Preparation method and application of Chinese herbal medicine hydrogel

Abstract

The invention discloses a preparation method and application of Chinese herbal medicine hydrogel. The preparation method of the Chinese herbal medicine hydrogel comprises the following steps: (1) preparing keratinocyte vesicles by taking keratinocytes as a raw material, then adding asiaticoside, and extruding back and forth through a cellulose acetate membrane to obtain keratinocyte loaded asiaticoside vesicles; and (2) diluting the keratinocyte loaded asiaticoside vesicles with a buffer solution, then adding calcium carbonate powder, then adding atriplex obscura leaf powder, uniformly mixing, and then shaping with a mold to obtain the Chinese herbal medicine hydrogel. According to the Chinese herbal medicine hydrogel, the asiaticoside is combined with the keratinocyte bionic nano-vesicles, so that the medicine can be promoted to better reach the interior of skin cells, and the Chinese herbal medicine hydrogel is particularly suitable for treating burn wounds, diabetic wounds and other wounds difficult to heal.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a method for preparing and applying a hydrogel made from traditional Chinese medicine. Background Technology

[0002] Traditional Chinese medicine (TCM) has a long history in wound treatment in China. TCM herbs are derived from nature, have numerous components, and are difficult to prepare artificially. In recent years, TCM has shown significant advantages in regulating inflammation, promoting angiogenesis, and stimulating cell proliferation and collagen synthesis. Currently widely used burn treatment drugs, such as silver sulfadiazine, antibiotic ointments, and skin substitutes, are mainly used to fight infection and relieve inflammation. However, they suffer from problems such as cytotoxicity, allergic reactions, increased drug resistance, or limited repair effects, making it difficult to fully meet the clinical needs for wound healing. Therefore, there is an urgent need to develop safe, effective, and cost-effective new wound treatment drugs.

[0003] *Atriplex laciniata*, also known as Tofu Wood Leaf, mainly grows in East, Central, and South China. Its leaves are rich in flavonoids, triterpenoids, and polyphenols, exhibiting good anti-inflammatory, antioxidant, and antibacterial potential; however, research on its application in burn wound repair is limited. Asiaticoside, the main triterpenoid active ingredient extracted from *Centella asiatica*, has been shown to accelerate wound repair by promoting fibroblast proliferation, stimulating type I collagen synthesis, and angiogenesis, and possesses multiple pharmacological effects including anti-inflammatory and antioxidant properties; however, its wound repair efficacy is relatively poor. Summary of the Invention

[0004] The primary objective of this invention is to overcome the shortcomings and deficiencies of the prior art and provide a method for preparing hydrogels made from traditional Chinese medicine.

[0005] Another object of the present invention is to provide a hydrogel of traditional Chinese medicine prepared by the method described above.

[0006] Another object of the present invention is to provide the application of the herbal hydrogel.

[0007] The objective of this invention is achieved through the following technical solution: A method for preparing a hydrogel made from traditional Chinese medicine includes the following steps: (1) Preparation of keratinocyte-loaded asiaticoside vesicles: After lysing and grinding keratinocytes (Hacat), centrifuging was performed, and the supernatant was collected to obtain a cell membrane solution. Then, the cell membrane solution was squeezed back and forth using a cellulose acetate membrane to prepare keratinocyte vesicles. A asiaticoside solution was then added to the keratinocyte vesicles, and after ultrasonic mixing, the mixture was squeezed back and forth using a cellulose acetate membrane to obtain keratinocyte vesicles loaded with asiaticoside. (2) Preparation of keratinocyte-loaded asiaticoside vesicle hydrogel: ① After sterilizing the leaves of *Trifolium repens*, dehydrate and dry them using a freeze dryer, then grind them to obtain *Trifolium repens* leaf powder; ② After diluting the keratinocyte-loaded asiaticoside vesicles obtained in step (1) with a buffer solution, add calcium carbonate powder to obtain a calcium carbonate solution containing cell vesicles; or first prepare a calcium carbonate solution with a buffer solution, and then add the keratinocyte-loaded asiaticoside vesicles obtained in step (1) to obtain a calcium carbonate solution containing cell vesicles. ③ Add the small-leaved ginseng leaf powder obtained in step ① to the calcium carbonate solution containing cell vesicles, mix evenly, and then shape it with a mold to obtain keratinocyte-loaded asiaticoside vesicle hydrogel, namely the herbal hydrogel.

[0008] The density of keratinocytes (Hacat) mentioned in step (1) is preferably 5 million / ml.

[0009] The lysis described in step (1) is performed by adding cell lysis buffer.

[0010] The grinding described in step (1) is performed using a grinding rod; preferably, the grinding is performed by grinding with a grinding rod about 20 times.

[0011] The centrifugation speed in step (1) is 3000-3500 g / min; preferably 3200 g / min.

[0012] The keratinocyte vesicles described in step (1) are preferably prepared by the following steps: pressing back and forth 30 times each using cellulose acetate membranes with pore sizes of 400 nm and 200 nm.

[0013] The concentration of keratinocyte vesicles mentioned in step (1) is 100-500 mg / ml.

[0014] The concentration of the asiaticoside solution in step (1) is 0.8–1.2 mmol / L; preferably 1 mmol / L.

[0015] The keratinocyte-loaded asiaticoside vesicles described in step (1) are preferably prepared by the following steps: pressing back and forth 30 times each using a cellulose acetate membrane with a pore size of 400 nm.

[0016] The small-leaved schefflera leaves mentioned in step (2) ① are small-leaved schefflera leaves that grow from May to October.

[0017] The disinfection mentioned in step (2) ① is carried out using 75% alcohol.

[0018] The conditions for dehydration and drying in the freeze dryer described in step (2) ① are: temperature -20℃ to -80℃, time 4 to 12 hours.

[0019] The buffer solution mentioned in step (2) ② is preferably physiological saline.

[0020] In step (2) ②, the concentration of calcium carbonate in the calcium carbonate solution containing cell vesicles is 0.5-1% by mass (preferably 1% by mass), the concentration of vesicles is 100-1000 ng / ml (preferably 100 ng / ml), and the concentration of asiaticoside is 10-100 μmol / L (preferably 100 μmol / L).

[0021] The amount of *Platycladus orientalis* leaf powder mentioned in step (2) ③ is calculated as 6-10g of *Platycladus orientalis* leaf powder per 100ml of calcium carbonate solution containing cell vesicles (accounting for 6%-10% (w / v) of the calcium carbonate solution containing cell vesicles); preferably, it is calculated as 10g of *Platycladus orientalis* leaf powder per 100ml of calcium carbonate solution containing cell vesicles.

[0022] The mold mentioned in step (2) ③ is a conventional mold in the art, such as a round or other shaped mold.

[0023] A hydrogel made from traditional Chinese medicine is prepared by any of the methods described above.

[0024] The application of the herbal hydrogel in the preparation of wound treatment (wound repair) drugs.

[0025] The wound treatment mentioned includes the treatment of ordinary wounds, or difficult-to-heal wounds such as burns and diabetic wounds.

[0026] The present invention has the following advantages and effects compared with the prior art: 1. The herbal hydrogel of this invention promotes better drug delivery to skin cells by combining asiaticoside with keratinocyte-mimetic nanovesicles. Drug delivery is achieved through a keratinocyte-loaded asiaticoside vesicle hydrogel. Compared to ordinary clinical dressings or other types of hydrogels, this composite hydrogel exhibits better synergistic effects. This method is expected to provide new ideas for the modernization of traditional Chinese medicine and also offers potential novel topical drug options for burn treatment.

[0027] 2. Based on the pharmacological characteristics of *Trifolium repens* leaves and asiaticoside, this invention provides a herbal hydrogel that is particularly suitable for treating difficult-to-heal wounds such as burns and diabetic lesions. This hydrogel combines traditional Chinese medicine, its effective components, and cell-mimetic nanovesicle components, maximizing the molecular function of the herbs while providing non-invasive drug delivery, reducing pain during dressing changes, adding scientific basis for the clinical application of traditional Chinese medicine, and providing better conditions for wound treatment. Attached Figure Description

[0028] Figure 1 These are the characterization results of keratinocytes loaded with asiaticoside vesicles; where A is the SDS-PAGE result of keratinocytes (Hacat) and keratinocyte vesicles (VEs); B is the particle size of keratinocyte vesicles; C is the particle size of keratinocytes loaded with asiaticoside; D is the transmission electron microscopy image of keratinocyte vesicles; E is the DIO fluorescence staining result of keratinocyte vesicles; F is the transmission electron microscopy image of asiaticoside; and G is the transmission electron microscopy image of keratinocytes loaded with asiaticoside.

[0029] Figure 2 The figure shows the characterization results of keratinocyte-loaded asiaticoside vesicular hydrogel; where A and B are both *Acer negundo* leaves; C is *Acer negundo* leaf hydrogel (simple hydrogel); D is keratinocyte-loaded asiaticoside vesicular hydrogel; E: mechanical property test results of three hydrogels with different concentrations.

[0030] Figure 3 The results of the cell vesicle screening experiment are shown in Figure 1. A shows the depth of penetration of keratinocyte vesicles and adipose-derived stem cell vesicles into the skin, compared using a rat skin depth test. B shows the number of vesicles entering cells at different time points, using keratinocyte vesicles and adipose-derived stem cell vesicles. C shows the statistical analysis of the number of cell vesicles entering the cell interior in Figure 2 (* represents P<0.05, ** represents P<0.01). D shows the expression of adhesion proteins in different experimental groups. E shows the statistical analysis of D (** represents P<0.01).

[0031] Figure 4 This is a diagram showing the antibacterial test results of keratinocytes loaded with asiaticoside vesicle hydrogel.

[0032] Figure 5 This is a comparison of the therapeutic effects of different drugs on burn wounds in rats. Detailed Implementation

[0033] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Test methods in the following embodiments that do not specify specific experimental conditions are generally performed according to conventional experimental conditions or experimental conditions recommended by the manufacturer. Unless otherwise specified, the reagents and raw materials used in the present invention are commercially available.

[0034] Example 1 1. Preparation and characterization of asiaticoside-loaded vesicles on keratinocytes: The keratinocytes used in this experiment were purchased from the Ruijing Mall Hacat Cells (brand: Pronosai). They were digested with 2.5% trypsin as the main digesting enzyme, and then counted at a density of 5 million / ml. Subsequent processing mainly included the following steps: (1) After the Hacat cells were treated with cell lysis buffer, they were ground 20 times with a grinding rod, and then centrifuged at 3200g / min to obtain the cell membrane supernatant. (2) Take the cell membrane solution from step (1), and squeeze it back and forth 30 times each using cellulose acetate membranes with pore sizes of 400 nm and 200 nm, and then prepare keratinocyte vesicles with a concentration of 100-500 mg / ml for later use. (3) Take the keratinocyte vesicles prepared in step (2), add 1 mmol / L of asiaticoside solution, mix with ultrasound, and squeeze back and forth 30 times with a cellulose acetate membrane with a pore size of 400 nm to obtain vesicles containing the material, namely keratinocyte vesicles loaded with asiaticoside. Before use, they can be diluted with physiological saline to make the concentration of the vesicles 100-1000 ng / ml and the concentration of asiaticoside 10-100 μmol / L.

[0035] (4) Perform SDS-PAGE analysis (with Hacat cells as control) and DIO fluorescence staining on the keratinocyte vesicles prepared in step (2), and perform particle size and transmission electron microscopy analysis (with asiaticoside as control) on the keratinocyte vesicles and keratinocyte asiaticoside-loaded vesicles prepared in steps (2) and (3).

[0036] The results are as follows Figure 1 As shown: The results revealed that keratinocyte vesicles (VEs) contain protein types similar to those of keratinocytes (Hacat), which proves that vesicles can perform some of the functions of cells. The diameter of keratinocyte vesicles and keratinocyte vesicles containing asiaticoside is 150-250 nm, which is similar to the size of cell exosomes. Electron microscopy results further confirmed the size and morphology of the vesicles.

[0037] 2. Preparation and characterization of keratinocyte-loaded asiaticoside vesicle hydrogels: The keratinocyte-loaded asiaticoside hydrogel used in this experiment utilizes the principle that keratinocyte vesicles have good skin cell adhesion properties, and incorporates traditional Chinese herbal medicines used in burn treatment, delivering the drug in hydrogel form. Its preparation process mainly consists of the following steps: (1) Preparation of small-leaved schistose leaf powder: First, about 500g of small-leaved schistose leaves grown in Anhui from May to October were picked. The leaves were disinfected with 75% alcohol and then dehydrated using a freeze dryer (the freeze dryer was set between -20℃ and -80℃ for 4-12 hours) until the leaves were dry (the leaves were dry enough that they could easily form powder when subjected to external force (such as a grinding rod). The powder was then prepared and stored at 4℃ for the next experiment.

[0038] (2) Preparation of hydrogel (a) Prepare keratinocyte-loaded asiaticoside vesicular hydrogels containing 6%-8%-10% (w / v) of each cell, using any of the following methods: ① Prepare keratinocyte-loaded asiaticoside vesicles using physiological saline (prepared in step 1 (3) above), so that the final concentration of the vesicles is 100 ng / ml and the final concentration of asiaticoside is 100 μmol / ml. Then weigh calcium carbonate powder and add it to this saline system to obtain a calcium carbonate solution containing vesicles with a concentration of 0.5-1% (w / v) (1% was added in this example). Then mix the small-leaved ginseng leaf powder prepared in step 2 (1) above with the calcium carbonate solution containing vesicles evenly, wherein the amount of small-leaved ginseng leaf powder accounts for 6%, 8%, and 10% (w / v) of the calcium carbonate solution containing vesicles. Then use a round (or other shape) mold to wait for gelation (this process usually occurs within 1-2 minutes) to obtain 6%, 8%, and 10% keratinocyte-loaded asiaticoside vesicle hydrogels.

[0039] ② First, prepare a calcium carbonate solution with physiological saline, and then add the keratinocyte-loaded asiaticoside vesicles prepared in step 1 (3) above to obtain a mixed solution containing calcium ions (the final concentration of the vesicles is 100 ng / ml, the final concentration of asiaticoside is 100 μmol / ml, and the final concentration of calcium carbonate is 0.5-1% (W / V)); then mix the small-leaved ginseng leaf powder prepared in step 2 (1) above with the mixed solution containing calcium ions evenly, wherein the amount of small-leaved ginseng leaf powder accounts for 6%, 8%, and 10% (w / v) of the calcium carbonate solution containing vesicles, respectively. Then, use a round (or other shape) mold to wait for gelation (this process usually occurs within 1-2 minutes) to obtain 6%, 8%, and 10% keratinocyte-loaded asiaticoside vesicle hydrogels.

[0040] (b) Preparation of small-leaved schistose leaf hydrogel (simple hydrogel) Following the method in step ① above, the difference is that keratinocytes loaded with asiaticoside vesicles are not added to prepare small-leaved ginseng leaf hydrogel (the active substance is only small-leaved ginseng).

[0041] (3) The mechanical properties of the keratinocyte-loaded asiaticoside vesicle hydrogels prepared in step ① at different concentrations were tested using a RapidTA® texture analyzer. The test conditions were set to a deformation of 30%, and the mechanical force required to deform to 30% was observed to compare the stress of different hydrogels. Meanwhile, the *Acer negundo* leaf hydrogel (simple hydrogel) (10% *Acer negundo* leaf) was used as a control. The experiment was repeated three times.

[0042] The results are as follows Figure 2 As shown, there was no significant difference in appearance between the small-leaved asiaticus leaf hydrogel (10%) and the keratinocyte-loaded asiaticoside vesicle hydrogel (10%). As the concentration of small-leaved asiaticus leaf increased, the stress also increased. However, as the concentration of small-leaved asiaticus leaf continued to increase, the solubility also increased. Therefore, we selected the 10% keratinocyte-loaded asiaticoside vesicle hydrogel for subsequent experiments.

[0043] Example 2 1. Screening of cell vesicles: (1) Experimental materials: ① SD rats, male, purchased from the Experimental Animal Center of Southern Medical University, 4-6 weeks old, weighing 100g-150g; ② Keratinocyte glycoside vesicles (concentration 500 ng / ml): prepared in step 1 (2) of Example 1; ③Adipose-derived stem cell vesicles (concentration 500ng / ml): The preparation method is the same as in Example 1, except that keratinocytes are replaced with adipose-derived stem cells, and the rest of the preparation method is the same to prepare adipose-derived stem cell vesicles; wherein the adipose-derived stem cells of rats are obtained by primary culture of fat from SD rats.

[0044] (2) Experimental methods 1) Animal experiment on vesicle penetration into the skin: Six SD rats were anesthetized with 2% isoflurane. The hair on the rats' backs was removed, and the backs were divided into two sections. The left side was treated with 0.01 ml of adipose-derived stem cell vesicles (500 ng / ml after DAPI staining), while the right side was treated with the same concentration of keratinocyte vesicles. After 30 minutes, skin sections were excised from the rats' backs for staining and observation. Blue represents DAPI staining of cell nuclei, and red represents the depth of vesicle penetration into the skin. The control group is shown as the fluorescence image of DAPI-stained skin from rats that did not undergo vesicle treatment.

[0045] 2) Cellular assays of vesicle entry into the skin: Human skin fibroblasts were purchased from Ragen iCell (iCell-h337). Fibroblasts were seeded at a density of 10,000 cells / well in 24-well plates. Keratinocyte vesicles and adipose-derived stem cell vesicles were stained with DIL, and then added to each well for treatment with 500 ng / ml of stained vesicles. Fluorescence was observed at 3, 6, and 9 hours. The cytoskeleton was stained with α-SMA (abcam, 1:250), and the cell nuclei were stained with DAPI. The experiment was performed in triplicate.

[0046] The results are as follows Figure 3 As shown in AC, compared with adipose stem cell vesicles, keratinocyte vesicles can reach the deeper layers of the skin faster and in greater quantities. Therefore, keratinocyte vesicles were selected as the carrier of asiaticoside.

[0047] Furthermore, the keratinocyte vesicles prepared in Example 1 and the keratinocyte vesicles loaded with asiaticoside were co-cultured with commercially available human skin fibroblasts. The difference in the rate at which the two types of cell vesicles entered the cells was observed, and the cell adhesion protein β-Catenin was detected in the fibroblasts. The specific steps are as follows: Asiaticoside (100 μg / ml), keratinocyte vesicles (500 ng / ml) (vesicle group), and keratinocytes loaded with Asiaticoside vesicles (100 μg / ml Asiaticoside and 500 ng / ml vesicles) (asiaticoside + vesicle group) were co-cultured with human skin fibroblasts. Human skin fibroblasts were seeded in 24-well plates at a density of 10,000 cells / ml. After 24 hours of cell seeding, the Asiaticoside group, vesicle group, and Asiaticoside + vesicle group were treated with physiological saline as a control for 24 hours. Western blotting (WB) was performed on each experimental group after 24 hours to detect the expression of cell adhesion proteins (n=3).

[0048] The results are as follows Figure 3 As shown in D and E, the addition of keratinocyte vesicles increases the expression of the adhesion protein β-Catenin, which can enhance the cell's adhesion function.

[0049] Example 3 We conducted an antibacterial experiment against methicillin-resistant Staphylococcus aureus (BNCC388949) (purchased from Ruijing Mall), using other herbal plants for comparison. The specific steps are as follows: Take 100 μL of Staphylococcus aureus culture and incubate it in 10 mL of LB medium for 24 hours. Then repeat this step to obtain relatively stable bacteria, and count the bacteria using a colorimetric method.

[0050] Experimental groups: keratinocyte-loaded asiaticoside vesicle hydrogel (prepared according to the method of preparing hydrogel (10% of small-leaved ginseng leaf) in step (2)① of Example 1, the difference being that calcium ion solution was not added to obtain a liquid product), simple hydrogel group (prepared according to the method of preparing small-leaved ginseng leaf hydrogel (10%) in step (2) (b) of Example 1, the difference being that calcium ion solution was not added to obtain a liquid product), mulberry leaf group (mulberry leaves were freeze-dried and ground into powder, then added to physiological saline to prepare a concentration of 10% (w / v) for use), asiaticoside group (1 mmol / L), with physiological saline as a blank control (Ctrl). 1 ml of the liquid from each experimental group was added to the bacteria to be diluted (final bacterial concentration was 1*10). 9 The solution was added to a container (cfu / ml) and then incubated overnight (8-12 hours) at 4°C. The antibacterial activity was then examined by plating the sample the next day. The experiment was conducted in triplicate.

[0051] The results are as follows Figure 4 As shown, the results showed that neither hydrogel nor mulberry leaf alone had a significant antibacterial effect, while asiaticoside could achieve a better antibacterial effect, and the hydrogel loaded with asiaticoside on keratinocytes had the best effect.

[0052] Example 4 A comparison of the therapeutic effects of different drug treatments on burn wounds in rats was conducted, with the specific steps as follows: Eight SD rats (4-6 weeks old, weighing 100-150g) were purchased from the Experimental Animal Center of Southern Medical University. A burn model was created on the rats' backs using a burn instrument. Each rat was anesthetized with isoflurane, and after skin preparation and disinfection, four burn points (approximately 1.2cm in diameter, circular, and approximately 1.1cm in area) were made on the rat's back. 2 Four rats were observed and analyzed on day 3, and four rats were observed and analyzed on day 7. After the burn model on the back of each rat was prepared, the scab was removed, and then four groups of experiments were conducted: ① Saline blank control group: The burn wound was treated with physiological saline at a dose of 0.1 ml; ② Simple vesicle group: Keratinocytes loaded with asiaticoside vesicles (prepared in step 1 (3) of Example 1) were mixed with physiological saline to prepare a concentration of 500 ng / ml, and then 0.1 ml was used for wound treatment; ③ Simple hydrogel group: The wound was covered with 10% small-leaved thorn leaf hydrogel (prepared in step (2) (b) of Example 1), and the dosage was 0.1 ml; ④ Hydrogel vesicle group: The keratinocyte-loaded asiaticoside vesicle hydrogel (10% of small-leaved quinquefolia leaf) prepared in step (2) ① of Example 1 was used at a dosage of 0.1 ml; Photos were taken on days 0, 3, and 7.

[0053] The results are as follows Figure 5 As shown: Hydrogel vesicle combination therapy can better promote wound repair.

[0054] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a hydrogel made from traditional Chinese medicine, characterized in that, Includes the following steps: (1) Preparation of keratinocyte-loaded asiaticoside vesicles: After lysing and grinding keratinocytes, centrifugation was performed to obtain a cell membrane solution. The cell membrane solution was then squeezed back and forth using a cellulose acetate membrane to prepare keratinocyte vesicles. A asiaticoside solution was then added to the keratinocyte vesicles, and after ultrasonic mixing, the mixture was squeezed back and forth using a cellulose acetate membrane to obtain keratinocyte vesicles loaded with asiaticoside. (2) Preparation of keratinocyte-loaded asiaticoside vesicle hydrogel: ① After sterilizing the leaves of *Trifolium repens*, dehydrate and dry them using a freeze dryer, then grind them to obtain *Trifolium repens* leaf powder; ② After diluting the keratinocyte-loaded asiaticoside vesicles obtained in step (1) with a buffer solution, add calcium carbonate powder to obtain a calcium carbonate solution containing cell vesicles; or first prepare a calcium carbonate solution with a buffer solution, and then add the keratinocyte-loaded asiaticoside vesicles obtained in step (1) to obtain a calcium carbonate solution containing cell vesicles. ③ Add the small-leaved ginseng leaf powder obtained in step ① to the calcium carbonate solution containing cell vesicles, mix evenly, and then shape it with a mold to obtain keratinocyte-loaded asiaticoside vesicle hydrogel, namely the herbal hydrogel.

2. The method for preparing the herbal hydrogel according to claim 1, characterized in that: In step (2) ②, the concentration of calcium carbonate in the calcium carbonate solution containing cell vesicles is 0.5-1% by mass, the concentration of vesicles is 100-1000 ng / ml, and the concentration of asiaticoside is 10-100 μmol / L. The amount of *Trifolium repens* leaf powder mentioned in step (2) ③ is calculated as 6-10g of *Trifolium repens* leaf powder per 100ml of calcium carbonate solution containing cell vesicles.

3. The method for preparing herbal hydrogel according to claim 2, characterized in that: In step (2) ②, the calcium carbonate solution containing cell vesicles has a calcium carbonate concentration of 1% by mass, a vesicle concentration of 100 ng / ml, and a asiaticoside concentration of 100 μmol / L. The amount of *Trifolium repens* leaf powder mentioned in step (2) ③ is calculated as 10g of *Trifolium repens* leaf powder per 100ml of calcium carbonate solution containing cell vesicles.

4. The method for preparing the herbal hydrogel according to claim 1, characterized in that: The concentration of keratinocyte vesicles mentioned in step (1) is 100-500 mg / ml; The concentration of the asiaticoside solution mentioned in step (1) is 0.8–1.2 mmol / L.

5. The method for preparing herbal hydrogel according to claim 1, characterized in that: The keratinocyte vesicles described in step (1) are prepared by the following steps: extruding cellulose acetate membranes with pore sizes of 400 nm and 200 nm back and forth 30 times each; The keratinocyte-loaded asiaticoside vesicles described in step (1) were prepared by the following steps: pressing back and forth 30 times each using a cellulose acetate membrane with a pore size of 400 nm.

6. The method for preparing the herbal hydrogel according to claim 1, characterized in that: The buffer solution mentioned in step (2) ② is physiological saline.

7. The method for preparing the herbal hydrogel according to claim 1, characterized in that: The centrifugation speed in step (1) is 3000-3500 g / min; The disinfection mentioned in step (2) ① is carried out using 75% alcohol; The conditions for dehydration and drying in the freeze dryer described in step (2) ① are: temperature -20℃ to -80℃, time 4 to 12 hours.

8. A hydrogel made from traditional Chinese medicine, characterized in that: It is prepared by the method described in any one of claims 1 to 7.

9. The use of the herbal hydrogel according to claim 8 in the preparation of wound treatment drugs.

10. The application according to claim 9, characterized in that: The wound treatment mentioned includes the treatment of ordinary wounds or wounds that are difficult to heal; The aforementioned difficult-to-heal wounds include burns or diabetic wounds.

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