribed amount with 6 to 8 times the total weight of the extracts in water for injection, slowly add them to the above gel solution under light protection and stirring at 60 to 80 r / min. Add the remaining water for injection to the total volume, and continue stirring for 20 to 30 minutes until the mixture is uniform.

8. The preparation method according to claim 7, characterized in that, After step S5, the following will also be executed: S6. Fill the well-mixed adhesive solution into a medical-grade brown light-proof container and seal it. Sterilize it by moist heat at 115℃~121℃ for 25~30 minutes.

9. The preparation method according to claim 8, characterized in that, After sterilization in step S6, a gradient cooling method is used: first, the temperature is reduced to 80°C at a rate of 1°C / min, and then naturally cooled to room temperature.