Hyaluronic acid composite hydrogel and preparation method and application thereof

Abstract

The present application relates to the technical field of hydrogel, and discloses a hyaluronic acid composite hydrogel, a preparation method and application thereof. First, fat-soluble gallic acid is dispersed and dissolved by glycerol mediation; then, hyaluronic acid with different molecular weights is compounded to form a gel network and a three-dimensional multi-dimensional moisturizing network, and tannic acid (TA) is used as a composite base, and the polyphenol structure of tannic acid is used as a natural cross-linking node and a functional enhancement unit; finally, asiaticoside (AS) is added, and a three-dimensional network hyaluronic acid composite hydrogel is formed through the chain segment winding of hyaluronic acid with different molecular weights, strong hydrogen bonds between polyphenol molecules and between polyphenol and polysaccharide molecules, and hydrophobic interaction. The method is simple in process, mild in condition, green and safe, and avoids the introduction of organic solvents and external catalysts, and the obtained hydrogel has excellent adhesion, self-healing, injectability, moisturizing, antibacterial, antioxidant, wound repair promotion and anti-scarring functions.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of hydrogel, in particular to a hyaluronic acid composite hydrogel and a preparation method and application thereof. BACKGROUND

[0002] Hyaluronic acid (HA) is a commonly used hydrogel matrix material, but single HA hydrogel has poor mechanical properties, no antibacterial properties, and single function, etc. Therefore, it is often modified to improve its performance. For example, a Chinese patent application for invention with publication date of December 16, 2022 and publication number CN115477764A discloses a hyaluronic acid hydrogel and a preparation method and application thereof. The preparation method comprises the following steps: (1) adding a buffer solution to hyaluronic acid, stirring to dissolve, then adding an activator to activate, then adding 5-norbornene-2-methylamine, stirring to react, and post-treatment to obtain modified hyaluronic acid (HA-NB); (2) adding a solvent to the modified hyaluronic acid of step (1) to dissolve, then adding a solution containing Ca 2+ The above-mentioned application can improve the mechanical properties of HA hydrogel through chemical cross-linking, but toxic cross-linking reagents may be left; and additional antibacterial agents are required to improve the antibacterial properties.

[0003] Tannic acid (TA) is a natural plant polyphenol containing a large number of phenolic hydroxyl groups, which has antibacterial, antioxidant, anti-inflammatory and other properties, and can bind to biological macromolecules such as proteins and polysaccharides through various non-covalent bonds (such as hydrogen bonds and hydrophobic interactions). In recent years, it has become a research hotspot to use TA as a cross-linking agent or functional component to construct multifunctional hydrogel. However, the existing technology mostly uses TA to modify polymers (such as gelatin and polyvinyl alcohol) or coordinates with metal ions for cross-linking.

[0004] Gallic acid (GA) is the hydrolysis product of TA and also an effective antibacterial and antioxidant agent, but its solubility in aqueous solution at room temperature is not high and it needs to be dissolved in hot water, and after cooling, it will partially precipitate. Asiaticoside (AS) is a recognized active ingredient for promoting fibroblast proliferation, collagen ordered regeneration and inhibiting scar formation. Therefore, in order to improve the antibacterial properties, mechanical properties and biological activity of hyaluronic acid gel, TA, GA and AS can be introduced into the gel system.

[0005] However, in the preparation process of multi-component composite hydrogel, the traditional method often uses the way of adding components step by step, which leads to long preparation process, low efficiency, and easy to appear the problem of uneven dispersion of components. For example, gallic acid as a natural polyphenol has excellent antibacterial and antioxidant properties, but due to its poor water solubility, it is easy to agglomerate when directly added into the hyaluronic acid system, which affects the performance of the gel; asiaticoside can promote skin repair and scar fading, but in the prior art, its compounding with hyaluronic acid often needs the help of additional dispersants, which increases the product cost and potential safety risk. At present, there is no report on the hyaluronic acid-tannic acid-gallic acid-asiaticoside composite hydrogel which has the functions of scar removal, antibacterial and promoting skin wound repair. SUMMARY

[0006] The purpose of the present application is to provide a method for preparing a hyaluronic acid composite hydrogel with the functions of moisturizing, antibacterial, antioxidant, promoting repair and anti-scarring by "one-pot method". In order to solve one or more technical problems existing in the prior art, at least provide a beneficial choice or create conditions.

[0007] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows.

[0008] The preparation method of the hyaluronic acid composite hydrogel provided by the present application uses glycerol to mediate the dispersion and dissolution of gallic acid (GA), uses hyaluronic acid (HA) and tannic acid (TA) with different molecular weights as a composite substrate, and mixes with asiaticoside (AS), and through the action of different molecular weight hyaluronic acid chain segment winding, strong hydrogen bond between polyphenol molecules and between polyphenol and polysaccharide molecules, and hydrophobic interaction, a three-dimensional network is formed, and then a hyaluronic acid composite hydrogel with uniform structure is prepared by one-pot method.

[0009] The preparation method of the hyaluronic acid composite hydrogel provided by the present application uses glycerol as a dispersion and dissolution solvent for GA, and uses hyaluronic acid with different molecular weights to form a gel network and a three-dimensional multi-dimensional moisturizing network, and a hyaluronic acid-tannic acid-gallic acid-asiaticoside composite hydrogel is prepared by one-pot method. The raw materials of the hyaluronic acid composite hydrogel are all medical grade or natural extracts, there is no chemical crosslinking agent residue, and it can be widely used in infected wound dressings, scar prevention and treatment preparations and drug delivery systems, etc. It provides a safe and efficient solution for wound repair.

[0010] The application provides a preparation method of a hyaluronic acid composite hydrogel, and specific steps are as follows: 1) gallic acid is weighed and added into a glycerol solution, and magnetic stirring is conducted until the gallic acid is completely dissolved, so that a gallic acid-glycerol mixture is obtained; 2) medium molecular weight hyaluronic acid (m-HA, a molecular weight is 200-400 kDa), high molecular weight hyaluronic acid (h-HA, a molecular weight is 1800-2200 kDa), tannic acid and asperosaponin are respectively weighed and added into the gallic acid-glycerol mixture prepared in step 1); 3) deionized water is continuously added into the gallic acid-glycerol mixture in step 2), and stirring is conducted at room temperature until complete dissolution, so that a hyaluronic acid-tannic acid-gallic acid-asperosaponin hydrogel precursor solution is obtained; and 4) the hyaluronic acid-tannic acid-gallic acid-asperosaponin hydrogel precursor solution is transferred into a mold or a well plate, and after standing for a certain time, the hyaluronic acid composite hydrogel is formed.

[0011] In the application, the glycerol solution is a mixture of medical glycerol and water, the concentration of the glycerol solution is 4%-9% (V / V), and further preferably 6%-8% (V / V), wherein the concentration refers to a volume concentration.

[0012] In the application, the concentration of the gallic acid (GA) is 10 mg / mL-50 mg / mL, and further preferably 10 mg / mL-30 mg / mL, wherein the concentration refers to the concentration of the gallic acid uniformly dissolved in the gel precursor solution.

[0013] In the application, the concentration of the medium molecular weight hyaluronic acid (m-HA) is 20 mg / mL-50 mg / mL, and further preferably 30 mg / mL-40 mg / mL, the concentration of the high molecular weight hyaluronic acid (h-HA) is 20 mg / mL-50 mg / mL, and further preferably 30 mg / mL-40 mg / mL, and the mass ratio of m-HA to h-HA is 1:2-2:1, wherein the concentration refers to the concentration of the hyaluronic acid uniformly dissolved in the gel precursor solution.

[0014] In the application, the concentration of the tannic acid (TA) is 10 mg / mL-50 mg / mL, and further preferably 10 mg / mL-30 mg / mL, wherein the concentration refers to the concentration of the tannic acid uniformly dissolved in the gel precursor solution.

[0015] In the application, the concentration of the asperosaponin (AS) is 10 mg / mL-30 mg / mL, and further preferably 10 mg / mL-20 mg / mL, wherein the concentration refers to the concentration of the asperosaponin uniformly dissolved in the gel precursor solution.

[0016] On the other hand, the application also provides a hyaluronic acid composite hydrogel prepared by the preparation method.

[0017] In another aspect, the present application also provides a use of the hyaluronic acid composite hydrogel as described above in a biomedical material.

[0018] The present application provides a hyaluronic acid composite hydrogel preparation method with process innovation and function synergy. In view of the three core pain points of high risk of chemical crosslinking agent residue, uneven dispersion of multiple components, and single function in the preparation of existing hyaluronic acid composite hydrogel, the present application breaks through the traditional technical path and provides a preparation scheme in which medical glycerol is used to mediate the dispersion and dissolution of gallic acid, the poor water solubility and easy agglomeration of gallic acid are solved, and a hyaluronic acid-tannic acid composite with two different molecular weights is used as a fixed substrate, and then a one-pot reaction is performed with asiaticoside. This method does not require additional chemical crosslinking agents, relies on the physical crosslinking of hyaluronic acid with two different molecular weights, tannic acid and gallic acid to form a gel, to improve the mechanical properties, moisturizing properties, adhesion properties, antibacterial properties, antioxidant properties, repair promotion properties and anti-scarring properties of the hyaluronic acid gel, and to improve the preparation efficiency, avoid impurity pollution introduced by step-by-step reaction, and ensure the green preparation and biological safety of the product.

[0019] In addition, the hyaluronic acid composite hydrogel prepared by the present application has excellent function integration while considering safety in use. From the functional level, the hydrogel realizes the trinity effect of "antibacterial- wound repair-scar lightening" through component synergy mechanism: the hyaluronic acid with different molecular weights can provide a three-dimensional long-acting moisturizing network and biocompatibility foundation, the synergistic effect of tannic acid and gallic acid can effectively kill Staphylococcus aureus and Escherichia coli, and effectively reduce the risk of wound infection; asiaticoside can promote fibroblast proliferation and collagen orderly arrangement, accelerate wound healing, and reduce scar tissue formation. From the use characteristics, the hydrogel is in a uniform state without stratification and agglomeration, and can be made into gel dressings, smearing gels, patches and other dosage forms according to actual needs, to adapt to different scenes such as postoperative scar repair, burn wound care, and chronic wound healing; and the raw materials are all medical grade or natural extracts (hyaluronic acid purity ≥98%, gallic acid purity ≥99%, asiaticoside purity ≥95%, glycerol purity ≥99.5%), which are verified by skin irritation test to have no adverse reactions such as redness and itching, and can be safely used on sensitive skin and open wounds.

[0020] The hyaluronic acid composite hydrogel preparation method and application provided by the application have four core values and can promote the development of the medical dressing field. (1) The preparation mode of "glycerol-mediated dispersion dissolution + one-pot reaction" is innovatively used, the traditional step-by-step process is simplified, the energy consumption and industrialization cost are reduced, the risk of chemical crosslinking agent residue is avoided, and the product safety is improved; (2) The concentrations of hyaluronic acid, tannic acid, gallic acid and asiaticoside are precisely controlled, the functions of various components are complementary, and the defects of single function of existing products are solved; (3) The prepared hydrogel has high antibacterial rate (≥90%), fast repair efficiency and strong scar lightening effect, and the comprehensive performance is better than that of existing similar products; (4) It is suitable for various dosage forms and scenes, and can meet the needs of medical skin dressings, scar repair products, wound care products and other fields, and has good clinical application prospect and market value. Through systematic innovation of preparation process, raw material ratio and function design, the application provides a hyaluronic acid composite hydrogel technical scheme with practicability, safety and high efficiency, and provides a new idea and direction for the research and development of functional hydrogel in the field of biological medical materials.

[0021] Compared with the prior art, the application has at least the following advantages and beneficial effects.

[0022] 1) The raw materials of the application are selected from medical grade or natural extracts: the purity of hyaluronic acid is ≥98%, the purity of tannic acid is ≥98%, the purity of gallic acid is ≥99%, the purity of asiaticoside is ≥95%, and the purity of glycerol is ≥99.5%, without adding chemical crosslinking agents and synthetic dispersants.

[0023] 2) The application innovatively uses the mode of "glycerol-mediated dispersion dissolution + one-pot reaction": only through "glycerol pretreatment of gallic acid - multi-component one-pot mixing reaction", the preparation can be completed, the steps are simplified and the preparation efficiency is improved; at the same time, one-pot method avoids the impurity pollution risk caused by multiple transfers and stirring in step-by-step reaction, and improves the product purity. In addition, the process does not require high temperature and high pressure equipment, which can reduce energy consumption, and does not need to add chemical crosslinking agents and dispersants, which can reduce the cost of raw materials and is more suitable for large-scale industrial production.

[0024] 3) The application can form a base support by hyaluronic acid and tannic acid to solve the problem of poor mechanical properties of pure hyaluronic acid; the antibacterial rate of gallic acid and tannic acid to staphylococcus aureus and escherichia coli is ≥90%; under the hyaluronic acid moisturizing environment, the proliferation rate of fibroblasts is improved, the wound healing time is shortened, and the postoperative scar area is reduced, realizing the functions of "antibacterial - repair - scar removal", and the function integration degree is significantly higher than that of existing similar products.

[0025] 4) The prepared hyaluronic acid composite hydrogel is in a uniform gel state without stratification and agglomeration, and the dosage form can be flexibly adjusted according to the clinical requirements, such as a smear gel, a gel dressing, a patch, etc. The gel forming speed is fast without the need of adding an initiator and a crosslinking agent, and the hyaluronic acid composite hydrogel has good antibacterial performance, scar removal, repair promotion, adhesion performance, mechanical performance, cell compatibility, biological activity and self-healing performance, etc. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 Fig. 1 shows a physical sample diagram of the hyaluronic acid composite hydrogel prepared in Example 1 of the present application.

[0027] Figure 2 Fig. 2 shows a scanning electron microscope diagram of the hyaluronic acid composite hydrogel prepared in Example 1 of the present application.

[0028] Figure 3 Fig. 3 shows an adhesion performance test diagram of the hyaluronic acid composite hydrogel prepared in Example 1 of the present application.

[0029] Figure 4 Fig. 4 shows a self-healing performance test diagram of the hyaluronic acid composite hydrogel prepared in Example 1 of the present application.

[0030] Figure 5 Fig. 5 shows an antibacterial performance test comparison diagram of the hyaluronic acid composite hydrogel. DETAILED DESCRIPTION

[0031] The specific embodiments of the present application are further described below to make the technical scheme of the present application and its beneficial effects more clear and explicit. The following description of the embodiments is exemplary and is intended to explain the present application, but cannot be understood as a limitation of the present application.

[0032] Additional aspects and advantages of the present application will become apparent in the description that follows, or will be appreciated by practice of the present application.

[0033] The basic idea of the present application is that: a three-dimensional moisturizing gel network is formed by compounding hyaluronic acid (HA) with medium and high molecular weights, and then the gel base is formed by mixing with tannic acid (TA), and physical cross-linking is further realized through hydrogen bonding, which effectively improves the mechanical properties of the gel and retains the biocompatibility; in view of the comprehensive repair requirements of the wound, functional components are introduced: glycerol solution is innovatively used to pre-disperse and dissolve gallic acid (GA) to strengthen the antibacterial performance and moisturizing capacity, and ascorbic acid (AS) is used to improve the tissue repair and scar inhibition capacity, so as to realize the multi-functional synergistic effect.

[0034] The application completes material mixing and crosslinking reaction under mild conditions through the mode of "glycerol-mediated dispersion dissolution + one-pot method", and obtains a hydrogel system with uniform structure and good wound adhesion. The gel has sustained high moisturizing property, broad-spectrum antibacterial property, significant healing promotion ability and scar removal effect, etc., and can flexibly adapt to various application scenarios such as postoperative wounds, burns, chronic ulcers and daily injuries, to meet the diversified needs of clinical and consumer markets for efficient and safe wound repair materials.

[0035] The hyaluronic acid composite hydrogel prepared by the glycerol-mediated dispersion dissolution and one-pot method has good adhesion and biological activity, and can provide a new research direction, research foundation and idea for the preparation and application of biomedical materials in the field of tissue engineering and regenerative medicine.

[0036] Example 1.

[0037] A preparation method of a hyaluronic acid composite hydrogel, the preparation steps of which are as follows: 1) 150 mg of gallic acid is added to 0.4 mL of a glycerol solution, and stirred at room temperature until completely dissolved to obtain a gallic acid-glycerol mixture; 2) 150 mg of medium molecular weight hyaluronic acid (m-HA, molecular weight 200-400 kDa), 150 mg of high molecular weight hyaluronic acid (h-HA, molecular weight 1800-2200 kDa), 150 mg of tannic acid, and 50 mg of asiaticoside are added to the gallic acid-glycerol mixture; 3) 4.6 mL of deionized water is further added to the gallic acid-glycerol mixture, and stirred at room temperature until completely dissolved to obtain a hyaluronic acid-tannic acid-gallic acid-asiaticoside hydrogel precursor solution; 4) the hyaluronic acid-tannic acid-gallic acid-asiaticoside hydrogel precursor solution is transferred to a mold or a well plate, and left to form a hyaluronic acid composite hydrogel sample 1. The hyaluronic acid composite hydrogel sample 1 prepared by the present application can be completely adsorbed on the bottom of an inverted bottle, and has no obvious flow, as shown in Figure 1 .

[0038] The scanning electron microscope image of the hyaluronic acid composite hydrogel sample 1 prepared in the present example is shown in Figure 2 , from which it can be seen that the hyaluronic acid composite hydrogel sample 1 has a uniform structure and good wound adhesion. Figure 2

[0039] Example 2.

[0040] ​The preparation method of the hyaluronic acid composite hydrogel comprises the following steps: 1) 100 mg of gallic acid is added to 0.4 mL of a glycerol solution, and stirring is performed at room temperature until the gallic acid is completely dissolved, to obtain a gallic acid-glycerol mixture; 2) 150 mg of medium molecular weight hyaluronic acid (m-HA, with a molecular weight of 200-400 kDa), 150 mg of high molecular weight hyaluronic acid (h-HA, with a molecular weight of 1800-2200 kDa), 150 mg of tannic acid and 50 mg of asiaticoside are added to the gallic acid-glycerol mixture; 3) 4.6 mL of deionized water is continuously added to the gallic acid-glycerol mixture, and stirring is performed at room temperature until the hyaluronic acid-tannic acid-gallic acid-asiaticoside hydrogel precursor solution is completely dissolved; and 4) the hyaluronic acid-tannic acid-gallic acid-asiaticoside hydrogel precursor solution is transferred into a mold or a well plate, and is left to stand to form a hyaluronic acid composite hydrogel sample 2. The hyaluronic acid composite hydrogel sample 2 prepared in the application can also be completely adsorbed on the bottom of an inverted bottle without obvious flow, and the structure of the hyaluronic acid composite hydrogel sample 2 is uniform and has good wound adhesion.

[0041] Example Three.

[0042] The preparation method of the hyaluronic acid composite hydrogel comprises the following steps: 1) 100 mg of gallic acid is added to 0.4 mL of a glycerol solution, and stirring is performed at room temperature until the gallic acid is completely dissolved, to obtain a gallic acid-glycerol mixture; 2) 150 mg of medium molecular weight hyaluronic acid (m-HA, with a molecular weight of 200-400 kDa), 150 mg of high molecular weight hyaluronic acid (h-HA, with a molecular weight of 1800-2200 kDa), 150 mg of tannic acid and 50 mg of asiaticoside are added to the gallic acid-glycerol mixture; 3) 4.6 mL of deionized water is continuously added to the gallic acid-glycerol mixture, and stirring is performed at room temperature until the hyaluronic acid-tannic acid-gallic acid-asiaticoside hydrogel precursor solution is completely dissolved; and 4) the hyaluronic acid-tannic acid-gallic acid-asiaticoside hydrogel precursor solution is transferred into a mold or a well plate, and is left to stand to form a hyaluronic acid composite hydrogel sample 2. The hyaluronic acid composite hydrogel sample 2 prepared in the application can also be completely adsorbed on the bottom of an inverted bottle without obvious flow, and the structure of the hyaluronic acid composite hydrogel sample 2 is uniform and has good wound adhesion.

[0043] To better reflect the technical progress of the application, the performance of the hyaluronic acid composite hydrogel prepared in the application is tested. The performances of the hyaluronic acid composite hydrogels prepared in Example One, Example Two and Example Three are basically consistent.

[0044] 1. Physicochemical performance test of the hyaluronic acid composite hydrogel.

[0045] 1) Adhesion test of hyaluronic acid composite hydrogel.

[0046] Test method: As shown in Figure 3 , the prepared hyaluronic acid composite hydrogel sample 1 was attached to objects made of different materials such as paper, rubber, plastic, wood, glass and iron, which covered various interface types from hydrophilic to hydrophobic and from rigid to flexible.

[0047] As can be seen from Figure 3 , the hyaluronic acid composite hydrogel can be tightly contacted on various substrates by external pressure, without obvious slipping or falling phenomenon, which preliminarily proves that the hydrogel has universal adhesion ability. Especially on high smoothness surface and low surface energy material, it still maintains good wettability and adhesion effect, which confirms that the hydrogel can adapt to various application scenarios required by wound dressings, and provides experimental basis for attaching irregular shaped and different material wounds.

[0048] 2) Self-healing test of gel.

[0049] Test method: The hyaluronic acid composite hydrogel sample 1 was cut into two equal parts, which were dyed with rhodamine B and methylene blue respectively, then the cut surfaces of the two cut parts were carefully overlapped, and finally the self-healing of the hydrogel was observed. As shown in Figure 4 .

[0050] As can be seen from Figure 4 , the two cut surfaces of the hyaluronic acid composite hydrogel can be effectively bonded together and self-healed well, and the two cut surfaces are fused together, which indicates that it can form self-healing through the dynamic nature of physical crosslinking, and has the performance of self-healing.

[0051] 2, antibacterial performance test of hyaluronic acid composite hydrogel.

[0052] Hyaluronic acid composite hydrogel sample 1, hyaluronic acid composite hydrogel sample 2, hyaluronic acid composite hydrogel sample 3, blank control group, HA control group, TA-free control group and GA-free control group (TA-free control group and GA-free control group refer to comparative test three and comparative test four respectively) were cultured at 37℃ under Staphylococcus aureus, and the results are shown in Figure 5 .

[0053] As can be seen from Figure 5It can be known that the antibacterial circle diameters of the hyaluronic acid composite hydrogel sample 1, the hyaluronic acid composite hydrogel sample 2 and the hyaluronic acid composite hydrogel sample 3 are large, indicating that the hydrogel prepared in the application has a significant and uniform diffusion inhibition ability to Staphylococcus aureus. The size difference of the antibacterial circle can reflect the strength trend of the antibacterial activity under different formulations or concentration gradients. In the concentration range of 1-3% of GA, the higher the concentration of GA, the more obvious the antibacterial effect. Compared with the blank control group, the HA control group, the TA-free control group and the GA-free control group, the hyaluronic acid composite hydrogel prepared in the application all shows a stronger antibacterial effect, and the combination of GA and TA has a synergistic effect of enhancing the antibacterial effect.

[0054] In order to better reflect the synergistic effect of each combination in the application and the technical progress, the inventors also made the following comparative tests.

[0055] Comparative test one: all the medium molecular weight hyaluronic acid in example one is replaced by high molecular weight hyaluronic acid, and other ingredients and processes remain unchanged. The sample prepared in the comparative test one cannot be attached to the bottom of the inverted bottle and fails to form a hydrogel similar to the application.

[0056] Comparative test two: all the high molecular weight hyaluronic acid in example one is replaced by medium molecular weight hyaluronic acid, and other ingredients and processes remain unchanged. The sample prepared in the comparative test two cannot be attached to the bottom of the inverted bottle and fails to form a hydrogel similar to the application.

[0057] Comparative test three: all the tannic acid in example one is replaced by gallic acid, and other ingredients and processes remain unchanged. The sample prepared in the comparative test three cannot be attached to the bottom of the inverted bottle and fails to form a hydrogel similar to the application.

[0058] Comparative test four: all the gallic acid in example one is replaced by tannic acid, and other ingredients and processes remain unchanged. Although the sample prepared in the comparative test four can be attached to the bottom of the inverted bottle, it will flow to a certain extent.

[0059] Comparative test five: the glycerol in step 1) of example one is replaced by deionized water, and other ingredients and processes remain unchanged. The sample prepared in the comparative test five cannot be attached to the bottom of the inverted bottle and fails to form a hydrogel similar to the application.

[0060] From the comparative test one and the comparative test two, it can be seen that single high molecular weight hyaluronic acid or single medium molecular weight hyaluronic acid cannot successfully prepare a hydrogel, indicating that the combination of high molecular weight hyaluronic acid and medium molecular weight hyaluronic acid is the key to successfully preparing a hydrogel in the application, and both are indispensable.

[0061] From the comparison test three, the lack of tannin acid also failed to form a hydrogel, indicating that if a suitable hyaluronic acid molecular weight combination is one of the basic conditions for forming a hydrogel, other ingredients also need to play a synergistic role, and tannin acid plays a key role in the formation of hydrogel. This may be that tannin acid plays a hydrogen bond cross-linking role of polyphenol hydroxyl group, and the lack of tannin acid makes it difficult to form a hydrogel. In addition, it is found that the lack of tannin acid will make the antibacterial performance of the sample worse after comparing the antibacterial performance of the sample prepared in the comparison test three with the sample prepared in the first embodiment of the present application, indicating that the addition of tannin acid can improve the antibacterial performance of the gel.

[0062] From the comparison test four, the lack of gallic acid also failed to form a non-flowing hydrogel, and there was some flow, but less than the sample lacking tannin acid. It can be seen that gallic acid is also essential in the formation of hydrogel. In addition, it is found that the lack of gallic acid will make the antibacterial performance of the sample significantly worse after comparing the antibacterial performance of the sample prepared in the comparison test three with the sample prepared in the first embodiment of the present application, indicating that the addition of gallic acid can significantly improve the antibacterial performance of the gel.

[0063] From the comparison test five, it is also impossible to form a hydrogel after replacing glycerol with deionized water to dissolve gallic acid, which may be because gallic acid cannot be completely dissolved in water, and the mediated dispersion and dissolution effect of glycerol on gallic acid has an important influence on the formation of hydrogel.

[0064] From the comparison test one to the comparison test five, it can be seen that the successful preparation of hydrogel requires the combination of different molecular weight of hyaluronic acid, and tannin acid and gallic acid are indispensable, and gallic acid needs the mediated dispersion and dissolution of glycerol, and finally through the mutual synergistic effect of each component, the effective formation of hydrogel can be realized.

[0065] It should be noted that any combination of the technical features of the above embodiments can be combined, and in order to make the description simple, all possible combinations of the technical features in the above embodiments are not described, but as long as the combination of these technical features does not exist contradictory, it should be considered as the scope of the description.

[0066] The above embodiments only express several embodiments of the present application, and the description is more specific and detailed, but it should not be understood as limiting the scope of the patent. It should be noted that for ordinary skilled in the art, without departing from the concept of the present application, a number of variations and improvements can be made, which are within the scope of the present application. The parts not described in the specific embodiments are prior art or common knowledge.

Claims

1. A method for preparing a hyaluronic acid composite hydrogel, characterized in that, The preparation steps are as follows: 1) Dissolve gallic acid in glycerol solution to obtain gallic acid-glycerol mixture; 2) Add medium molecular weight hyaluronic acid, high molecular weight hyaluronic acid, tannic acid and asiaticoside to gallic acid-glycerol mixture respectively; 3) Continue to add deionized water to gallic acid-glycerol mixture and stir at room temperature until completely dissolved to obtain hyaluronic acid-tannic acid-gallic acid-asiaticoside hydrogel precursor solution; 4) Transfer hyaluronic acid-tannic acid-gallic acid-asiaticoside hydrogel precursor solution to mold or well plate and let stand for a certain time until hyaluronic acid composite hydrogel is formed; the molecular weight of the medium molecular weight hyaluronic acid is 200-400 kDa, and the molecular weight of the high molecular weight hyaluronic acid is 1800-2200 kDa.

2. The method for preparing a hyaluronic acid composite hydrogel according to claim 1, characterized in that, The glycerol solution is a mixture of medical glycerol and deionized water, and the volume concentration of glycerol in the glycerol solution is 4%-9%.

3. The method for preparing a hyaluronic acid composite hydrogel according to claim 1, characterized in that, In the hyaluronic acid-tannic acid-gallic acid-asiaticoside hydrogel precursor solution, the concentration of gallic acid is 10 mg / mL-50 mg / mL.

4. The method for preparing a hyaluronic acid composite hydrogel according to claim 1, characterized in that, In the hyaluronic acid-tannic acid-gallic acid-asiaticoside hydrogel precursor solution, the mass ratio of the medium molecular weight hyaluronic acid to the high molecular weight hyaluronic acid is 1:2-2:1, the concentration of the medium molecular weight hyaluronic acid is 20 mg / mL-50 mg / mL, and the concentration of the high molecular weight hyaluronic acid is 20 mg / mL-50 mg / mL.

5. The method for preparing a hyaluronic acid composite hydrogel according to claim 1, characterized in that, In the hyaluronic acid-tannic acid-gallic acid-asiaticoside hydrogel precursor solution, the concentration of tannic acid is 10 mg / mL-50 mg / mL.

6. The method for preparing a hyaluronic acid composite hydrogel according to claim 1, characterized in that, In the hyaluronic acid-tannic acid-gallic acid-asiaticoside hydrogel precursor solution, the concentration of asiaticoside is 10 mg / mL-30 mg / mL.

7. The method for preparing a hyaluronic acid composite hydrogel according to claim 1, characterized in that, The purity of the medium molecular weight hyaluronic acid and the high molecular weight hyaluronic acid is ≥98%, the purity of the tannic acid is ≥98%, and the purity of the gallic acid is ≥99%.

8. A hyaluronic acid composite hydrogel, characterized in that, It is prepared using the preparation method of a hyaluronic acid composite hydrogel as described in any one of claims 1-7.

9. The application of the hyaluronic acid composite hydrogel as described in claim 8 in biomedical materials.

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