B-CGT hydrogel as well as preparation method and application thereof

Abstract

The invention discloses a B-CGT hydrogel as well as a preparation method and application thereof. The B-CGT hydrogel is prepared from a rhizoma bletillae 2-isobutyl malic acid glucose oxybenzyl ester extract serving as a main raw material, carbomer-940 serving as a gel matrix, glycerol serving as a cross-linking agent (also can be used as a humectant) and triethanolamine serving as a pH regulator. The prepared B-CGT hydrogel has good oxidation resistance, remarkable free radical scavenging capacity, good blood compatibility and cell proliferation promoting effect, the wound healing speed is remarkably increased, new epithelial tissue and collagen are formed more perfectly, and the B-CGT hydrogel is a promising wound dressing candidate product and has wide application prospects. The hydrogel has excellent physicochemical characteristics and drug controlled release capability, can effectively promote the wound healing process, and especially has a remarkable curative effect on healing of diabetic wounds.

Description

Technical Field

[0001] The invention relates to a traditional Chinese medicine gel preparation, in particular to a B-CGT hydrogel and a preparation method and application thereof. Background Art

[0002] Bletilla striata is the dried tuber of Bletilla striata (Thunb.) Reichb.f., an orchid plant. It is also known as "Bletilla striata", "Bai smilax", "Bai Gen", "Gan Gen", etc. It tastes bitter, sweet, and astringent, and is slightly cold in nature. It enters the lung, liver, and stomach meridians, and has the effects of astringent hemostasis, swelling, and tissue regeneration. It is mainly used to treat hemoptysis, hematemesis, traumatic bleeding, sores, swelling, and skin chapped skin. It is widely used in various fields such as medicine, health food, daily chemical industry, cosmetics, gastroscope protective agent, natural additives, and membrane materials. Modern research shows that Bletilla striata has diverse chemical components, mainly bibenzyl, 2-isobutylmalic acid glucose oxybenzyl ester, glycosides, phenanthrene, and ester components, which have pharmacological effects such as hemostasis, protection of gastric mucosa, anti-ulcer, anti-inflammatory, antibacterial, anti-cancer, and immunomodulation.

[0003] Wound management is an important area in modern medical practice, and the development of wound treatment materials is an active research direction, especially in the search for effective treatment strategies for acute and chronic wounds. Wound healing is a complex biological process involving a series of interactions among cells, cytokines, and biomolecules. The basic stages of this process include hemostasis, inflammatory response, tissue proliferation, and tissue remodeling. In this process, appropriate wound dressings can provide a moist environment, prevent infection, and promote cell migration and the formation of new blood vessels, thereby accelerating the healing process.

[0004] Hydrogels have attracted much attention due to their high water content, good biocompatibility, and ability to mimic the extracellular matrix (ECM). Hydrogels can provide an ideal moist environment for the wound surface, help maintain humidity, promote cell migration, proliferation, and the formation of new blood vessels. Its biocompatibility and degradability reduce irritation and side effects to human tissues. In recent years, research has focused on developing new hydrogels with antibacterial, anti-inflammatory, and healing-promoting functions to improve wound healing. However, existing hydrogels still have some shortcomings in practical applications. For example, some hydrogels are prone to dehydration during use, affecting their ability to maintain a moist environment. Although other hydrogels have good biocompatibility, their mechanical properties are insufficient and cannot meet the needs of specific wound sites. For example, hydrogels containing hyaluronic acid perform well in maintaining a moist environment and promoting cell migration, but their rapid degradation results in a short residence time at the wound site, requiring frequent replacement, increasing treatment costs and patient discomfort. In addition, the degradation products of alginate hydrogels may cause local acidification, causing wound irritation and discomfort, thereby affecting the healing process.

[0005] Therefore, the present invention aims to develop a novel hydrogel based on 2-isobutyl glucosyl malate extract from Bletilla striata and explore its application potential in promoting wound healing. 2-isobutyl glucosyl malate extract was separated and enriched from Bletilla striata to prepare a hydrogel preparation, and the wound healing effect of the developed hydrogel was evaluated by in vitro cell experiments and animal trauma models. Summary of the invention

[0006] The purpose of the present invention is to provide a B-CGT hydrogel and a preparation method and application thereof. The prepared B-CGT hydrogel has good antioxidant properties, significant free radical scavenging ability, good blood compatibility and cell proliferation promoting effect, significantly accelerates the wound healing speed, and the formation of new epithelial tissue and collagen is more perfect. It is a promising wound dressing candidate with excellent physical and chemical properties and drug controlled release ability, can effectively promote the wound healing process, and has a significant therapeutic effect on the healing of diabetic wounds.

[0007] The technical solution of the present invention is a B-CGT hydrogel, which is composed of a 2-isobutylmalic acid glucose oxybenzyl ester extract of Bletilla striata, a hydrogel matrix, a cross-linking agent and a pH regulator; the hydrogel matrix is ​​carbomer-940; the cross-linking agent is glycerol; and the pH regulator is triethanol.

[0008] The aforementioned B-CGT hydrogel contains 1.0-1.5 g / 100 mL of the Bletilla striata 2-isobutyl glucosylmalate extract, 0.3-0.9 g / 100 mL of carbomer-940, 12-16 mL / 100 mL of glycerol, and 0.45-0.65 mL / 100 mL of triethanolamine.

[0009] Specifically, in the aforementioned B-CGT hydrogel, the content of the Bletilla striata 2-isobutylmalic acid glucose oxybenzyl ester extract is 1.25 g / 100 mL, the content of Carbomer-940 is 0.71 g / 100 mL, the content of glycerol is 13.89 mL / 100 mL, and the content of triethanolamine is 0.57 mL / 100 mL.

[0010] The preparation method of the aforementioned B-CGT hydrogel is carried out according to the following steps:

[0011] (1) Pretreatment of PRP-512B reverse resin: Soak PRP-512B resin in 95% ethanol for 24 hours and then pack it into the column so that the filler occupies half of the total column length. Rinse with 95% ethanol until the fraction is mixed with water in a ratio of 1:5 and is not turbid. Then wash with water until there is no alcohol smell. Set aside.

[0012] (2) Preparation of 2-isobutylmalic acid glucose oxybenzyl ester extract of Bletilla striata: Add 55% ethanol to the dried and crushed Bletilla striata medicinal material at a solid-liquid ratio of 1:10 g / mL, place in an ultrasonic machine for extraction, the ultrasonic power is 344 W, the ultrasonic frequency is 40 kHz, the extraction is performed 3 times, each time for 30 minutes, the residue is filtered, the medicinal liquid is collected and concentrated by a rotary evaporator under reduced pressure, and the solvent is evaporated in a water bath to obtain an extract; weigh the pretreated PRP-512B reverse resin at a mass ratio of 1:15 to the PRP-512B reverse resin, use a dry method to load the sample, and perform gradient elution in the order of washing with water until colorless, washing with 20% ethanol until colorless, and washing with 40% ethanol until colorless, collect the fraction of 40% ethanol, and concentrate under reduced pressure until the water content reaches 10-11%, thereby obtaining 2-isobutylmalic acid glucose oxybenzyl ester extract of Bletilla striata, which is set aside;

[0013] (3) Preparation of hydrogel matrix: Weigh 0.3-0.9 g of Carbomer-940 and sprinkle it evenly on 60-80 mL of water, let it stand for 10-14 h to allow it to fully swell, stir and exhaust, and obtain a hydrogel matrix for later use;

[0014] (4) B-CGT hydrogel: Measure 12-16 mL of glycerol and add it to the hydrogel matrix while stirring. After mixing, add 1.0-1.5 g of Bletilla striata 2-isobutylmalic acid glucose oxybenzyl ester extract while stirring. Add 0.45-0.65 mL of triethanolamine to adjust the pH value to 5.0-6.5. Finally, add purified water to make the volume to 100 mL to prepare B-CGT hydrogel.

[0015] In the above step (3), the hydrogel matrix is ​​prepared by weighing 0.6-0.8 g of Carbomer-940, evenly spreading it on 70-80 mL of water, letting it stand for 11-13 hours to allow it to fully swell, stirring to remove bubbles, and obtaining a hydrogel matrix for later use.

[0016] Specifically, in the above step (3), the preparation of the hydrogel matrix is ​​as follows: 0.71 g of Carbomer-940 is weighed and evenly sprinkled on 75 mL of water, and allowed to stand for 12 hours to allow it to fully swell, and stirred to remove bubbles to obtain a hydrogel matrix for use.

[0017] In the above step (4), the preparation of B-CGT hydrogel is as follows: 13-15 mL of glycerol is measured and added to the hydrogel matrix while stirring, and after mixing, 1.20-1.30 g of 2-isobutylmalic acid glucose oxybenzyl ester extract of Bletilla striata is added while stirring, and the mixture is evenly mixed, and then 0.5-0.6 mL of triethanolamine is added to adjust the pH value to 5.0-6.5, and finally purified water is added to make the volume to 100 mL to prepare B-CGT hydrogel.

[0018] Specifically, in the above step (4), the preparation of B-CGT hydrogel is as follows: 13.89 mL of glycerol is measured and added to the hydrogel matrix while stirring, and after mixing, 1.25 g of 2-isobutylmalic acid glucose oxybenzyl ester extract of Bletilla striata is added while stirring, and mixed evenly, and then 0.57 mL of triethanolamine is added to adjust the pH value to 5.0-6.5, and finally purified water is added to make the volume to 100 mL to prepare B-CGT hydrogel.

[0019] The aforementioned B-CGT hydrogel is used in the preparation of medicines for promoting wound healing.

[0020] The aforementioned wound healing includes diabetic wound healing.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. The B-CGT hydrogel prepared by the present invention is the first attempt to prepare a gel containing Bletilla striata-2-isobutyl glucoside oxybenzyl malate extract, and the hydrogel has excellent biocompatibility and suitable physical properties, such as porosity, biodegradability and adjustable mechanical properties. These properties effectively meet the requirements of certain special wounds for mechanical properties and can regulate the inflammatory microenvironment of the wound without the need for additional therapeutic agents.

[0023] 2. The present invention uses carbomer-940 as a hydrogel matrix, glycerol as a cross-linking agent, and triethanolamine as a pH regulator, uses fewer auxiliary materials, has no toxic side effects, and the hydrogel can maximize the wound healing effect of the Bletilla striata-2-isobutylmalic acid glucoside oxybenzyl ester extract.

[0024] 3. The excellent hygroscopicity of the hydrogel can not only maintain the moist environment of the wound, but also absorb the exudate of the wound tissue, thereby effectively avoiding secondary trauma and promoting timely wound cleaning. The antioxidant capacity of B-CGT hydrogel can balance the oxidative stress response and effectively manage the excessive generation of free radicals caused by enhanced cell metabolic activity and inflammatory response. At the same time, B-CGT hydrogel significantly promoted the proliferation, adhesion and migration of NIH / 3T3 cells in in vitro experiments, showing good cell compatibility. In vivo studies further confirmed that B-CGT hydrogel promotes wound healing by regulating the inflammatory microenvironment, and contributes to the formation of new blood vessels (CD31 expression) and proper collagen deposition (CD163 positive area ratio is significant). The safety assessment includes in vivo irritation tests and in vitro blood compatibility tests. The results show that B-CGT hydrogel does not cause tissue reactions and has good blood compatibility, further confirming its safety. These research results support its application value in wound treatment.

[0025] 4. Its superior performance in the treatment of common wounds and diabetic wounds was verified through systematic in vitro and in vivo experiments. The results show that B-CGT hydrogel provides a safe and effective potential solution for wound treatment with its excellent physicochemical properties, biocompatibility and wound healing promotion. At the same time, this study also provides new ideas for the development of hydrogel dressings based on plant extracts in the medical field. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 : Screening of hydrogel matrices (A: swelling effects of different hydrogel matrices; B: drug-loaded hydrogels);

[0027] Figure 2 : Response surface design results (A: normal probability distribution of experimental residual response value; B: disturbance of each factor; C: comparison of actual value and predicted value; D: response surface plot of factors A and B; E: response surface plot of factors A and C; F: response surface plot of factors B and C);

[0028] Figure 3 : Physical properties and characterization of B-CGT hydrogel (A: stability test; B: infrared spectroscopy analysis; C: scanning electron microscopy (SEM) inspection; D: hydrogel swelling test; E: swelling curve of B-CGT hydrogel within 12 h);

[0029] Figure 4 : Cytocompatibility of B-CGT hydrogel (A: Adhesion experiment of NIH / 3T3 cells on B-CGT hydrogel; B: Effect of B-CGT hydrogel on proliferation of NIH / 3T3 cells, ***P<0.001; C: Cell scratch experiment; D: Changes in scratch area of ​​NIH / 3T3 cells under the intervention of B-CGT hydrogel; smaller scratch area indicates higher migration rate, ***P<0.001; E: Toxicity test of B-CGT hydrogel on cells);

[0030] Figure 5 : Blood compatibility and safety test (A: B-CGT hydrogel hemolysis test; B: B-CGT hydrogel coagulation test; C: skin irritation test; D: scratch irritation test);

[0031] Figure 6 :Effects of B-CGT on wound healing in rats (A: wound healing process in rats; B: graph of changes in wound healing area in rats; C: quantitative analysis of wound healing area at different time points; D: perimeter of wound cross section on the 13th day; E: section of epithelial layer of wound tissue; F: graph of wound epidermal thickness; G: thickness of wound granulation tissue on the 13th day);

[0032] Figure 7:Effects of B-CGT on wound healing; (A: morphological changes of skin layers at different time points; B: Masson staining analysis; C: Masson positive area ratio; D: fluorescence double labeling detection of wound tissue on the third day; E: fluorescence double labeling detection of wound tissue on the seventh day; F: fluorescence double labeling detection of wound tissue on the thirteenth day; G: number of microvessels in wound tissue at different time points; H: immunofluorescence positive area ratio; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001);

[0033] Figure 8 :Effects of B-CGT hydrogel on wound healing in diabetic mice (A: Wound healing process in mice; B: Changes in wound healing area in mice; C: Quantitative analysis of wound healing rate at different time points);

[0034] Fig. 9 :The effect of B-CGT on the repair of granulation tissue and epidermal growth on the wound surface of diabetic mice (A: morphological changes of the skin layer on the wound surface of diabetic mice at different time points; B: schematic diagram of the measurement of granulation tissue thickness and epidermal thickness on the wound surface of diabetic mice; C: statistical graph of epidermal thickness; D: statistical graph of granulation tissue thickness). DETAILED DESCRIPTION

[0035] The present invention will be further described below in conjunction with the embodiments, but they are not intended to limit the present invention.

[0036] Example 1: Preparation method of B-CGT hydrogel:

[0037] (1) Pretreatment of PRP-512B reverse resin: Soak PRP-512B resin in 95% ethanol for 24 hours and then pack it into the column so that the filler occupies half of the total column length. Rinse with 95% ethanol until the fraction is mixed with water in a ratio of 1:5 and is not turbid. Then wash with water until there is no alcohol smell. Set aside.

[0038] (2) Preparation of 2-isobutylmalic acid glucose oxybenzyl ester extract of Bletilla striata: Add 55% ethanol to the dried and crushed Bletilla striata medicinal material at a solid-liquid ratio of 1:10 g / mL, place in an ultrasonic machine for extraction, the ultrasonic power is 344 W, the ultrasonic frequency is 40 kHz, the extraction is performed 3 times, each time for 30 minutes, the residue is filtered, the medicinal liquid is collected and concentrated by a rotary evaporator under reduced pressure, and the solvent is evaporated in a water bath to obtain an extract; weigh the pretreated PRP-512B reverse resin at a mass ratio of 1:15 to the PRP-512B reverse resin, use a dry method to load the sample, and perform gradient elution in the order of washing with water until colorless, washing with 20% ethanol until colorless, and washing with 40% ethanol until colorless, collect the fraction of 40% ethanol, and concentrate under reduced pressure until the water content reaches 10-11%, thereby obtaining 2-isobutylmalic acid glucose oxybenzyl ester extract of Bletilla striata, which is set aside;

[0039] (3) Preparation of hydrogel matrix: Weigh 0.71 g of Carbomer-940 and sprinkle it evenly on 75 mL of water. Let it stand for 12 h to allow it to fully swell. Stir to remove bubbles to obtain a hydrogel matrix for later use.

[0040] (4) Preparation of B-CGT hydrogel: 13.89 mL of glycerol was measured and added to the hydrogel matrix while stirring. After mixing, 1.25 g of 2-isobutylmalic acid glucose oxybenzyl ester extract of Bletilla striata was added while stirring. After adding, 0.57 mL of triethanolamine was added to adjust the pH value to 5.0-6.5. Finally, purified water was added to make the volume to 100 mL to prepare B-CGT hydrogel.

[0041] Example 2: Preparation method of B-CGT hydrogel:

[0042] (1) Pretreatment of PRP-512B reverse resin: Soak PRP-512B resin in 95% ethanol for 24 hours and then pack it into the column so that the filler occupies half of the total column length. Rinse with 95% ethanol until the fraction is mixed with water in a ratio of 1:5 and is not turbid. Then wash with water until there is no alcohol smell. Set aside.

[0043] (2) Preparation of 2-isobutylmalic acid glucose oxybenzyl ester extract of Bletilla striata: Add 55% ethanol to the dried and crushed Bletilla striata medicinal material at a solid-liquid ratio of 1:10 g / mL, place in an ultrasonic machine for extraction, the ultrasonic power is 344 W, the ultrasonic frequency is 40 kHz, the extraction is performed 3 times, each time for 30 minutes, the residue is filtered, the medicinal liquid is collected and concentrated by a rotary evaporator under reduced pressure, and the solvent is evaporated in a water bath to obtain an extract; weigh the pretreated PRP-512B reverse resin at a mass ratio of 1:15 to the PRP-512B reverse resin, use a dry method to load the sample, and perform gradient elution in the order of washing with water until colorless, washing with 20% ethanol until colorless, and washing with 40% ethanol until colorless, collect the fraction of 40% ethanol, and concentrate under reduced pressure until the water content reaches 10-11%, thereby obtaining 2-isobutylmalic acid glucose oxybenzyl ester extract of Bletilla striata, which is set aside;

[0044] (3) Preparation of hydrogel matrix: Weigh 0.3 g of Carbomer-940 and sprinkle it evenly on 80 mL of water. Let it stand for 14 h to allow it to fully swell. Stir to remove bubbles to obtain a hydrogel matrix for later use.

[0045] (4) Preparation of B-CGT hydrogel: 16 mL of glycerol was measured and added to the hydrogel matrix while stirring. After mixing, 1.5 g of 2-isobutylmalic acid glucose oxybenzyl ester extract of Bletilla striata was added while stirring. After adding, 0.65 mL of triethanolamine was added to adjust the pH value to 6.5. Finally, purified water was added to make the volume to 100 mL to prepare B-CGT hydrogel.

[0046] Example 3: Preparation method of B-CGT hydrogel:

[0047] (1) Pretreatment of PRP-512B reverse resin: Soak PRP-512B resin in 95% ethanol for 24 hours and then pack it into the column so that the filler occupies half of the total column length. Rinse with 95% ethanol until the fraction is mixed with water in a ratio of 1:5 and is not turbid. Then wash with water until there is no alcohol smell. Set aside.

[0048] (2) Preparation of 2-isobutylmalic acid glucose oxybenzyl ester extract of Bletilla striata: Add 55% ethanol to the dried and crushed Bletilla striata medicinal material at a solid-liquid ratio of 1:10 g / mL, place in an ultrasonic machine for extraction, the ultrasonic power is 344 W, the ultrasonic frequency is 40 kHz, the extraction is performed 3 times, each time for 30 minutes, the residue is filtered, the medicinal liquid is collected and concentrated by a rotary evaporator under reduced pressure, and the solvent is evaporated in a water bath to obtain an extract; weigh the pretreated PRP-512B reverse resin at a mass ratio of 1:15 to the PRP-512B reverse resin, use a dry method to load the sample, and perform gradient elution in the order of washing with water until colorless, washing with 20% ethanol until colorless, and washing with 40% ethanol until colorless, collect the fraction of 40% ethanol, and concentrate under reduced pressure until the water content reaches 10-11%, thereby obtaining 2-isobutylmalic acid glucose oxybenzyl ester extract of Bletilla striata, which is set aside;

[0049] (3) Preparation of hydrogel matrix: Weigh 0.9 g of Carbomer-940 and sprinkle it evenly on 60 mL of water. Let it stand for 10 h to allow it to fully swell. Stir to remove bubbles to obtain a hydrogel matrix for later use.

[0050] (4) Preparation of B-CGT hydrogel: 12 mL of glycerol was measured and added to the hydrogel matrix while stirring. After mixing, 1.0 g of 2-isobutylmalic acid glucose oxybenzyl ester extract of Bletilla striata was added while stirring. After adding, 0.45 mL of triethanolamine was added to adjust the pH value to 5.0. Finally, purified water was added to make the volume to 100 mL to prepare B-CGT hydrogel.

[0051] Example 4: Preparation method of B-CGT hydrogel:

[0052] (1) Pretreatment of PRP-512B reverse resin: Soak PRP-512B resin in 95% ethanol for 24 hours and then pack it into the column so that the filler occupies half of the total column length. Rinse with 95% ethanol until the fraction is mixed with water in a ratio of 1:5 and is not turbid. Then wash with water until there is no alcohol smell. Set aside.

[0053] (2) Preparation of 2-isobutylmalic acid glucose oxybenzyl ester extract of Bletilla striata: Add 55% ethanol to the dried and crushed Bletilla striata medicinal material at a solid-liquid ratio of 1:10 g / mL, place in an ultrasonic machine for extraction, the ultrasonic power is 344 W, the ultrasonic frequency is 40 kHz, the extraction is performed 3 times, each time for 30 minutes, the residue is filtered, the medicinal liquid is collected and concentrated by a rotary evaporator under reduced pressure, and the solvent is evaporated in a water bath to obtain an extract; weigh the pretreated PRP-512B reverse resin at a mass ratio of 1:15 to the PRP-512B reverse resin, use a dry method to load the sample, and perform gradient elution in the order of washing with water until colorless, washing with 20% ethanol until colorless, and washing with 40% ethanol until colorless, collect the fraction of 40% ethanol, and concentrate under reduced pressure until the water content reaches 10-11%, thereby obtaining 2-isobutylmalic acid glucose oxybenzyl ester extract of Bletilla striata, which is set aside;

[0054] (3) Preparation of hydrogel matrix: Weigh 0.6 g of Carbomer-940 and sprinkle it evenly on 70 mL of water. Let it stand for 11 h to allow it to fully swell. Stir to remove bubbles to obtain a hydrogel matrix for later use.

[0055] (4) Preparation of B-CGT hydrogel: 15 mL of glycerol was measured and added to the hydrogel matrix while stirring. After mixing, 1.3 g of 2-isobutylmalic acid glucose oxybenzyl ester extract of Bletilla striata was added while stirring. After adding, 0.55 mL of triethanolamine was added to adjust the pH value to 5.0-6.5. Finally, purified water was added to make up to 100 mL to prepare B-CGT hydrogel.

[0056] Example 5: Preparation method of B-CGT hydrogel:

[0057] (1) Pretreatment of PRP-512B reverse resin: Soak PRP-512B resin in 95% ethanol for 24 hours and then pack it into the column so that the filler occupies half of the total column length. Rinse with 95% ethanol until the fraction is mixed with water in a ratio of 1:5 and is not turbid. Then wash with water until there is no alcohol smell. Set aside.

[0058] (2) Preparation of 2-isobutylmalic acid glucose oxybenzyl ester extract of Bletilla striata: Add 55% ethanol to the dried and crushed Bletilla striata medicinal material at a solid-liquid ratio of 1:10 g / mL, place in an ultrasonic machine for extraction, the ultrasonic power is 344 W, the ultrasonic frequency is 40 kHz, the extraction is performed 3 times, each time for 30 minutes, the residue is filtered, the medicinal liquid is collected and concentrated by a rotary evaporator under reduced pressure, and the solvent is evaporated in a water bath to obtain an extract; weigh the pretreated PRP-512B reverse resin at a mass ratio of 1:15 to the PRP-512B reverse resin, use a dry method to load the sample, and perform gradient elution in the order of washing with water until colorless, washing with 20% ethanol until colorless, and washing with 40% ethanol until colorless, collect the fraction of 40% ethanol, and concentrate under reduced pressure until the water content reaches 10-11%, thereby obtaining 2-isobutylmalic acid glucose oxybenzyl ester extract of Bletilla striata, which is set aside;

[0059] (3) Preparation of hydrogel matrix: Weigh 0.8 g of Carbomer-940 and sprinkle it evenly on 65 mL of water. Let it stand for 11 h to allow it to fully swell. Stir and exhaust the air to obtain a hydrogel matrix for later use.

[0060] (4) Preparation of B-CGT hydrogel: 13 mL of glycerol was measured and added to the hydrogel matrix while stirring. After mixing, 1.4 g of 2-isobutylmalic acid glucose oxybenzyl ester extract of Bletilla striata was added while stirring. After adding, 0.55 mL of triethanolamine was added to adjust the pH value to 5.0-6.5. Finally, purified water was added to make up to 100 mL to prepare B-CGT hydrogel.

[0061] The inventors conducted a lot of experiments, and the following are some of the experimental studies

[0062] 1 Materials

[0063] Chitosan (lot number: 2230614002), DPPH (lot number: 2230505001) and ABTS (lot number: 3230821001) were purchased from Beijing Solebaugh Technology Co., Ltd. (Beijing, China); gelatin (lot number: C15218933) and carbomer 940 (lot number: C15626960) were purchased from Shanghai MacLean Biochemical Technology Co., Ltd. (Shanghai, China); glycerol (lot number: 23180341) was purchased from Lanjieke Technology Co., Ltd. (Beijing, China); triethanolamine (lot number: B2313574) and methylparaben (lot number: A2125255) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Co., Ltd. (Shanghai, China); NIH / 3T3 (lot number: WHAA24C281) special culture medium was purchased from Wuhan Punosai Life Science Technology Co., Ltd. (Wuhan, China); PBS (lot number: GA24020023047), trypsin (lot number: GA2406091), DAPI staining reagent (lot number: CR2404001), CCK-8 kit (lot number: CR2312062), Calcein-AM / PI live cell / dead cell double staining kit (lot number: MPC2402034), Triton X-100 (lot number: CR2210021), and normal saline (lot number: CR2210021). No.: GA24010090444), environmentally friendly dewaxing solution (lot number: G1128), universal tissue fixative (lot number: G1101), hematoxylin-eosin high-definition constant staining kit (lot number: G1076), iF488-Tyramide (lot number: G1231), CY3-Tyramide (lot number: G1223), iF647-Tyramide (lot number: G1232), FITC-Tyramide (lot number: G1222), citric acid antigen retrieval solution (lot number: G1202), EDTA antigen retrieval solution (lot number: G1203), tissue autofluorescence Quencher (lot number: G1221), bovine serum albumin (lot number: GC305010), DAPI staining reagent (lot number: G1012), and anti-fluorescence quenching mounting medium (G1401) were purchased from Wuhan Saiweier Biotechnology Co., Ltd. (Wuhan, China); isoflurane (lot number: 2024012501) was purchased from Shandong Ante Animal Husbandry Technology Co., Ltd. (Shandong, China); anhydrous ethanol (lot number: 100092683), xylene (lot number: 10023418), n-butanol (lot number: 100052190), and neutral gum (lot number: 10004160) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

[0064] 2. Methods

[0065] 2.1 Preparation of Bletilla striata 2-isobutyl glucosylmalate extract

[0066] The dried and crushed Bletilla striata medicinal material was added with 55% ethanol (solid-liquid ratio: 1:10 g / mL), and extracted in an ultrasonic machine (power: 344 W; frequency: 40 kHz), extracted 3 times, each time for 30 min, filtered the residue, collected the liquid and concentrated it under reduced pressure with a rotary evaporator, placed it in a water bath to evaporate the solvent to obtain an extract; weighed PRP-512B reverse resin according to the ratio of extract to PRP-512B reverse resin 1:15 (g / g) for pretreatment (Pretreatment: soak PRP-512B resin in 95% ethanol for 24 hours and then load it into the column so that the filler occupies about 1 / 2 of the total column length. Rinse with 95% ethanol until the fraction is mixed with water in a ratio of 1:5 and is not turbid. Then wash with water until there is no alcohol smell.) Dry loading is adopted, and gradient elution is performed with water (rinsed until colorless), 20% ethanol (rinsed until colorless), and 40% ethanol (rinsed until colorless). The fractions in the 40% ethanol section are collected and concentrated under reduced pressure (water content reaches 10-11%) to obtain extract B for standby use.

[0067] 2.2 Preparation of hydrogel

[0068] 2.2.1 Screening of hydrogel matrix

[0069] 2.2.1.1 Screening methods for hydrogel matrices

[0070] Three commonly used excipients, namely, Carbomer-940, chitosan and gelatin, were selected as gel matrix screening objects and prepared according to the following three methods: (1) Take an appropriate amount of hydrogel matrix, sprinkle it evenly on the water surface in batches, and let it stand for 12 hours to fully swell; (2) Take an appropriate amount of hydrogel matrix, place it in a beaker, gradually add water, and swell for 12 hours; (3) Direct method, take an appropriate amount of hydrogel matrix, slowly add it to the stirring water in batches, and stir for 4 hours (1000r / min) until no white substance is seen. The state of each matrix under different methods can be obtained, and the preliminary screening of gel matrix is ​​carried out according to the comprehensive sensory indicators such as the molding condition, texture, fluidity and viscosity of each matrix. The method of step (1) is preferred, a certain amount of Carbomer-940 is weighed and swelled for 12 hours, placed in a stirrer and stirred (1000r / min) to remove bubbles generated during the swelling process, take an appropriate amount of glycerol for cross-linking, add triethanolamine to adjust the pH, and finally make the volume to 100mL.

[0071] 2.2.1.2 Screening results of hydrogel matrix

[0072] The gel matrix is ​​a monodisperse system, which can be divided into water-based and oil-based. However, through drug property analysis, we found that B-CGT is more suitable for preparing aqueous gels. Aqueous gel matrices are usually composed of water, propylene glycol or glycerol (glycerol) and carbomer / alginate / gelatin / cellulose derivatives / chitosan, etc. After consulting relevant literature and preliminary experiments, this experiment selected commonly used chitosan, gelatin, and carbomer as alternative hydrogel matrices. The results are shown in the figure. Figure 1 A and Table 1 show that Carbomer-940 was selected as the gel matrix after comprehensive investigation. It has a white semi-solid appearance, is transparent and delicate, has few bubbles, good fluidity, and appropriate viscosity. Carbomer itself is a three-dimensional network structure composed of hydrophilic polymers, with special properties such as high water content and structure similar to extracellular matrix (ECM), and is a good carrier material for external drug delivery. Comprehensive scoring was performed based on the appearance, fluidity, and viscosity of the gel. The results are shown in Table 2. The 0.3% to 0.9% Carbomer-940 blank gel obtained by preparation method ① is better, easily soluble in water, and has no special odor. The prepared hydrogel is smooth and delicate, has strong adhesion, and the drug has a longer retention time on the skin. Compared with other external matrices, it is easy to clean, has no greasy feeling, and is more comfortable to apply on the skin. Carbomer-940 was selected as the gel matrix after comprehensive consideration. It appears as a white semi-solid with moderate viscosity, good formability, and good stability without stratification. 0.3%, 0.6%, and 0.9% Carbomer-940 were selected as the matrix to add drugs and mix experiments. The hydrogel with B-CGT added was brown-yellow semi-solid, with a uniform and fine texture and easy to spread. It is more suitable for preparing into aqueous gels. The aqueous gel matrix is ​​usually composed of water, propylene glycol or glycerol (glycerol) and carbomer / alginate / gelatin / cellulose derivatives / chitosan, etc. After consulting relevant literature and preliminary experiments, this experiment selected commonly used chitosan, gelatin, and carbomer as alternative hydrogel matrices. The results are as follows Figure 1 As shown in Table 1, after comprehensive investigation, Carbomer-940 was selected as the gel matrix, which has a white semi-solid appearance, is transparent and delicate, has few bubbles, good fluidity and appropriate viscosity.

[0073] Table 1 Results of the test for the selection of aqueous gel preparation methods

[0074]

[0075] " / " indicates no such feature; "-" indicates poor (visible bubbles>20, difficult to flow, no viscosity); "+" indicates fair (visible bubbles n≤20; difficult to flow; slightly viscous and easy to separate); "++" indicates good (visible bubbles n≤5; easy to flow; strong viscosity and difficult to separate)

[0076] Table 2 Matrix dosage screening results

[0077]

[0078]

[0079] "-" means no (no stickiness; no formation; delamination); "+" means fair (no stickiness; formation; not easy to delaminate); "++" means good (good stickiness; good stability; no delamination)

[0080] 2.2.2 Single factor investigation

[0081] In the single factor test, the total water consumption of each prescription was 100mL, and the mass percentage was (g / 100mL).

[0082] 2.2.2.1 Screening of the amount of cross-linking agent glycerol

[0083] When making hydrogel, glycerol can be used not only as a crosslinking agent, but also as a moisturizing agent to achieve a moisturizing effect. According to the 1.0g formula under "2.2.1.1", a blank carbomer gel is prepared, and 1.25g of extract B and 0, 5%, 10%, 15%, and 20% glycerol are added while stirring, and then 0.6mL of triethanolamine is added, and finally pure water is added to make the volume to 100mL to prepare B-CGT self-adhesive water-based gel. It is evaluated by its appearance, viscosity, spreadability, stability (high temperature, low temperature, centrifugation) and other indicators.

[0084] 2.2.2.2 Screening of pH regulator triethanolamine dosage

[0085] Prepare a blank carbomer gel according to the method under "2.2.1.1", add 1.25g of extract B and 10mL of glycerol while stirring, then add different amounts of triethanolamine to adjust the pH value, and finally add purified water to make the volume to 100mL to prepare B-CGT aqueous gel. The amount of pH adjuster is screened based on its appearance, viscosity, spreadability, stability (high temperature, low temperature, centrifugation), pH value, etc.

[0086] 2.2.2.3 Screening results of dosage of cross-linking agent and pH regulator

[0087] The amount of cross-linking agent was screened, and the results are shown in Table 3. 15% and 20% glycerol were too greasy, and there was no moisturizing property without glycerol. The moisturizing property was poor at 5%. When glycerol was 10%, the hydrogel showed a brown-yellow semi-solid, good spreadability, moderate viscosity, good stability, and a comfortable cool feeling when applied to the skin. The results of pH adjustment agent screening are shown in Table 4. 0.6mL triethanolamine was added to 0.6% carbomer-940 and 10mL glycerol to make a volume of 100mL gel, and the pH was 5.62, which is close to the pH range of human skin (5.0-6.5). It showed a brown-yellow semi-solid, good spreadability, moderate viscosity, no stratification, and good stability.

[0088] Table 3 Screening results of the amount of glycerin used as a moisturizer in B-CGT

[0089]

[0090] "-" means no (difficult to spread and apply; no stickiness; delamination; no greasy feeling); "+" means fair (spreadable, difficult to apply; slightly sticky; stable, moderate greasy feeling); "++" means good (easy to spread and apply; good stickiness; good stability; slightly greasy feeling)

[0091] Table 4 Screening results of the amount of triethanolamine, a pH regulator, in CGT matrix

[0092]

[0093]

[0094] "-" means no (difficult to spread and apply; no stickiness; delamination; no greasy feeling); "+" means fair (spreadable, difficult to apply; slightly sticky; stable, moderate greasy feeling); "++" means good (easy to spread and apply; good stickiness; good stability; slightly greasy feeling)

[0095] 2.2.3 Optimization of hydrogel formulation by response surface methodology

[0096] On the basis of the single factor experiment, the content of carbomer 940, glycerol and triethanolamine were selected as the investigation factors, and the comprehensive score of the hydrogel sensory index was used as the response value. The Box-Behnkend in the Design-Expert 13 software was used to perform a three-factor three-level response surface design on B-CGT hydrogel to screen the optimal amount of each excipient in B-CGT hydrogel. The response surface design is shown in Table 5. The scoring criteria are shown in Table 6.

[0097] Table 5 Response surface design factor level table

[0098]

[0099] Table 6 Scoring criteria for hydrogel sensory indexes

[0100]

[0101] The optimal dosage of each auxiliary material in the hydrogel was screened out by orthogonal experimental design, and the experimental results are shown in Tables 7 and 8. The data were processed and analyzed using Design-Expert 13 software to obtain the response surface contour map and curve diagram, as shown in Figure 2 As shown, the fitting equation obtained through data analysis is:

[0102] Y=33.40+0.8750A+2.63B+2.50C+1.25AB+0AC+0.5BC-2.58A 2-2.08B 2 -4.825C 2 (7)

[0103] The optimized formula is carbomer 940 content of 0.71g / 100mL, glycerol content of 13.89mL / 100mL, and triethanolamine concentration of 0.57mL / 100mL. The hydrogel prepared after optimization is brown-yellow semi-solid, uniform and stable, and has good adhesion.

[0104] Table 7 Response surface experimental design and results

[0105]

[0106] Table 8 ANOVA results of hydrogel preparation process screening

[0107]

[0108] (P<0.01)high significant, (P<0.05)significant, (P<0.1)not important.

[0109] A is the percentage of Carbomer 940, B is the percentage of glycerol, and C is the percentage of triethanolamine.

[0110] Physical property testing of 3B-CGT hydrogel

[0111] 3.1 Characterization of B-CGT hydrogel

[0112] 3.1.1 Characterization and detection methods of B-CGT hydrogel

[0113] The microstructure and morphology of the B-CGT hydrogel (Example 1) were observed using a TESCAN CLARA scanning electron microscope. The test environment was to use liquid nitrogen to quickly freeze a large amount of water in the hydrogel, freeze-dry the hydrogel, plate a layer of conductive metal on the surface of the freeze-dried hydrogel to reduce charge accumulation and improve conductivity, and then place it on a scanning electron microscope (SEM) stage for scanning and observation.

[0114] Thermo Scientific Nicolet is5 Fourier transform infrared spectrometer (FT-IR) was used to analyze the -1 The chemical structure of B-CGT hydrogel was characterized within a wide range and the chemical bonds in the molecular network structure of the hydrogel were analyzed.

[0115] Take the B-CGT aqueous gel prepared in the example and test it according to the appearance under the general rule 0114 gel of Part IV of the 2020 edition of the Chinese Pharmacopoeia. Appropriate pH is conducive to promoting wound healing, and the pH value of the semisolid hydrogel is measured with a PHS-3C desktop pH meter. The stability test of the hydrogel is also a key factor to ensure its safety and effectiveness in practical applications (centrifugal stability, heat stability, cold stability). The swelling behavior of the B-CGT hydrogel was evaluated by investigating the swelling rate of the hydrogel.

[0116] 3.1.2 Appearance and centrifugal stability test results

[0117] The appearance inspection of the hydrogel meets the requirements of the 2020 edition of the Pharmacopoeia of the People's Republic of China under the general chapter 0114 of the gelling agent. It is uniform and delicate, remains gelatinous at room temperature, does not dry up or liquefy, has good stability, and meets the requirements of the pharmacopoeia. Figure 3 (B) shown.

[0118] The centrifugal stability of the hydrogel has a good effect of removing bubbles, and the thermal stability and low-temperature freeze-thaw stability of the hydrogel at different temperatures and humidities can be used to examine the storage conditions of the preparation and provide a theoretical basis for determining the later drug transportation methods and storage conditions. Figure 3 As shown in result A, the morphological properties of the hydrogel did not change. At the same time, there was no stratification, drying up or liquefaction, and good uniformity was maintained, which met the requirements for gelling agents in General Chapter 0114 of Part 4 of the 2020 edition of the "Pharmacopoeia of the People's Republic of China".

[0119] 3.1.3 Infrared and morphological detection results

[0120] The Fourier transform infrared spectrum (FTIR) of B-CGT hydrogel is shown in Figure 3 (B) shows the spectrum of B-CGT hydrogel at 3300-500 cm -1 The strong absorption band near 2900 cm is related to the stretching vibration of OH bonds, which is usually seen in the abundant hydroxyl groups in hydrogels, indicating the presence of alcohol or phenolic compounds; -1 The peaks near 1700 cm-1 are usually the stretching vibrations of the CH bonds, indicating the presence of alkanes; -1 The nearby sharp peaks are attributed to the stretching vibration of the C=O bond, and there are ketones, aldehydes or carboxylic acids; the peak at 1066cm-1 represents the stretching vibration of CO. These data are consistent with the chemical bond characteristics of the B-type components in Bletilla striata.

[0121] After freeze-drying, the hydrogel was observed using a scanning electron microscope (SEM). Figure 3(C) The interior of the hydrogel is loose and porous, presenting irregular microcavities. The microcavities have a porous structure and a three-dimensional network structure, which can absorb and retain a large amount of water, provide a moist environment for the wound, and avoid secondary damage. At the same time, it simulates the extracellular matrix (ECM) well, providing support and guidance for cells, and the three-dimensional network structure has a certain mechanical strength, which can provide physical protection for the wound. Not only that, it can also effectively absorb wound exudate and lock it in the network structure to prevent overflow, reduce the breeding environment for bacteria, reduce the risk of infection, and facilitate wound healing.

[0122] 3.1.4 Swelling performance test results

[0123] like Figure 3 (D) is a real picture of the swelling of B-CGT hydrogel. The hydrogel swells due to hydration. It can be observed that the hydrogel absorbs water and expands, and the volume increases. The hydrogel did not dissolve even after 12 hours. Figure 3 (E) As shown in the results, the swelling rate of the hydrogel increased significantly with the increase of time, and did not dissolve within a certain period of time. It quickly rose to 203.71% within the initial 1 hour, and gradually tended to equilibrium at 10 to 12 hours, showing good water absorption capacity. It can absorb a large amount of liquid within a certain period of time. It can solve the tissue fluid exuded from the wound during wound healing, clean the wound in time, prevent the infection risk caused by bacteria breeding in the tissue fluid, and accelerate wound healing. At the same time, the soft and moist hydrogel provides a moist environment for the wound, avoiding secondary trauma caused by wound dressing changes.

[0124] 3.2 Antioxidant activity (ROS) detection

[0125] The freeze-dried B-CGT hydrogel (Example 1) was extracted with PBS at a ratio of 1:50 for 24 hours, and the extract was used for later use. DPPH detection was used, and the working solution was prepared according to the DPPH instruction manual for later use, and the absorbance value was measured at 517 nm using an ELISA reader.

[0126]

[0127] (A1 is the control group, only water + DPPH solution, A2 is the hydrogel extract)

[0128] The ABTS method was further verified. The preparation was also prepared according to the instructions, and the absorbance value was detected at a wavelength of 734 nm by an enzyme marker.

[0129]

[0130] (A1 is the control group, only water + ABTS solution, A2 is the hydrogel extract)

[0131] The results showed that the free radical scavenging rate of the hydrogel under the DPPH method was 69.28±1.31%, showing strong antioxidant capacity. The free radical scavenging rate of the hydrogel was further detected by the ABTS method, which was 62.82±12.73%. The results of both detection methods showed that the prepared hydrogel had significant antioxidant properties and could reduce the oxidative stress generated during wound healing.

[0132] Biocompatibility of 4B-CGT hydrogel

[0133] 4.1 Cytocompatibility of B-CGT hydrogel

[0134] 4.1.1 Cytocompatibility testing method of B-CGT hydrogel

[0135] The B-CGT hydrogel (Example 1) was extracted with culture medium (1:30) for 24 h and then filtered with a 0.45 μm filter. The cell proliferation ability was detected using a cell counting kit (CCK-8 kit). NIH / 3T3 cells were revived and cultured at 37°C and 5% CO2. The culture density in a 96-well plate was 2×10 3 NIH / 3T3 cells. After 4 hours of culture, the culture medium was aspirated, 80 μL of the extract was added, and 20 μL of the culture medium was added to continue the culture. At 24h, 36h and 48h, CCK-8 (10% CCK-8 solution) was added according to the kit protocol. After incubation for 2h in the dark, the absorbance value of each well was detected at 450nm using an ELISA reader.

[0136]

[0137] OD a Representative of B-CGT experimental group, OD b represents the control group (without cells), OD c Represents the blank group (without drug-loaded hydrogel and cells).

[0138] To assess cell adhesion, 3 × 10 5 NIH / 3T3 cell suspensions were inoculated on 24-well plates that were not treated with TCP, and 50 μL of B-CGT extract and CGT (hydrogel without drug loading) extract were added to the 24-well plates and cultured for 4 hours. The same number of cells were then directly inoculated on the surface of a 24-well TCP cell culture dish as a positive control. After 4 hours, the culture medium was gently aspirated from the wells, and the cells were gently rinsed twice with PBS. After staining with DAPI dye for 5 minutes, the cells were observed under an inverted microscope (DMI 8, LIGHTE).

[0139] In order to study the effect of hydrogel on fibroblast migration, a scratch test was performed. 6NIH / 3T3 cells were inoculated in a 6-well plate and cultured for 24 hours. A scratch of the same width was made on the bottom of each well, and then the culture medium was removed and the hydrogel extract was added for co-culture. After a certain period of incubation, the cells were observed under an inverted microscope and images were taken to monitor the migration of cells on the scratched area.

[0140] The cell viability staining method was used to evaluate the cytotoxicity of the hydrogel. 1.5×10 4 The cell suspension was inoculated into a 24-well plate, the culture medium was removed after culturing the cells (12h, 24h, 36h), 100 μl of live-dead stain was added to each well, the plate was protected from light for 15 min, the stain was removed, the plates were washed twice with sterile PBS, and observed under an inverted fluorescence microscope (1, 3, 5d).

[0141] 4.1.2 Cytocompatibility test results of B-CGT hydrogel

[0142] Cell adhesion is the basis of communication between cells and their microenvironment and determines cell migration, proliferation, differentiation and other basic cell behaviors. Cell adhesion and migration are crucial in the wound healing process. Figure 4 As shown in (A), after NIH / 3T3 cells were incubated in culture medium for 4 h, NIH / 3T3 cells effectively attached to CGT and B-CGT, and their behavior was similar to that of cells on TCP culture dishes, indicating that CGT and B-CGT can promote the adhesion of fibroblasts and their distribution is uniform, while the CGT group is significantly less than the B-CGT group.

[0143] The CCK-8 method was further used to quantitatively detect the effect of B-CGT hydrogel on the proliferation of NIH / 3T3 cells. Figure 4 As shown in (B), cells cultured on B-CGT hydrogels showed good proliferation ability after 24 hours. In the B-CGT group, the number of cells increased significantly after 48 hours. With continued culture, the OD value of the B-CGT group was higher than that of the control group. Figure 4 (C) In the cell migration experiment, the distance moved by cells in the B-CGT group was significantly greater than that in the control group (P < 0.05). Figure 4 The results shown in (D) also confirmed this result.

[0144] Live / dead cell staining was used to detect the cytotoxic effect of B-CGT hydrogel. Figure 4(E) Live / dead cell staining analysis showed that the survival rate of NIH / 3T3 cells in B-CGT and the control group (containing only culture medium without gel) after 5 days of culture reached more than (76.74±5.58)%. According to the International Organization for Standardization (ISO) 10993-5 standard, live / dead staining was performed to detect cytotoxicity, and the standard usually used is cell survival rate. If the cell survival rate remains above 70%, it is considered to have no potential toxicity to the cells and is suitable for further biocompatibility evaluation.

[0145] These results further prove that compared with the control group, B-CGT hydrogel has good cell compatibility, proliferation ability, good migration ability and adhesion, and B-CGT hydrogel provides more adhesion sites and environment for cell adhesion, diffusion and proliferation.

[0146] 4.2 Blood compatibility of B-CGT hydrogel

[0147] 4.2.1 Blood compatibility test method of B-CGT aqueous gel

[0148] The blood compatibility of B-CGT hydrogel was studied by hemolysis test. Fresh rat blood was centrifuged at 2000rpm for 10min to separate RBC, and then washed 3 times with 0.9% NaCl solution to obtain purified RBC. Prepare RBC suspension (5%, v / v). 200μL RBC suspension was mixed with 800μL 0.9% NaCl solution as a negative control, 200μL RBC was mixed with 800μL 0.1% Triton X-100 as a positive control, and 200μL RBC was mixed with 100μL hydrogel extract and 700μL 0.9% NaCl solution as an experimental group. After incubation at 37°C for 2h, centrifuge for 10min (2000rpm), and take photos and record. Then take the supernatant in a 96-well plate, use an enzyme reader to measure its absorbance value at 540nm, and calculate the hemolysis rate (HR).

[0149]

[0150] A h , A t and A n Represent the absorbance of the experimental group, positive group and negative group at 540nm respectively.

[0151] 4.2.2 Blood coagulation effect of B-CGT aqueous gel

[0152] The in vitro coagulation effect of the hydrogel was evaluated according to the coagulation method. The hydrogel was placed in a 96-well plate and placed in a 37°C water bath for 10 minutes to allow the hydrogel to reach the temperature required for the experiment. Then 45 μL of anticoagulated fresh rat blood was added, and then 9 μL of CaCl2 solution (25 mol / L) was immediately added. The blood coagulation was observed and recorded at different time points, and the uncoagulated blood was gently aspirated at different time points, and gently rinsed twice with 0.9% NaCl solution to remove the uncoagulated blood and impurities on the surface. The formation of clots in each well was photographed.

[0153] According to the international standard ISO 10993-4, a hemolysis rate HR ≤ 5% is considered to be no hemolysis rate. The HR value of B-CGT hydrogel is less than 3% (2.48 ± 0.40%, n = 6). Figure 5 (A) shows that B-CGT hydrogel has good blood compatibility. What cannot be ignored in the wound healing process is the small amount of continuous bleeding after debridement. At the same time, whether the bleeding can be effectively stopped will also affect the normal healing process of the wound. By testing the coagulation time of blood treated with B-CGT hydrogel, the hemostatic ability of the hydrogel was preliminarily evaluated. Figure 5 (B) The clotting time of whole blood alone was 12 minutes, while the B-CGT hydrogel formed a stable blood clot after about 3 minutes of contact, which showed that the B-CGT hydrogel had a good clotting effect compared with the NS group.

[0154] Safety evaluation of 5B-CGT aqueous gel

[0155] According to ISO 10993-10 standard, the back of SD rats was shaved, and normal saline (NS) and B-CGT were applied to the back twice a day. The skin condition was observed and scored on the 1st, 2nd, 3rd, 4th, 5th, 6th and 7th day after application. The PII index of each group was calculated.

[0156]

[0157] (N is the sum of the total scores at each time point, and n represents the number of animals). The lower the PII index, the less irritating it is.

[0158] The same grouping was used for further experiments. A “#” was scratched with a needle on the shaved part of the mouse back. The model was considered successful if there was slight bleeding. The mouse was cleaned once with NS and then smeared with hydrogel twice a day. The skin condition was observed and scored at 1h, 12h, 24h and 48h after the application of the drug, and photographed and recorded. The scoring criteria are shown in Tables 9 and 10.

[0159] Table 9 Scoring criteria for hydrogel skin irritation test

[0160]

[0161] Table 10 Hydrogel primary irritation index PII reaction type standard

[0162]

[0163] According to ISO 10993-10 standard, the skin irritation test of B-CGT hydrogel was evaluated. Figure 5 (C) The B-CGT group was the same as the NS group, with no erythema or edema on the back of the mice. The results are shown in Table 11 (n=4). The primary irritation index (PII) of the B-CGT group was low, almost the same as that of the NS group, indicating that the hydrogel was not irritating. Figure 5 (D) The safety of the hydrogel was further evaluated by the scratch test. The B-CGT hydrogel had no erythema or edema, and no eschar formation. The results are shown in Table 12 (n=4). The B-CGT hydrogel had no irritation, further indicating that the hydrogel was safe and non-irritating.

[0164] Table 11 PII score of irritation test

[0165]

[0166] Table 12 Scoring of “#” on the back of irritation test

[0167]

[0168] Experimental study on wound healing of 6B-CGT hydrogel

[0169] SD rats were selected for this experiment and purchased from Hunan Jiatai Experimental Animal Co., Ltd. All animals were housed in an SPF-grade animal facility with a light-dark cycle of 12 hours. Twenty-four SD rats weighing 180-220 g were randomly divided into NS group (normal saline), CGT group (hydrogel without drug loading), and B-CGT group (hydrogel loading), and each rat was in a single cage, with 8 rats in each group. The back of the rat was shaved and washed with normal saline the day before the experiment. On the day of modeling, isoflurane was used for gas anesthesia for several minutes to enter the anesthesia state, and then mask anesthesia was continued. The surgical area was disinfected with iodine tincture, and a 10 mm full-thickness skin wound was created with a skin biopsy needle. NS (control group), CGT, and B-CGT were applied evenly to the entire wound range. Digital images of the wound were collected on the 3rd, 7th, 13th, and 18th days after surgery, and measured using ImageJ software.

[0170]

[0171] A0 and A (3,7,13,18) They represent the unhealed areas on days 0, 3, 7, 13, and 18, respectively.

[0172] like Figure 6(A) B-CGT hydrogel was applied to the full-thickness acute wound of SD rats, and the wound healing status at different time points was recorded. Figure 6 As shown in (B), the wound size diagrams of different groups showed obvious changes on days 0, 3, 7, 13, and 18. Figure 6 As shown in (C), the wound healed faster after B-CGT hydrogel treatment. On the 3rd day, the wound of B-CGT hydrogel had healed by 37.09%, which was significantly higher than that of CGT group (8.43±0.03%) and NS group (8.05±0.06%). On the 7th day, the healing rate of B-CGT group (59.04±0.07%) was significantly higher than that of CGT group (24.47±0.07%) and NS group (23.66±0.17%). CGT group and NS group healed slowly due to the longer inflammatory period and stronger inflammatory response, and the healing rate did not exceed 50%. As time went on, the wound treated with B-CGT hydrogel maintained the best healing state among all groups. On the 13th day, the healing rates of CGT group and NS (control group) were about 80%. In contrast, the wound healing rate of B-CGT was over 90% (98.22±0.02%), basically healed, and the healing rate was significantly higher than that of NS group (P<0.05). Figure 6 (D) Quantification of the length of histological wound cross sections also confirmed this result.

[0173] In the process of skin wound healing, re-epithelialization is the first and most important step, which occurs before the repair of the dermis. This process can quickly rebuild the functional barrier of the wound and effectively prevent excessive transepidermal water loss and infection risk

[53] . In order to evaluate the effect of hydrogel on wound re-epithelialization and granulation tissue formation in more detail, observations were conducted on the 7th and 13th days after treatment. Figure 6 As shown in (E), the wound tissue sections of the B-CGT group showed a relatively complete epithelial layer. In contrast, the wounds of the CGT group and the NS group (control group) showed an incomplete epithelial layer. In the NS group (control group), the wound had not closed on the 7th day. Figure 6 (F), By day 13, epithelialization was significantly more pronounced in the B-CGT group compared with the control group.

[0174] The growth of granulation tissue plays a key role in the wound regeneration and repair process accompanied by inflammation. It provides structural support, fills the wound cavity, and provides a matrix for the migration of epidermal cells. At the same time, the angiogenesis process of granulation tissue ensures the supply of nutrients to the wound site and the removal of metabolic waste. The contraction of the wound and the formation of scar tissue are closely related to granulation tissue. Therefore, the thickening of granulation tissue during wound healing is an important indicator for evaluating the repair effect. Figure 6(G) After 13 days, the granulation tissue produced by the B-CGT group showed a thicker trend than that of the blank gel matrix group, while that of the NS group (control group) was thinner and less uniform. B-CGT had a significant effect in promoting wound repair.

[0175] Overall, the healing process of full-thickness wounds is a complex biological process involving the coordinated action of multiple stages and cell types. In this process, the epidermis and granulation tissue interact in full-thickness wound healing and work together to complete the wound healing process. The epidermis provides coverage and protection, while the granulation tissue provides support and nutrition. The coordinated development and remodeling of these two tissues is key to ensuring smooth wound healing and restoration of normal skin function.

[0176] 7B-CGT hydrogel histopathology experiment

[0177] For histological and immunofluorescence analysis, the regenerated skin on the back was collected on the 3rd, 7th, and 13th days after surgery. It was fixed with 4% paraformaldehyde solution and embedded in paraffin for subsequent staining experiments.

[0178] 7.1 HE staining

[0179] Paraffin sections are dewaxed to water: the tissue sections embedded in paraffin are placed in an environmentally friendly dewaxing solution for dewaxing twice (20 minutes / time), then washed with anhydrous ethanol twice (5 minutes / time), 75% alcohol for 5 minutes, and then washed with water. Pretreatment: the sections are placed in high-definition constant staining pretreatment solution for 1 minute. Hematoxylin staining: after gently absorbing water, the sections are immersed in hematoxylin staining solution for 3 minutes, rinsed with running water, differentiated with differentiation solution, rinsed with water, blued with blueing solution, and rinsed again. Eosin staining: the sections are dehydrated in 95% alcohol for 1 minute, and stained in eosin staining solution for 15 seconds. Finally, dehydrate and seal the sections: first dehydrate in anhydrous ethanol for 3 times (2 minutes / time), then in n-butanol for 2 times (2 minutes / time), and finally in xylene for 2 times (2 minutes / time) until transparent, and seal the sections with neutral gum). Use an upright microscope to collect images and measure the thickness of granulation tissue and epidermis at the same time. Image-Pro Plus 6.0 measured cross-sections of tissue wounds.

[0180] HE staining was used to observe the morphological changes of the skin layer at different time points during wound healing. Figure 7(A) shows that on the third day of drug treatment, each group showed incomplete epidermis and significant necrosis of the dermis (black arrow), accompanied by a large amount of necrotic cell debris and unstructured eosinophilic substances and a large amount of inflammatory exudates (purple arrows). The cortex at both ends of the injury became thicker (orange arrows), with a small amount of granulation tissue (grey arrows), new blood vessels (green arrows), fibroblasts (brown arrows) and a large number of lymphocytes (red arrows), and a small amount of bleeding was visible (yellow arrows). On the 7th day, the skin tissue of the NS group and the CGT group was thin and there was a large amount of inflammatory exudate. The CGT group also had a large area of ​​necrosis in the epidermis, with only a small area of ​​granulation tissue hyperplasia. In contrast, the B-CGT group had a large area of ​​granulation tissue hyperplasia, containing a large number of fibroblasts, a large number of new blood vessels and a large number of lymphocytes. On the 13th day, the skin structure of all groups recovered further, especially the B-CGT group, which showed a relatively complete epidermal structure and more granulation tissue proliferation, rich collagen fibers and new blood vessels. A small number of cells were seen gathering at both ends of the granulation tissue (dark red arrows). However, although there was a large area of ​​granulation tissue proliferation in the NS and CGT groups, the overall structure recovered slowly. A small number of spinous cells with loose cytoplasm (light blue arrows) and a small amount of hyperkeratosis (dark red) were seen in the epidermis of the NS group. Overall, B-CGT can effectively promote the transformation from the inflammatory phase to the proliferative phase and promote the repair effect of accelerating wound healing.

[0181] 7.2 Masson trichrome staining

[0182] Dewax the paraffin sections prepared above and the paraffin sections in HE staining to water, use the Masson trichrome staining kit for staining, and place them in the Masson dye set for staining according to the instructions. Soak in Masson A solution overnight and rinse with running water. Soak in a dye solution mixed with Masson B and Masson C solutions in equal proportions for 1 minute, rinse with running water, differentiate for a few seconds with differentiation solution, and rinse. Then put it in Masson D solution for 6 minutes, rinse with water, and finally soak it in Masson E solution for 1 minute, take it out until there is no dripping liquid, directly put it in Masson F solution for 2-30 seconds, then rinse with 1% acetic acid for differentiation, and dehydrate with anhydrous ethanol. Finally, it is transparent sealing: put the slices in anhydrous ethanol again for 5 minutes, xylene for 5 minutes for transparency, and neutral resin glue for sealing. Microscopic examination, image acquisition, and analysis and measurement with Image J software.

[0183] Masson staining was used to observe the distribution of collagen fibers in the wound area. Collagen (blue), muscle fibers (red) and cell nuclei (black) in the tissue. Figure 7(B) On the third day after drug treatment, in the early stage of healing, only a small amount of myofibrils were generated in the NS group (control group), mainly red muscle fibers, and the tissue structure was loose with slight fibrosis. The CGT group and the B-CGT group generated more collagen fibers, with signs of fibrosis. Compared with the control group and the BH matrix group, the tissue structure of the B-CGT group was denser, and the fibrosis was obvious with a few early signs of angiogenesis. After 7 days of treatment, the generation of collagen fibers in each group increased significantly in the B-CGT group, and the tissue density was also denser than that in the control group, which helped to provide structural support for the wound and promote tissue repair and regeneration. The fibrosis effects of the CGT group and the B-CGT group were significant, and angiogenesis increased, but not significantly. In the late stage of healing, on the 13th day, the B-CGT group was the most prominent, with a large amount of collagen fibers generated, obvious fibrosis, high tissue density, significant tissue reconstruction effect, and more angiogenesis. Figure 7 (C) The positive area ratio of the B-CGT group was higher than that of the control group and the CGT group at each time point. On the 7th day, the positive area ratio of the CGT group was slightly higher than that of the B-CGT group. Overall, the positive ratio of the control group was always low, and the overall positive area ratio of the CGT group was lower than that of the B-CGT group, which proved that B-CGT had a significant effect in promoting wound healing and might accelerate the healing process through the production of collagen fibers.

[0184] 7.3 Immunofluorescence analysis

[0185] Immunofluorescence analysis, after dewaxing (environmentally friendly dewaxing solution) and hydration (absolute ethanol), antigen retrieval (EDTA pH = 8.0, do not dry the slices), the skin tissue sections were treated with PBS at pH = 7.4 for 3 times at room temperature, 5 minutes each time, circled at room temperature (the hydrogen peroxide blocking group brush drew circles around the tissue), incubated in 3% hydrogen peroxide solution for 25 minutes in the dark, blocked endogenous peroxidase, treated with PBS for 3 times for 5 minutes each time. And blocked with 3% BSA for 30 minutes. The sections were incubated with CD31 (1:2000, GB11063-2, servicebio) in a humidified box at 4°C overnight. After being treated with PBS for 3 times (5 minutes / time), HRP-labeled secondary antibody was added dropwise, incubated at room temperature for 50 minutes, treated with PBS in the same washing method, CY3 was added dropwise, incubated at room temperature for 10 minutes in the dark, placed in TBST for 3 times (5 minutes / time), and microwave heating was performed. Add CD163 (1:200, GB113751, servicebio) and incubate overnight at 4°C in a humidified box, wash with PBS for 5 minutes, treat 3 times, add fluorescent secondary antibody labeled with Alexa Fluor 488, incubate at room temperature in the dark for 50 minutes, treat with PBS, add DAPI staining solution, incubate at room temperature in the dark for 10 minutes. Finally, add an appropriate amount of autofluorescence quencher, incubate for 5 minutes, and collect fluorescence images of the slices using a fluorescence microscope.

[0186] The expression and distribution of CD31 and CD163 in wound tissue were detected by fluorescent double labeling. At the same time, the number of microvessels of CD31 (red light), the positive area ratio (expression range) of CD163 (green light), the average optical density (the depth of positive signal), the surface density (the average depth of positive signal in the tissue area to be measured) and the distribution of cell nuclei were observed in the inflammatory stage, proliferation stage and tissue remodeling stage.

[0187] Depend on Figure 7 (D) It can be seen that on the third day, there was almost no microangiogenesis in the NS and CGT groups, while the red color of the B-CGT group was obvious, indicating that the B-CGT hydrogel can promote angiogenesis during the inflammatory period. Figure 7 (E) The B-CGT group had significantly more blood vessels (CD31) and green light (CD163) than the NS and CGT groups, which had a very good trend for wound healing, indicating that more mature capillaries were formed in the wound of the B-CGT group. After 13 days, compared with the NS (control group), the CD31 density in the B-CGT group increased significantly. The generation and expression of CD31 and CD163 in the NS group were the worst, while the expression of CD31 and CD163 in the B-CGT group was better, as shown in Figure 1. Figure 7(F). Overall, the wound healing process was slower in the NS and CGT groups, and the B-CGT group accelerated wound healing. Figure 7 (G), the number of microvessels generated at different time points, Figure 7 (H) The positive expression of CD163 was consistent with these results (P < 0.001).

[0188] 8 Statistical analysis

[0189] All experimental results were obtained from at least 3 replicates. Statistical analysis was performed using X software. Data are presented as mean ± standard deviation (Mean ± SD). Experimental data were analyzed using t-test or one-way / two-way ANOVA according to different experimental designs. Finally, the data were plotted using Graphpad prism 9.0. P value < 0.05 was considered statistically significant.

[0190] 9 Experimental study on wound healing in diabetic mice

[0191] 9.1 Animals

[0192] The ob / ob mouse is a well-established animal model of obesity and type 2 diabetes and is also a homozygous animal with characteristics similar to those of adult-onset diabetes. C57BL / 6J controls have the same genetic background as ob / ob mice. All mice (male, 6 weeks old) were purchased from Beijing Huafukang Biotechnology Co., Ltd. (Beijing, China). The mice were housed in an SPF-grade room at 22°C, with a 12:12 light-dark cycle and airflow regulation, one mouse per cage, with unlimited food and water, and were adaptively fed for 2 weeks. All experiments and procedures involving mice were approved by the Experimental Animal Ethics Review Committee of Guizhou University of Traditional Chinese Medicine.

[0193] 9.2 Full-thickness excision wound

[0194] The hair on the back of the mice was shaved and disinfected one day before the operation. The next day, the surgical area was disinfected after anesthesia with isoflurane (2024012501, Shandong Ante Animal Husbandry Technology Co., Ltd.), and a full-thickness wound with a diameter of about 10 mm was excised. The wound was rinsed with saline, and the mice were raised individually, and the weight and blood glucose level of the mice were monitored.

[0195] 9.3 Animal grouping and treatment

[0196] The mice were divided into 4 groups, including C57BL / 6J mice as the control group (CG), ob / ob mice as the model group (MG), Beifuxin (bovine basic fibroblast growth factor gel produced by Zhuhai Yisheng Biopharmaceutical Co., Ltd.) as the positive group (PC) and B-CGT hydrogel group. These drugs were applied once a day, thickly applied to the wound area on the first and second days after injury, in order to absorb the exudate in the early stage of the inflammatory response of the wound. After that, the wound was covered once a day. The remaining control group and model group were smeared with normal saline. Wound photos were collected on days 0, 3, 7, 15, and 20 after surgery, and the pixel area and the perimeter of the wound edge were measured using ImageJ 10.0. The percentage of the area of ​​non-healing rate was calculated as follows:

[0197]

[0198] A0 and A(0, 3, 7, 15, 20) represent the unhealed areas on day 0 and days 3, 7, 15, and 20, respectively.

[0199] Dynamic changes of different groups of mice during wound healing Figure 8 As shown in A. The healing progress of CG and MG was slow throughout the experiment. On the 7th day, there was still a large unhealed area on the wound surface. After the 15th day, there was still an obvious open wound surface, and it was not completely healed until the 20th day. The wounds in the PC group improved and the wound surface gradually shrank, but it was not completely healed. In contrast, the drug group (B-CGT) showed a significant accelerated healing effect from the 7th day, the wound area was significantly reduced on the 14th day, and the wound surface was basically completely closed on the 20th day, showing a better healing effect.

[0200] The wound healing area change chart directly reflects the difference in healing speed among the groups. Figure 8 B. The wound area of ​​the CG and MG groups decreased slightly, and the healing process was relatively slow; PC showed a certain degree of wound area reduction, but the rate of change was not as fast as that of the B-CGT group. The wound area treated with B-CGT hydrogel decreased significantly, especially from the 7th to the 15th day, when the rate of decrease was the fastest. On the 20th day, the wound area was close to zero, which also proved that it has a strong healing-promoting effect.

[0201] Quantitative analysis results of healing rate ( Figure 8 C), further verified the significant therapeutic effect of B-CGT hydrogel. The healing rates of the control group and the model group were low on the 7th, 15th and 20th days, and the MG group had a slightly delayed healing progress; the healing rate of the PC group increased during the experiment, but it still did not reach a high healing rate level in the later period. The B-CGT group was significantly higher than the other groups at all time points. On the 20th day of treatment, the healing rate was close to 100%, which was significantly better than the other treatment groups, showing a significant therapeutic effect.

[0202] In summary, B-CGT hydrogel has a significant effect on wound healing in diabetic mice. It has important application potential and clinical value in the treatment of chronic wounds related to diabetes by accelerating wound contraction, promoting tissue repair and improving the overall healing rate. This shows that B-CGT hydrogel can provide a safe and efficient treatment strategy for patients with diabetic wounds.

[0203] 9.4 Wound granulation tissue repair and epidermal growth

[0204] Granulation tissue plays a key role in the regeneration and repair of wounds accompanied by inflammation. With the inflammatory response, fibroblasts begin to proliferate and form granulation tissue, providing growth matrix and new blood vessels. Therefore, in the process of wound healing, the thickness of granulation tissue is an important indicator for evaluating the effect of wound repair. The histological changes of the skin layer of the wounds of diabetic mice in different groups at different time points showed significant differences ( Fig. 9 AB). In the CG and MG groups, wound repair was poor, skin tissue rupture was obvious, no complete epidermal structure was seen, and granulation tissue was sparse. The PC group showed a certain degree of repair effect, the skin tissue was more regular, but the thickness of the epidermis and granulation tissue was still insufficient. The wound skin tissue of the B-CGT hydrogel group was significantly better than that of the other groups in the progress of repair. A continuous epidermal structure began to form on the 15th day. On the 20th day, the epidermal structure was thick and the granulation tissue was substantial and regularly arranged, close to normal skin.

[0205] The quantitative analysis results of epidermal thickness showed that ( Fig. 9 C), the epidermal thickness of the B-CGT group on the 15th and 20th days was significantly higher than that of the CG group, MG group and PC group, indicating that B-CGT has a significant promoting effect on epidermal hyperplasia. The quantitative analysis results of granulation tissue thickness showed that ( Fig. 9 D), the thickness of granulation tissue was lower in the CG and MG groups, and increased in the PC group, while the thickness of granulation tissue in the B-CGT group was significantly higher than that in the other groups at all time points, especially reaching peak values ​​on the 15th and 20th days, indicating that B-CGT accelerates the generation of granulation tissue and tissue repair.

[0206] In summary, B-CGT hydrogel has significant advantages in the formation of granulation tissue and epidermal hyperplasia in the wound of diabetic mice. Histological observation and thickness quantitative analysis both showed that B-CGT significantly improved the repair of wound skin tissue and accelerated the reconstruction of the wound, showing significant therapeutic effects and clinical application potential.

Claims

1. A B-CGT hydrogel, characterized in that: The B-CGT hydrogel is composed of Bletilla striata 2-isobutyl malic acid glucose oxybenzyl ester extract, a hydrogel matrix, a crosslinking agent and a pH regulator; the hydrogel matrix is ​​carbomer-940; the crosslinking agent is glycerol; and the pH regulator is triethanolamine.

2. The B-CGT hydrogel according to claim 1, characterized in that: The content of the Bletilla striata 2-isobutylmalic acid glucose oxybenzyl ester extract is 1.0-1.5 g / 100 mL, the content of carbomer-940 is 0.3-0.9 g / 100 mL, the content of glycerol is 12-16 mL / 100 mL, and the content of triethanolamine is 0.45-0.65 mL / 100 mL.

3. The B-CGT hydrogel according to claim 1 or 2, characterized in that: The content of the Bletilla striata 2-isobutylmalic acid glucose oxybenzyl ester extract is 1.25 g / 100 mL, the content of carbomer-940 is 0.71 g / 100 mL, the content of glycerol is 13.89 mL / 100 mL, and the content of triethanolamine is 0.57 mL / 100 mL.

4. The method for preparing the B-CGT hydrogel according to any one of claims 1 to 3, characterized in that: The preparation method of the B-CGT hydrogel is carried out according to the following steps: (1) Pretreatment of PRP-512B reverse resin: Soak PRP-512B resin in 95% ethanol for 24 hours and then pack it into the column so that the filler occupies half of the total column length. Rinse with 95% ethanol until the fraction is mixed with water in a ratio of 1:5 and is not turbid. Then wash with water until there is no alcohol smell. Set aside. (2) Preparation of 2-isobutylmalic acid glucose oxybenzyl ester extract of Bletilla striata: Add 55% ethanol to the dried and crushed Bletilla striata medicinal material at a solid-liquid ratio of 1:10 g / mL, place in an ultrasonic machine for extraction, the ultrasonic power is 344 W, the ultrasonic frequency is 40 kHz, the extraction is performed 3 times, each time for 30 minutes, the residue is filtered, the medicinal liquid is collected and concentrated by a rotary evaporator under reduced pressure, and the solvent is evaporated in a water bath to obtain an extract; weigh the pretreated PRP-512B reverse resin at a mass ratio of 1:15 to the PRP-512B reverse resin, use a dry method to load the sample, and perform gradient elution in the order of washing with water until colorless, washing with 20% ethanol until colorless, and washing with 40% ethanol until colorless, collect the fraction of 40% ethanol, and concentrate under reduced pressure until the water content reaches 10-11%, thereby obtaining 2-isobutylmalic acid glucose oxybenzyl ester extract of Bletilla striata, which is set aside; (3) Preparation of hydrogel matrix: Weigh 0.3-0.9 g of Carbomer-940 and sprinkle it evenly on 60-80 mL of water, let it stand for 10-14 h to allow it to fully swell, stir to remove bubbles, and obtain a hydrogel matrix for later use; (4) B-CGT hydrogel: Measure 12-16 mL of glycerol and add it to the hydrogel matrix while stirring. After mixing, add 1.0-1.5 g of Bletilla striata 2-isobutylmalic acid glucose oxybenzyl ester extract while stirring. Add 0.45-0.65 mL of triethanolamine to adjust the pH value to 5.0-6.

5. Finally, add purified water to make the volume to 100 mL to prepare B-CGT hydrogel.

5. The method for preparing the B-CGT hydrogel according to claim 4, characterized in that: In the step (3), the hydrogel matrix is ​​prepared by weighing 0.6-0.8 g of Carbomer-940, evenly spreading it on 70-80 mL of water, letting it stand for 11-13 hours to allow it to fully swell, stirring to remove bubbles, and obtaining a hydrogel matrix for later use.

6. The method for preparing the B-CGT hydrogel according to claim 5, characterized in that: In the step (3), the hydrogel matrix is ​​prepared by weighing 0.71 g of Carbomer-940, evenly spreading it on 75 mL of water, leaving it to stand for 12 hours to allow it to fully swell, stirring to exhaust air, and obtaining a hydrogel matrix for later use.

7. The method for preparing the B-CGT hydrogel according to claim 4, characterized in that: In the step (4), the preparation of B-CGT hydrogel is as follows: 13-15 mL of glycerol is measured and added to the hydrogel matrix while stirring, and after mixing, 1.20-1.30 g of Bletilla striata 2-isobutyl malic acid glucose oxybenzyl ester extract is added and stirred while adding to mix evenly, and then 0.5-0.6 mL of triethanolamine is added to adjust the pH value to 5.0-6.5, and finally purified water is added to make the volume to 100 mL to prepare B-CGT hydrogel.

8. The method for preparing the B-CGT hydrogel according to claim 7, characterized in that: In the step (4), the preparation of B-CGT hydrogel is as follows: 13.89 mL of glycerol is measured and added to the hydrogel matrix while stirring, and after mixing, 1.25 g of Bletilla striata 2-isobutyl malic acid glucose oxybenzyl ester extract is added and stirred while adding to mix evenly, and then 0.57 mL of triethanolamine is added to adjust the pH value to 5.0-6.5, and finally purified water is added to make the volume to 100 mL to prepare B-CGT hydrogel.

9. Use of the B-CGT hydrogel according to any one of claims 1 to 3 in the preparation of medicines for promoting wound healing.

10. The use according to claim 9, characterized in that: The wound healing comprises diabetic wound healing.

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