Bletilla striata compound gel for promoting wound healing as well as preparation method and application of bletilla striata compound gel

Abstract

The invention relates to the technical field of biological medicine, in particular to bletilla striata compound gel for promoting wound healing and a preparation method and application thereof. Mixing chitosan, carboxymethyl chitosan, oxidized regenerated fibers and a sodium hydroxide solution to obtain a carrier substrate glue solution; mixing the carrier substrate gel solution with the human epidermal growth factor, and storing to obtain carrier gel; mixing the carrier gel with the bletilla striata polysaccharide solution to obtain composite gel; mixing the compound gel with bletilla striata plant essential oil, and storing to obtain the bletilla striata compound gel. The bletilla striata compound gel provided by the invention can promote wound healing, reduce the content of inflammatory factors IL-1beta of injured skin, promote generation of endovascular epidermal factors of the injured skin, promote regeneration of I-type collagen of the injured skin, reduce the content of IV-type collagen of the injured skin and promote generation of fibronectin of the injured skin.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to a bletilla striata compound gel for promoting wound healing, and a preparation method and application thereof. Background Art

[0002] Wound healing is an evolutionarily conserved, complex, multicellular process that, in the skin, aims to restore the barrier. This process involves the coordinated efforts of several cell types, including keratinocytes, fibroblasts, endothelial cells, macrophages, and platelets. The migration, infiltration, proliferation, and differentiation of these cells will ultimately lead to an inflammatory response, the formation of new tissue, and ultimately closure of the wound. This complex process is executed and regulated by an equally complex signal transduction network involving many growth factors, cytokines, and proteins. Of particular importance are vascular endothelial growth factor (VEGF), the interleukin (IL) family, type I collagen (CoI-I), α-SMA, type IV collagen (CoI-IV), and fibronectin.

[0003] Bletilla is a perennial herbaceous plant of the Orchidaceae family and the genus Bletilla. Its rhizomes contain a variety of secondary metabolites such as polysaccharides, mucilage, steroids, terpenes, esters, ethers, and trace elements such as boron, molybdenum, and manganese. It has multiple functions such as hemostasis, antibacterial, swelling, and mucosal protection. Bletilla polysaccharide is also called "Bletilla glue" because of its high viscosity. It is a natural polysaccharide substance that exists in large quantities in the rhizomes of Bletilla. It has the advantages of being non-toxic, low in irritation, and non-antigen specific.

[0004] There is no report on preparing Bletilla striata polysaccharide into gel in the prior art. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a Bletilla striata compound gel for promoting wound healing, and a preparation method and application thereof. The Bletilla striata compound gel provided by the present invention has a promoting effect on skin wound healing.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a method for preparing a Bletilla striata compound gel for promoting wound healing, comprising the following steps:

[0008] 1) mixing chitosan, carboxymethyl chitosan, oxidized regenerated fiber and sodium hydroxide solution to obtain a carrier-based adhesive solution;

[0009] 2) mixing the carrier base gel solution obtained in step 1) with human epidermal growth factor and storing them to obtain a carrier gel;

[0010] 3) mixing the carrier gel obtained in step 2) with the bletilla striata polysaccharide solution to obtain a composite gel;

[0011] The mass percentage of Bletilla striata polysaccharide in the Bletilla striata polysaccharide solution is 1-5%;

[0012] 4) mixing the composite gel obtained in step 3) with Bletilla striata plant essential oil and storing them to obtain Bletilla striata composite gel.

[0013] Preferably, in step 1), the volume ratio of the mass of chitosan, the mass of carboxymethyl chitosan, the mass of oxidized regenerated fiber and the sodium hydroxide solution is 10g:10g:0.2g:25mL;

[0014] The mass percentage of the sodium hydroxide solution is 1%.

[0015] Preferably, in step 2), the mass ratio of the carrier base gel solution to human epidermal growth factor is 50:1.

[0016] Preferably, the mixing in step 2) is carried out under stirring, and the stirring time is 30 to 60 minutes;

[0017] The storage temperature is 4° C. and the storage time is 20 to 40 hours.

[0018] Preferably, in step 3), the volume ratio of the carrier gel to the Bletilla striata polysaccharide solution is 1:1.

[0019] Preferably, the volume ratio of the composite gel to the Bletilla striata plant essential oil in step 4) is 10:1;

[0020] The mixing is carried out under stirring, and the stirring time is 30 to 60 minutes;

[0021] The storage temperature is 4° C. and the storage time is 20 to 40 hours.

[0022] The present invention also provides a Bletilla striata compound gel prepared by the preparation method described in the above technical scheme.

[0023] The present invention also provides the use of the bletilla striata compound gel described in the above technical solution in the preparation of a medicine for promoting wound healing.

[0024] The present invention also provides the use of the bletilla striata compound gel described in the above technical solution in the preparation of a drug for reducing the content of inflammatory factor IL-1β in damaged skin and / or promoting the production of epidermal factor in damaged skin blood vessels.

[0025] The present invention also provides the use of the bletilla striata compound gel described in the above technical solution in the preparation of a drug for promoting the regeneration of type I collagen in damaged skin, reducing the content of type IV collagen in damaged skin and promoting the production of fibronectin in damaged skin.

[0026] Beneficial effects of the present invention:

[0027] The bletilla striata compound gel provided by the present invention promotes wound healing, reduces the content of IL-1β, an inflammatory factor in damaged skin, promotes the production of epidermal factor in damaged skin blood vessels, promotes the regeneration of type I collagen in damaged skin, reduces the content of type IV collagen in damaged skin, and promotes the production of fibronectin in damaged skin. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0029] Figure 1 Schematic diagram of the SD rat skin full-section model;

[0030] Figure 2 This is a real picture of the SD rat skin full-cut model during the experiment;

[0031] Figure 3 The VAS evaluation of Bletilla striata compound gel promoting skin wound repair in SD rats, where I: blank group; II: model group; III: Bletilla striata compound gel group; IV: vehicle gel group; V: Buck group;

[0032] Figure 4 The results of HE staining of Bletilla striata compound gel promoting skin tissue repair in SD rats, where I: blank group; II: model group; III: Bletilla striata compound gel group; IV: vehicle gel group; V: Buck group;

[0033] Figure 5 The effect of Bletilla striata compound gel on interleukin IL-1β in the serum of SD rats;

[0034] Figure 6 The level of interleukin-1L-1β in the serum of SD rats treated with Bletilla striata compound gel on the 25th day (P<0.05);

[0035] Figure 7 The effect of Bletilla striata compound gel on VEGF in the serum of SD rats;

[0036] Figure 8 The level of interleukin VEGF in the serum of SD rats treated with Bletilla striata compound gel (P<0.05);

[0037] Figure 9-17It is the detection of proteins type I collagen (Col-I), α-smooth muscle actin (α-SMA), type IV collagen (Col-IV), and fibronectin (FN) in the serum of SD rats, where I: blank group; II: model group; III: Buck group; IV: Bletilla striata compound gel group; V: carrier gel group. DETAILED DESCRIPTION

[0038] The present invention provides a method for preparing a Bletilla striata compound gel for promoting wound healing, comprising the following steps:

[0039] 1) mixing chitosan, carboxymethyl chitosan, oxidized regenerated fiber and sodium hydroxide solution to obtain a carrier-based adhesive solution;

[0040] 2) mixing the carrier base gel solution obtained in step 1) with human epidermal growth factor and storing them to obtain a carrier gel;

[0041] 3) mixing the carrier gel obtained in step 2) with the bletilla striata polysaccharide solution to obtain a composite gel;

[0042] The mass percentage of Bletilla striata polysaccharide in the Bletilla striata polysaccharide solution is 1-5%;

[0043] 4) mixing the composite gel obtained in step 3) with Bletilla striata plant essential oil and storing them to obtain Bletilla striata composite gel.

[0044] The invention mixes chitosan, carboxymethyl chitosan, oxidized regenerated fiber and sodium hydroxide solution to obtain a carrier-based base glue solution.

[0045] In the present invention, the mass of the chitosan, the mass of the carboxymethyl chitosan, the mass of the oxidized regenerated fiber and the volume ratio of the sodium hydroxide solution are preferably 10g:10g:0.2g:25mL. In the present invention, the chitosan and carboxymethyl chitosan can form a three-dimensional pore structure through a cross-linking agent. As a gel scaffold, chitosan itself also has antibacterial activity and the effect of promoting wound healing. The oxidized regenerated fiber is used to adsorb wound extravasated tissue fluid, which is beneficial to maintaining a sterile environment on the wound surface. The innovation of the present invention is the different ratios of these elements, and such a combination has the best effect in the present invention. In the present invention, the mass percentage of the sodium hydroxide solution is preferably 1%. The present invention preferably dissolves the chitosan and carboxymethyl chitosan in a sodium hydroxide solution and then mixes them with the oxidized regenerated fiber. The present invention does not specifically limit the sources of the chitosan, carboxymethyl chitosan and oxidized regenerated fiber, and conventional commercial products can be used, such as chitosan from TCI Chemical Industry Development Co., Ltd., carboxymethyl chitosan from TCI Chemical Industry Development Co., Ltd., and oxidized regenerated fiber from Duolemeisong Trading Co., Ltd.

[0046] The present invention mixes and stores the obtained carrier base gel solution with human epidermal growth factor to obtain a carrier gel. In the present invention, the volume ratio of the carrier base gel solution to the human epidermal growth factor is preferably 50:1. In the present invention, the human epidermal growth factor is preferably an external human epidermal growth factor, and the function of the human epidermal growth factor is to promote the proliferation and differentiation of normal epidermal cells. In the present invention, the human epidermal growth factor is purchased from Shanghai Wuhai Biotechnology Co., Ltd., item number S20010099. In the present invention, the mixing is preferably carried out under stirring, and the stirring time is preferably 30 to 60 minutes. In the present invention, the storage temperature is 4°C, and the time is preferably 20 to 40 hours.

[0047] The present invention mixes the obtained carrier gel with a bletilla polysaccharide solution to obtain a composite gel; the mass percentage of bletilla polysaccharide in the bletilla polysaccharide solution is 1-5%. In the present invention, the volume ratio of the carrier gel to the bletilla polysaccharide solution is preferably 1:1. In the present invention, the bletilla polysaccharide is purchased from Nantong Feiyu Biotechnology Co., Ltd., with the item number FY3714-Y10, and the bletilla polysaccharide powder is purchased. As the basic material in this research system, bletilla polysaccharide is also called "bletilla glue" because of its rich viscosity. It is a natural polysaccharide substance that exists in large quantities in the rhizome of bletilla striata, and has the advantages of non-toxicity, low irritation, and no antigen specificity. Modern scientific research has found that bletilla striata contains rich active ingredients such as flavonoids, alkaloids and polysaccharides, and these ingredients have the most significant pharmacological effects on hemostasis, detumescence, and myogenesis. It is favored by the market due to its significant effect and strong applicability, and the developed series of products have been widely used in clinical practice. Published research results show that Bletilla striata gum can promote blood coagulation, reduce bleeding, and show excellent hemostatic effects. At the same time, through in vitro antibacterial experiments, it can be found that Bletilla striata gum can significantly inhibit the growth of Gram-positive bacteria (Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli), relieve clinical symptoms such as redness, swelling, heat, and pain caused by inflammatory reactions, and thus reduce the occurrence of adverse symptoms during wound healing. In addition, according to existing studies, Bletilla striata gum can promote wound healing and the formation of related collagen, such as type I collagen and fibronectin, thereby accelerating the regeneration of skin cortical tissue and having a good therapeutic effect on various wounds such as burns and trauma. At the same time, it can also eliminate swelling and reduce edema symptoms; the Bletilla striata polysaccharide gel matrix meets the standards of the wound moist healing theory, can avoid bacterial infection, absorb wound secretions, remove obstacles to wound repair, and provide a stable protective barrier for the wound.

[0048] The invention mixes and stores the obtained composite gel with bletilla striata plant essential oil to obtain the bletilla striata compound gel.

[0049] In the present invention, the volume ratio of the composite gel to the Bletilla striata plant essential oil is preferably 10:1. In the present invention, the mixing is preferably carried out under stirring, and the stirring time is preferably 30 to 60 minutes. In the present invention, the storage time is preferably 20 to 40 hours. In the present invention, the functions of the Bletilla striata plant essential oil are antibacterial, aromatherapy, lubrication and moisturizing. The present invention does not specifically limit the source of the Bletilla striata plant essential oil, and it can be prepared by a conventional preparation method or a commercially available product. For example, the Bletilla striata plant essential oil is extracted by supercritical carbon dioxide extraction, and the process conditions are extraction pressure 16MPa, ultrasonic frequency 20KHz, ultrasonic power 240W, extraction temperature 50°C, extraction time 80min, and the average extraction rate of essential oil is 1.92%.

[0050] The present invention also provides a Bletilla striata compound gel prepared by the preparation method described in the above technical scheme.

[0051] The present invention also provides the use of the Bletilla striata compound gel described in the above technical solution in the preparation of a drug for promoting wound healing. The present invention has no special limitation on the dosage form and preparation method of the drug, and those skilled in the art can follow the routine method.

[0052] The present invention also provides the use of the Bletilla compound gel described in the above technical solution in the preparation of a drug for reducing the content of IL-1β, an inflammatory factor in damaged skin, and / or promoting the production of epidermal factor in damaged skin blood vessels. The present invention does not specifically limit the dosage form and preparation method of the drug, and those skilled in the art can follow the routine method.

[0053] The present invention also provides the use of the Bletilla striata compound gel described in the above technical solution in the preparation of a drug for promoting the regeneration of type I collagen in damaged skin, reducing the content of type IV collagen in damaged skin, and promoting the production of fibronectin in damaged skin. The present invention does not specifically limit the dosage form and preparation method of the drug, and those skilled in the art can follow the routine method.

[0054] In order to further illustrate the present invention, the present invention is described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0055] Example 1

[0056] A method for preparing a Bletilla striata compound gel for promoting wound healing, comprising the following steps:

[0057] Step 1, preparation of bletilla striata gum: accurately weigh 5 g of bletilla striata polysaccharide powder and dissolve it in 1% acetic acid solution to prepare a bletilla striata gum solution with a mass percentage of 5%.

[0058] Step 2, preparation of carrier-based matrix glue: weigh 10 g of chitosan powder, 10 g of carboxymethyl chitosan powder, and 0.2 g of oxidized regenerated cellulose respectively, and dissolve them in 0.1 mol / L NaOH solution.

[0059] Step 3, preparation of carrier gel: add external human epidermal growth factor to the carrier base gel at a ratio of 20% by mass (the volume ratio of the carrier base gel solution to the human epidermal growth factor is preferably 50:1), stir at room temperature for 30 minutes, and store dry and refrigerated at 4°C for 20 hours to obtain the carrier gel.

[0060] Step 4, preparation of Bletilla striata compound gel: adding the prepared Bletilla striata gel solution to the carrier gel to obtain a composite gel mixture, adding Bletilla striata essential oil according to a volume ratio of the composite gel mixture to Bletilla striata essential oil of 10:1, magnetically stirring at room temperature for 50 minutes, and storing in a cold and dry state at 4°C for 20 hours to obtain a Bletilla striata compound gel.

[0061] Effect Example 1

[0062] 1. Methods and steps

[0063] 1. Experimental grouping of SD rats

[0064] Thirty healthy SD rats, half male and half female, weighing 200±20g, were selected and purchased from the Experimental Animal Center of the Fourth Military Medical University of the Chinese People's Liberation Army. The animal license number is SCXX(Shaanxi)2023-002. They were divided into 5 groups by random number method: blank group, model group, Bletilla compound gel group, carrier gel group, and Buck group (Buck is the Chinese transliteration trade name, purchased from Advanced Biotechnology Co., Ltd., item number 20162140383), as shown in Table 1:

[0065] Table 1 Grouping of SD rats

[0066] Group Alcohol Iodine Bletilla striata polysaccharide gel Buck Blank Group - - - - Model Group + + - - Bletilla striata compound gel group + - + - Buck Group + - - +

[0067] Note: “+” indicates that the wounds of SD rats were treated with the substance, and “-” indicates that the wounds of SD rats were not treated with the substance.

[0068] 2. Construction of the Skin Dermis Full-cut Model

[0069] After one week of adaptive feeding, the SD rats were fasted for 4 hours and anesthetized by subcutaneous intravenous injection of 30 mg / kg sodium pentobarbital. The rats' toes were gently pinched and stimulated with tweezers. If there was no reaction, the rats were considered to be anesthetized successfully. Two circular wounds with a diameter of d = 2 ± 0.5 cm were cut along both sides of the rat spine with surgical scissors and tweezers to form full-thickness skin defect wounds, avoiding visible blood vessels. Figure 1 shown.

[0070] After the model was successfully established, a skin incision model was made for SD rats to cause skin trauma. Treatment began on the first day. 0.1 ml was used for each wound. The gauze was changed every 12 hours and the drug was applied once. The wound healing of the rats, including the changes in wound color, area, and bleeding, was observed and recorded until the evaluation results of the Bletilla Compound Gel Group were all healed. The experiment was terminated after the evaluation results of the Bletilla Compound Gel Group were healed. Photos were taken every day during the experiment, and the experiment was terminated after the wounds of the Bletilla Compound Gel Group were healed.

[0071] 3. Visual analogue scales (VAS) for wound pain

[0072] During the experiment, the dressing was changed regularly and the wound was observed for flatness, roughness, pigmentation, hypertrophic scars, and other indicators. The wound area and the time of complete wound healing were recorded. The efficacy of gel in promoting skin wound repair was evaluated according to the "Standards for Diagnosis and Efficacy of Chinese Medicine Diseases and Syndromes", as shown in Table 2:

[0073] Table 2 Evaluation criteria for wound healing efficacy

[0074] Wound healing rate Wound condition Evaluation results 90％-100％ Wound healed or almost healed get well >50％ The granulation tissue is light red, without pus, and the redness and swelling around the wound surface have basically disappeared. Significant effect 20％-50％ The granulation tissue is bright red, with a small amount of pus, and the wound surface is slightly red and swollen efficient <20％ No significant change or even worsening invalid

[0075] V 伤口愈合 =(S 原始伤口 -S 治疗后伤口 )÷S 原始伤口 ×100%

[0076] Wound healing efficiency calculation formula (V: rate; S: area)

[0077] BE=(N 痊愈 +N 显效 +N 有效 )÷N total×100%

[0078] Wound treatment total effective rate calculation formula: (BE: overall effective rate; N: total number of sample individuals)

[0079] 4. Tissue Sectioning and HE Staining

[0080] Tissue collection: After the wound repair of the experimental group reached the healing standard, the circular wound with full-thickness skin defect was completely removed to prepare the full-thickness skin (epidermis, dermis and subcutaneous tissue) specimens.

[0081] Tissue embedding: The skin tissue samples were fixed with 4% paraformaldehyde for 24 hours, and the tissues were trimmed as needed to ensure that the wound was located in the center of the specimen. After rinsing with running water for 30 minutes, they were placed in a dehydration box, dehydrated and waxed with a dehydrator, and then embedded with paraffin.

[0082] Tissue preparation: Use a microtome to cut the tissue into slices with a thickness of 5 cm. Flatten the tissue in 42°C warm water and spread it on a glass slide. Bake the slices at 60°C for 120±30 min.

[0083] Dewaxing and entering water: dewaxing with xylene I for 15 min, dewaxing with xylene II for 15 min, anhydrous ethanol for 5 min, 90% ethanol for 5 min, 80% ethanol for 5 min, 70% ethanol for 5 min, and rinsing with distilled water for 5 min.

[0084] HE staining: hematoxylin staining for 30 minutes, washing with running water for 20 minutes, hydrochloric acid alcohol differentiation, eosin staining for 5 minutes, and then dehydration and transparency followed by sealing with resin glue. The histopathological changes were observed by electron microscopy, and image acquisition and analysis were performed using a microscopic imaging system.

[0085] 5. Elisa detection of cytokines in serum

[0086] On days 0, 5, 10, 15, 20, and 25 after the full-excision wound model was established, 2 mL of jugular venous blood was collected from all experimental mice, and the blood was centrifuged at 3000 rpm and 25°C for 10 min to obtain serum, which was tested using an ELISA kit in strict accordance with the instructions of the kit.

[0087] The kit uses a double antibody sandwich method to add samples, standards and P-labeled detection antibodies to the pre-coated antibodies, incubate and wash thoroughly. The color of the reaction with the substrates TMB and IMB is converted to blue under the catalysis of peroxidase and to yellow in the presence of acid.

[0088] After equilibration at room temperature for 20 minutes, remove the required strips from the aluminum foil bag and put the remaining strips back into the ziplock bag and place at 4°C. Set up standard wells and sample wells, standard concentration 50μL, sample well A 10μL, sample dilution 40μL, and no blank wells. Except for the blank wells, 100μL of horseradish peroxidase (HRP)-labeled detection antibody is added to each well of the sealed plate membrane and incubated in a 37°C water bath or constant temperature incubator for 60min. Remove the liquid, pat dry on absorbent paper, fill with washing solution, let stand for 1 minute, shake off the washing solution, pat dry on absorbent paper, and wash the plate repeatedly 5 times. 50μL of color development solution A and B are added to each well respectively and incubated at 37°C for 15min. Add 50μL of stop solution to each well for 15 minutes and measure the OD of each well at 450nm.

[0089] After adding the stop liquid, the color of the 96 wells changed from blue to yellow, and the color depth was positively correlated with the concentration of IL-1β and VEGF in the sample. The absorbance was measured at 450nm, and the concentration of IL-1β and VEGF in the sample was calculated using the standard curve, and the dynamic change trend of their concentrations was compared and analyzed.

[0090] Finally, draw the standard curve: In the Excel worksheet, use the standard concentration as the horizontal axis and the corresponding OD value as the vertical axis. Draw the linear regression curve of the standard concentration and calculate the concentration value of each sample according to the curve equation.

[0091] 6. Western Blot Quantitative Analysis of Wound Skin Tissue

[0092] Protein extraction: add skin samples to RIPAS lysis solution containing protease inhibitors, homogenize with tissue disruptor, place at 4°C for 15 minutes, centrifuge at 12000 rpm for 10 minutes at 4°C, and take the supernatant as the protein sample. Homogenize with tissue disruptor, place at 4°C for 15 minutes, centrifuge at 12000 rpm for 10 minutes at 4°C, and take the supernatant as the protein sample.

[0093] Protein quantification: Use BCA protein quantification kit for quantification. Dilute the sample 5 times for the experiment and perform quantification according to the kit instructions.

[0094] Protein denaturation: Use lysis buffer to adjust the concentration of all samples to the same level, add the corresponding volume of 5× loading buffer, boil for denaturation for 5 min, centrifuge at 12000 rpm and 4°C for 5 min, and store in aliquots and freeze.

[0095] Protein loading: about 15 g of sample was loaded, and the loading order of each group was: blank group, model group, Buck group, Bletilla striata compound gel group, and carrier gel group.

[0096] Electrophoresis: 6% concentrated gel at 80V for 25min, 8% separation gel at 120V for 70min

[0097] Transfer: Use 0.22 PVDF membrane and transfer at a constant current of 400 mA for 70 minutes. Take the membrane corresponding to the target protein for antibody incubation.

[0098] Primary antibody incubation: Use 5% skim milk powder to block at room temperature for 2.5 hours, wash the membrane with TBST (10 minutes / time, repeated three times) and then incubate with primary antibody (antibody dilution is the same as blocking solution). The dilution ratios of various antibodies are shown in the table below. The incubation conditions are incubation at 4°C overnight, and then incubation at room temperature for 1 hour. As shown in Table 3, antibody usage:

[0099] Table 3 Antibody usage

[0100] Antibody Name brand Part Number Molecular weight (KD) Species Dilution ratio BetaActin Proteintech 66009-1-Ig 42 mouse 1：10000 Co1-Ⅰ Proteintech 66761-1-Ig 130 mouse 1：2000 Co1-Ⅳ Proteintech 19674-1-AP 164 rabbit 1：1000 Fibronectin Proteintech 66042-1-Ig 263 mouse 1：2000 SMA Proteintech 67735-1-Ig 42 mouse 1：20000 Goat anti-rabbit Yike Biotechnology YK2231 Secondary Antibodies goat 1：10000 Goat anti-mouse Yike Biotechnology YK2232 Secondary Antibodies goat 1：10000

[0101] Secondary antibody incubation: After washing the membrane three times with TBST, incubate with secondary antibody (antibody dilution solution is the same as blocking solution) at room temperature for 1 hour. After washing the membrane three times with TBST, expose, develop and fix the membrane. β-actin was used as an internal reference to calculate the relative expression of each protein.

[0102] 2. Statistical Processing

[0103] SPSS25.0 software was used to analyze the data. The results were expressed as X±S. The significant differences among the groups were analyzed using the Duncan method. The results of multiple comparisons were marked with asterisks. ** indicates P<0.01, and * indicates P<0.05.

[0104] 3. Results and Analysis

[0105] 3.1 Visual analogue scale for wound pain

[0106] During the experiment, the rats' hair was shiny, and their mental state and daily activities remained within the normal range. The wound treatment effect evaluation after SD rat modeling was as follows: Figure 2 shown.

[0107] After 5 days of drug-mediated treatment, the wounds of all experimental rats began to be covered with scabs to varying degrees. At the same time, the skin wounds of the rats in the blank group and the model group showed different degrees of bleeding and wound tearing. No large-scale exudates were found on the skin wounds of the rats treated with the Bletilla compound group, the carrier gel group and the Buck group. None of the above adverse conditions occurred. Among them, on the 15th day after drug-mediated treatment, the treatment effect of the Bletilla compound group was the most significant, with a wound healing rate of 88.91±1.36%, and the evaluation result was cured. On the 25th day of treatment, the degree of wound skin healing of SD rats in each group was as follows: Bletilla compound gel group 98.40±0.97%> carrier gel group 94.37±5.42% Buck group 92.71±2.07%> model group 91.58±4.84%> blank group 89.83±2.31%.

[0108] A total of 60 wound tissue samples were collected, and the results of visual simulation assessment of the wounds showed that on the 15th day, 41 cases were significantly effective; 19 cases were cured, including 8 cases in the Bletilla striata compound gel group, 6 cases in the carrier gel group, 5 cases in the Buck group, and 1 case in the model group; all wounds were cured on the 25th day, and the total effective rate of wound treatment was 100%.

[0109] Table 4 Visual analogue score of SD rats in each group (X±S) (n=12) (P<0.01)

[0110] Grouping Maximum Minimum VAS% Blank Group 93.14％ 86.03％ 89.83±2.31 Model Group 98.40％ 82.36％ 91.58±4.84 Bletilla compound gel group 99.75％ 96.58％ 98.40±0.97 Carrier gel group 98.03％ 86.78％ 94.37±3.09 Buck Group 95.45％ 89.76％ 92.71±2.07

[0111] 3.2 Effects of gel on skin histopathology

[0112] After 25 days of drug-mediated treatment, when all the wounds of the SD rats in the compound Bletilla striata gel group were evaluated to be healed, the rats in each group were killed and the skin of the healed wounds on the back was cut for detection. Figure 3As shown in the figure, the skin cortical tissue cells of SD rats in the blank group and the model group were loose, the layers were unclear and there was no significant pattern; compared with the blank group, after the application treatment of the compound bletilla gel group and the buck group, the skin epidermis was thicker and distributed in a wavy pattern, the cells were distributed neatly, and the collagen fiber bundles in the dermis were tightly arranged with obvious patterns, indicating that the compound gel and buck had significant effects on the treatment of damaged skin.

[0113] 3.3 Effect of gel on serum cytokines

[0114] 3.3.1 Gel inhibits the expression of inflammatory factor IL-1β

[0115] Interleukin IL-1β in serum is an important indicator of the extent of inflammatory response during skin tissue repair. If it is overexpressed, a large amount of tissue exudate will accumulate on the wound surface, leading to tissue necrosis, pigmentation accumulation, and gradually forming scars.

[0116] In the inflammatory response stage after skin injury, i.e., day 0 to day 5, the blank group used a normal saline model to clean the wound surface stains. The interleukin IL-1β concentration in the serum of SD rats in the Bletilla compound gel group was 1.071±0.071μg / mL, which was significantly lower than that in the blank group (1.565±0.152μg / mL (P<0.05) (Table 5). In the cell proliferation and scar formation stage, i.e., day 20 to 25, the interleukin IL-1β concentration in the serum of SD rats in the Bletilla compound gel group was 1.071±0.071μg / mL, which was significantly lower than that in the blank group (1.565±0.152μg / mL (P<0.05) (Table 5). Figure 5 ), the interleukin IL-1β content in the Bletilla compound gel group was 0.480±0.038μg / mL, which showed an overall downward trend compared with the blank group 0.743±0.052μg / mL (P<0.05). This indicates that the Bletilla compound gel group can improve the local inflammatory response of the wound surface, inhibit the excessive inflammatory response, and effectively organize the production of inflammatory exudate to form scar connective tissue on the wound surface.

[0117] Table 5 Expression levels of interleukin IL-1β in serum of SD rats in each group (n=6) (P<0.01) (μg / mL)

[0118]

[0119]

[0120] 3.3.2 VEGF detection results

[0121] In the stage of angiogenesis and endothelial cell proliferation, VEGF is one of the more critical regulatory factors, which can promote angiogenesis and tissue repair and regeneration. When its expression level is too high, it will promote the rapid reproduction of collagen to achieve the effect of efficient wound repair.

[0122] Table 6 VEGF expression levels in serum of SD rats in each group (n=6) (P<0.05) (μg / mL)

[0123] Days / Groups Blank Group Model Group Bletilla compound gel group Carrier gel group Buck Group 0 0.228±0.085 0.380±0.070 0.230±0.152 0.263±0.112 0.310±0.108 5 0.150±0.118 0.269±0.167 0.314±0.131 0.311±0.146 0.211±0.187 10 0.316±0.118 0.290±0.113 0.329±0.117 0.376±0.123 0.367±0.066 15 0.322±0.134 0.318±0.173 0.444±0.067 0.384±0.030 .0328±0.291 20 0.332±0.228 0.472±0.288 0.530±0.182 0.460±0.362 0.449±0.272 25 0.391±0.275 0.453±0.219 0.652±0.240 0.585±0.245 0.593±0.273

[0124] The experimental results showed that the level of VEGF showed regular changes in the injured tissues of SD rats ( Figure 7 ). In the inflammatory response stage after skin injury, i.e., on the 5th day, the VEGF concentration in the serum of SD rats in the Bletilla striata compound gel group was 0.314±0.131μg / mL, which was significantly higher than that in the blank group (0.150±0.118μg / mL) (P<0.05). In the cell proliferation and scar formation stage, i.e., on the 25th day, the VEGF expression in the Bletilla striata compound gel group was 0.625±0.240μg / mL, which was higher than that in the blank group (0.314±0.131μg / mL) (P<0.05). This indicates that the Bletilla striata compound gel group can promote the production of epidermal factors in the blood vessels of the wound surface to ensure sufficient oxygen in the process of tissue repair, reduce the production of necrotic tissue, and thus quickly repair the wound surface.

[0125] 3.4 Effects of gel on key proteins in skin regeneration

[0126] The internal reference protein β-actin was selected, and the chemiluminescence imaging operation was performed on the protein detection results of each group. The exposure results were analyzed using the image analysis software GIS1D that came with the imager. Samples were loaded in order, and the net optical density values ​​of the final bands were as follows: Fig. 9 , respectively: blank group 416653, model group 397738, Buck group 368780, Bletilla striata compound gel group 334323, carrier gel group 357806. According to this value, the protein expression concentration of various proteins in rat skin tissue under different treatment conditions was calculated.

[0127] 3.4.1 Quantitative analysis results of type I collagen

[0128] The protein expression concentration of type I collagen (Col-I) in rat skin tissue under different treatment conditions (e.g. Fig.11 ) was 0.84μg / mL for compound Bletilla striata gel group>0.747μg / mL for vehicle gel group>0.719μg / mL for Buck group>0.508μg / mL for blank group>0.493μg / mL for model group. Among them, compound Bletilla striata gel had the most significant effect in promoting the regeneration of key protein Col-I in skin tissue, and had the effect of promoting skin repair.

[0129] 3.4.2 Quantitative analysis results of α-smooth muscle actin

[0130] Protein expression concentration of α-smooth muscle actin (α-SMA) in rat skin tissue under different treatment conditions (e.g. Fig.13 ) was blank group 0.654μg / mL> Bletilla compound gel group 0.495μg / mL> Buck group 0.421μg / mL> carrier gel group 0.190μg / mL> model group 0.132μg / mL. The results showed that the protein expression concentration of α-SMA in the blank group was the highest at 0.654μg / mL, followed by the Bletilla compound gel group at 0.495μg / mL, indicating that the inhibitory effect of Bletilla compound gel on α-SMA was not significant enough.

[0131] 3.4.3 Quantitative analysis results of type IV collagen

[0132] The protein expression concentration of type IV collagen (Col-IV) in rat skin tissue under different treatment conditions (e.g. Fig.15 ) was 0.693μg / mL in the model group>0.550μg / mL in the Buck group>0.176μg / mL in the blank group>0.164μg / mL in the carrier gel group>0.159μg / mL in the Bletilla compound gel group. The results showed that the protein expression concentration of Col-IV in the Bletilla compound gel group was the lowest, with a significant inhibitory effect, which could reduce skin tissue fibrosis and inhibit scar formation.

[0133] 3.4.4 Results of quantitative analysis of fibronectin

[0134] Fibronectin (FN) protein expression concentration in rat skin tissue under different treatment conditions (e.g. Fig.17 ) was carrier gel group 0.197μg / mL> Buck group 0.189μg / mL> Bletilla compound gel group 0.184μg / mL> model group 0.139μg / mL> blank group 0.058μg / mL. The results showed that the protein expression concentration of FN in the carrier gel group was the highest and that in the blank group was the lowest, indicating that it has a significant effect on promoting the production of FN and can promote the repair of skin cortical tissue.

[0135] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing a Bletilla striata compound gel for promoting wound healing, characterized in that: The following steps are involved: 1) mixing chitosan, carboxymethyl chitosan, oxidized regenerated fiber and sodium hydroxide solution to obtain a carrier-based glue solution; 2) mixing the carrier base gel solution obtained in step 1) with human epidermal growth factor and storing them to obtain a carrier gel; 3) mixing the carrier gel obtained in step 2) with the bletilla striata polysaccharide solution to obtain a composite gel; The mass percentage of Bletilla striata polysaccharide in the Bletilla striata polysaccharide solution is 1-5%; 4) mixing the composite gel obtained in step 3) with Bletilla striata plant essential oil and storing them to obtain Bletilla striata composite gel.

2. The preparation method according to claim 1, characterized in that: In the step 1), the volume ratio of the mass of chitosan, the mass of carboxymethyl chitosan, the mass of oxidized regenerated fiber and the sodium hydroxide solution is 10g:10g:0.2g:25mL; The mass percentage of the sodium hydroxide solution is 1%.

3. The preparation method according to claim 1, characterized in that: In the step 2), the mass ratio of the carrier base gel solution to human epidermal growth factor is 50:

1.

4. The preparation method according to claim 1, characterized in that: The step 2) mixing is carried out under stirring, and the stirring time is 30 to 60 minutes; The storage temperature is 4° C. and the storage time is 20 to 40 hours.

5. The preparation method according to claim 1, characterized in that: In the step 3), the volume ratio of the carrier gel to the bletilla striata polysaccharide solution is 1:

1.

6. The preparation method according to claim 1, characterized in that: In the step 4), the volume ratio of the composite gel to the Bletilla striata plant essential oil is 10:1; The mixing is carried out under stirring, and the stirring time is 30 to 60 minutes; The storage temperature is 4° C. and the storage time is 20 to 40 hours.

7. A Bletilla striata compound gel prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the Bletilla striata compound gel according to claim 7 in the preparation of a medicine for promoting wound healing.

9. Use of the Bletilla striata compound gel according to claim 7 in the preparation of a drug for reducing the content of inflammatory factor IL-1β in damaged skin and / or promoting the production of epidermal factor in blood vessels of damaged skin.

10. Use of the Bletilla striata compound gel according to claim 7 in the preparation of a drug for promoting the regeneration of type I collagen in damaged skin, reducing the content of type IV collagen in damaged skin, and promoting the production of fibronectin in damaged skin.

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