A chitosan-hyaluronic acid hydrogel composition based on astragaloside and salvianolic acid B, a preparation method and application thereof

Abstract

This invention relates to the field of biomedical materials technology, specifically to a chitosan-hyaluronic acid hydrogel composition based on astragaloside A and salvianolic acid B, its preparation method, and its applications. This hydrogel constructs a three-dimensional network of synergistic cross-linking between chitosan and hyaluronic acid, loading multiple natural active ingredients, including astragaloside A, salvianolic acid B, ferulic acid, and notoginsenoside R1, forming a composite hydrogel with good biocompatibility, controllable degradation, and anti-inflammatory and anti-adhesion functions. A maleimide-cysteine ​​system is used to achieve C-S covalent cross-linking, improving structural stability and component release efficiency. Animal experiments show that this hydrogel can significantly reduce postoperative adhesion scores and inflammatory factor levels, demonstrating broad potential for postoperative anti-adhesion applications.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a chitosan-hyaluronic acid hydrogel composition based on astragaloside A and salvianolic acid B, its preparation method, and its application. Background Technology

[0002] Intrauterine surgeries (such as adhesiolysis and endometrial polyp removal) are widely used in the treatment of female infertility and abnormal uterine bleeding. However, postoperative damage to the basal layer of the endometrium often leads to re-adhesion, significantly affecting the endometrial repair capacity and fertility prognosis. Even with traditional physical isolation methods such as balloons, intrauterine devices, or hyaluronic acid gels, it is still difficult to block the pathological cascade process of inflammation-fibrosis-adhesion. The main challenge of current postoperative interventions for intrauterine surgeries lies in how to form a biocompatible, degradable barrier system with anti-scarring function locally, continuously providing bioactive support for angiogenesis and tissue regeneration.

[0003] Traditional Chinese medicine active ingredients have been gradually incorporated into tissue engineering material design: Astragaloside IV, the main effective monomeric component of Astragalus membranaceus, has been proven to inhibit endometrial cell apoptosis and resist oxidative stress; Salvianolic acid B, a water-soluble phenolic acid component of Salvia miltiorrhiza, possesses biological functions such as anti-fibrosis, free radical scavenging, and inhibition of the TGF-β1 / Smad signaling pathway. However, as small-molecule natural products, Astragaloside IV and Salvianolic acid B, when used alone, have limitations such as short local retention time, low bioavailability, and uncontrollable release rate, which restrict their efficacy stability and clinical feasibility in postoperative repair.

[0004] Therefore, there is an urgent need to construct a drug delivery system that is controllable and sustained-release, has strong tissue adhesion, and has dual functions of anti-fibrosis and regeneration promotion, so as to achieve the precise delivery and long-term effect of the above-mentioned active ingredients of traditional Chinese medicine in the postoperative repair environment of the uterine cavity. Summary of the Invention

[0005] To address the above problems, the present invention aims to provide a chitosan-hyaluronic acid hydrogel composition based on astragaloside A and salvianolic acid B, comprising the following components by weight percentage:

[0006] 1.0%–5.0% chitosan; 0.2%–2.0% sodium hyaluronate; 0.05%–0.5% astragaloside A; 0.05%–0.5% salvianolic acid B; 0.01%–0.3% ferulic acid; 0.01%–0.2% notoginsenoside R1; 0.1%–0.5% lactate buffer, pH 6.2–6.8; 0.2%–1.0% cross-linking agent; balance water, make up to 100%.

[0007] The chitosan is a medium-viscosity chitosan with a degree of deacetylation greater than 85% and a molecular weight of 150–300 kDa.

[0008] The hyaluronic acid is a sodium salt type hyaluronic acid with a molecular weight of 100-200 kDa.

[0009] Astragaloside A, salvianolic acid B, ferulic acid and notoginsenoside R1 are added in a predetermined mass ratio.

[0010] The crosslinking agent is a reaction mixture of glutaraldehyde or aldehyde-modified hyaluronic acid and thiolated chitosan.

[0011] The composition is stirred evenly to form an injectable pregel liquid, which is then cross-linked to form a semi-solid three-dimensional hydrogel structure.

[0012] As a preferred technical solution, the addition of astragaloside A, salvianolic acid B, ferulic acid, and notoginsenoside R1 in a predetermined mass ratio specifically refers to:

[0013] The following single-component solutions were prepared by dissolving the following ingredients in phosphate buffer solution with pH 7.2–7.4 at concentrations of astragaloside A (0.5–2.0 mg / mL), salvianolic acid B (0.4–1.6 mg / mL), ferulic acid (0.2–1.0 mg / mL), and notoginsenoside R1 (0.2–0.8 mg / mL): 0.2–0.8 mg / mL.

[0014] The mixture was compounded according to the mass ratio of astragaloside A: salvianolic acid B: ferulic acid: notoginsenosides = 1:(0.8-1.2):(0.3-0.6):(0.2-0.4), and continuously stirred at room temperature (20-25°C) for 60 minutes under magnetic stirring to form a composite active solution.

[0015] The composite active solution was slowly added dropwise to the chitosan-hyaluronic acid base carrier liquid in a stable lactate buffer environment, and stirring was continued for 30 minutes to ensure that the four components were evenly distributed at the molecular level and to form a stable solution state, thus avoiding precipitation, crystallization or pH-induced decomposition reactions.

[0016] As a preferred technical solution, the chitosan is first dissolved in a lactic acid buffer solution with a mass concentration of 1.0-1.5% by stirring at room temperature (20-25°C) for 4-6 hours to ensure that it swells fully and forms a homogeneous polymer solution.

[0017] The hyaluronic acid is added to the chitosan solution by slow dropwise addition, and the mixture is stirred for 30-60 minutes to allow the composite network molecular chains to become fully entangled.

[0018] As a preferred technical solution, the crosslinking agent is a mixture of maleimide-modified hyaluronic acid and L-cysteine-modified chitosan, dissolved separately and then mixed in a 1:1 volume ratio. The crosslinking reaction is achieved through Michael addition reaction, with the reaction temperature controlled at 20-30°C and the reaction time at 15-30 minutes, forming a stable irreversible carbon-sulfur crosslinking structure.

[0019] As a preferred technical solution, the hydrogel formed by the composition is a non-self-flowing gel with a storage modulus of 200-600 Pa at a frequency of 1 Hz, and has a moderate degree of shape retention.

[0020] Its degradation rate in PBS body fluid simulation solution at pH 7.4 is 15% to 30% reduction in dry weight every 3 days, and it is completely degraded within 10 to 14 days; its hydration swelling rate is stable in the range of 500% to 800% at body temperature of 37°C.

[0021] The present invention also provides a method for preparing the chitosan-hyaluronic acid hydrogel composition based on astragaloside A and salvianolic acid B, comprising the following steps:

[0022] S1. Dissolve chitosan in lactate buffer to form a chitosan solution with a mass concentration of 1.2%, and stir at room temperature for 5 hours to fully swell and form a viscous homogeneous solution;

[0023] S2. Add sodium hyaluronate to the chitosan solution dropwise, while stirring and keeping the pH of the buffer solution between 6.4 and 6.6. Continue mixing for 30 minutes to obtain the composite carrier base solution.

[0024] S3. Weigh out astragaloside A, salvianolic acid B, ferulic acid and notoginsenoside R1, dissolve them separately in phosphate buffer solution with pH 7.2-7.4, mix them and add them to the solution obtained in step S2 in proportion, and stir for 60 minutes.

[0025] S4. Prepare a crosslinking agent solution separately, including glutaraldehyde solution or a multifunctional polymer solution containing reactive groups. Adjust the pH to 6.8-7.0 and add it to the mixture in step S3. Continue the reaction for 20-30 minutes to obtain the pre-formed hydrogel composition.

[0026] As a preferred technical solution, the active ingredients are added in the following order: first, Panax notoginseng saponin R1 and ferulic acid are added to the base carrier solution and stirred for 15 minutes; then, salvianolic acid B and astragaloside A are added sequentially and stirred for 45 minutes to prevent crystallization and precipitation between structurally similar components; during the reaction, a magnetic stirrer is used to control the speed at 300-500 rpm.

[0027] The present invention also provides the use of the chitosan-hyaluronic acid hydrogel composition based on astragaloside A and salvianolic acid B in the preparation of a medicament for preventing postoperative re-adhesion of the uterine cavity.

[0028] Beneficial effects:

[0029] The astragaloside A-tanshinone B complex chitosan-hyaluronic acid hydrogel composition provided by this invention possesses excellent physicochemical properties and biocompatibility. By optimizing the ratio and molecular weight range of chitosan and hyaluronic acid, the resulting hydrogel exhibits a stable structure, uniform gelation, moderate morphology retention, and a controllable degradation rate. A composite active liquid is formed by proportionally compounding astragaloside A, tanshinone B, ferulic acid, and notoginsenoside R1 in a pH-stable system, ensuring uniform distribution of each component, avoiding crystallization and precipitation, and enhancing the synergistic effect of the components. Stable carbon-sulfur bonds are constructed using the Michael addition reaction, achieving a mild cross-linking process and effectively preserving bioactive components. The hydrogel obtained by this invention is injectable, gels in situ at body temperature, and degrades within 10–14 days. It is suitable for filling and sealing wound sites after uterine surgery, reducing the risk of tissue re-adhesion and improving postoperative healing quality. Attached Figure Description

[0030] Figure 1 This is a schematic diagram of the comparative experimental results (AFS adhesion score) of the present invention;

[0031] Figure 2 This is a schematic diagram showing the comparative experimental results (gel degradation residue rate) of the present invention;

[0032] Figure 3 This is a schematic diagram of the comparative experimental results (level of inflammatory factors) of the present invention. Detailed Implementation

[0033] To enhance understanding of the present invention, the present invention will be further described in detail below with reference to embodiments. These embodiments are only used to explain the present invention and do not constitute a limitation on the scope of protection of the present invention.

[0034] Example 1 (T1):

[0035] This embodiment provides a chitosan-hyaluronic acid hydrogel composition based on astragaloside A and salvianolic acid B, the specific formulation and preparation steps of which are as follows:

[0036] The composition comprises, by weight percentage, the following components: 1.0% chitosan, 0.2% sodium hyaluronate, 0.05% astragaloside A, 0.05% salvianolic acid B, 0.01% ferulic acid, 0.01% notoginsenoside R1, 0.1% lactate buffer (pH 6.2), 0.2% crosslinking agent, and the balance being water for injection to bring the total to 100%.

[0037] The chitosan is a medium-viscosity chitosan with a degree of deacetylation >85% and a molecular weight of approximately 150 kDa; the sodium hyaluronate has a molecular weight of approximately 100 kDa.

[0038] The preparation steps are as follows:

[0039] S1. Weigh chitosan and dissolve it in lactic acid buffer (pH 6.2) at a mass concentration of 1.0%. Stir for 5 hours at room temperature (20℃) to fully swell and form a viscous, transparent, homogeneous solution.

[0040] S2. While maintaining the pH at 6.4, slowly add 0.2% sodium hyaluronate solution to the chitosan solution dropwise, and stir with a magnetic stirrer at 300 rpm for 30 minutes to form the basic carrier solution.

[0041] S3. Weigh out 0.05% astragaloside A, 0.05% salvianolic acid B, 0.01% ferulic acid, and 0.01% notoginsenoside R1, and prepare them according to the following concentrations: astragaloside A 0.5 mg / mL, salvianolic acid B 0.4 mg / mL, ferulic acid 0.2 mg / mL, and notoginsenoside R1 0.2 mg / mL. Dissolve all of them in phosphate buffer at pH 7.2 to form single-component solutions. Mix the four solutions sequentially according to the mass ratio of astragaloside A: salvianolic acid B: ferulic acid: notoginsenoside R1 = 1:0.8:0.3:0.2, and stir at 20℃ with magnetic stirring (400 rpm) for 60 minutes to form a composite active solution.

[0042] The above-mentioned composite active liquid is slowly added dropwise to the carrier liquid prepared in S2, and stirring is continued for 30 minutes to ensure uniform dispersion and avoid local precipitation and component decomposition.

[0043] S4. Prepare maleimide-modified hyaluronic acid and L-cysteine-modified chitosan solutions separately, control the pH to 6.9, mix them in a 1:1 volume ratio, and immediately add them dropwise to the S3 mixture. Maintain the reaction temperature at 25°C and stir magnetically for 20 minutes to allow it to form stable crosslinks through Michael addition reaction.

[0044] Example 2 (T2):

[0045] This embodiment provides a chitosan-hyaluronic acid hydrogel composition based on astragaloside A and salvianolic acid B, the formulation and preparation method of which are as follows:

[0046] The composition comprises, by weight percentage: 5.0% chitosan, 2.0% sodium hyaluronate, 0.5% astragaloside A, 0.5% salvianolic acid B, 0.3% ferulic acid, 0.2% notoginsenoside R1, 0.5% lactate buffer (pH 6.8), 1.0% crosslinking agent, and the balance being water for injection, to a total of 100%.

[0047] The chitosan used is a medium-viscosity chitosan with a degree of deacetylation >85% and a molecular weight of about 300kDa. The sodium hyaluronate has a molecular weight of about 200kDa. The active ingredients are added in a compound manner with the following fixed ratio: astragaloside A: salvianolic acid B: ferulic acid: notoginsenosides = 1:1.2:0.6:0.4.

[0048] The specific preparation steps are as follows:

[0049] S1. Chitosan dissolution: Weigh chitosan and add it to a lactic acid buffer solution with a mass concentration of 1.5% and a pH of 6.8. Stir for 6 hours at room temperature (25℃) to form a transparent and viscous chitosan solution.

[0050] S2. Hyaluronic acid mixing: Take a 2.0% sodium hyaluronate solution and slowly add it dropwise to the chitosan solution while maintaining the pH at 6.6. Stir at 500 rpm for 60 minutes using a magnetic stirrer to obtain a polymer-entangled composite carrier base solution.

[0051] S3. Dissolution and Combination of Active Ingredients: Astragaloside A, salvianolic acid B, ferulic acid, and notoginsenoside R1 were dissolved in phosphate buffer at pH 7.4 at concentrations of 2.0, 1.6, 1.0, and 0.8 mg / mL, respectively, to form four single-component solutions. The four solutions were then mixed sequentially according to the above mass ratio (1:1.2:0.6:0.4) and magnetically stirred for 60 minutes at 25°C to form a homogeneous composite active solution. Mixing of the composite active solution: First, the mixture of notoginsenoside R1 and ferulic acid was added to the carrier solution prepared in step S2 and stirred for 15 minutes; then, the mixture of salvianolic acid B and astragaloside A was added sequentially and stirred for 45 minutes, ensuring no precipitation or crystallization occurred; the magnetic stirring speed was controlled at 500 rpm throughout the mixing process.

[0052] S4. Addition of crosslinking agent and gel formation: Prepare 1.0% maleimide-modified hyaluronic acid and 1.0% L-cysteine-modified chitosan solutions respectively, dissolve them in buffer solution, adjust the pH to 6.9, mix them in a 1:1 volume ratio, and immediately add them to the mixed solution obtained in S3. Continue stirring and reacting at 25°C for 30 minutes to complete the Michael addition reaction and form a stable C-S covalent crosslinked structure.

[0053] Example 3 (T3):

[0054] This embodiment provides a chitosan-hyaluronic acid hydrogel composition based on astragaloside A and salvianolic acid B, the composition ratio, raw material processing and preparation method of which are as follows:

[0055] The composition comprises, by weight percentage, the following components: 3.0% chitosan, 1.0% sodium hyaluronate, 0.3% astragaloside A, 0.3% salvianolic acid B, 0.2% ferulic acid, 0.1% notoginsenoside R1, 0.3% lactate buffer (pH 6.6), 0.5% crosslinking agent, and the balance being water for injection, to a total of 100%.

[0056] The chitosan used is a medium-viscosity product with a degree of deacetylation >85% and a molecular weight of about 250kDa; the sodium hyaluronate has a molecular weight of about 150kDa; the crosslinking agent is a 1:1 volume reaction mixture of maleimide-modified hyaluronic acid and L-cysteine-modified chitosan.

[0057] The preparation steps are as follows:

[0058] S1: Chitosan dissolution: Weigh chitosan and dissolve it in a lactic acid buffer solution with a mass concentration of 1.2% at pH 6.6. Stir magnetically for 5 hours at room temperature (23℃) to form a transparent and uniform polymer solution.

[0059] S2: Sodium hyaluronate compound: Slowly add a 1.0% sodium hyaluronate solution to the chitosan solution at a rate of 2 mL per minute, while maintaining a stirring speed of 400 rpm and a temperature of 23°C. Continue mixing for 45 minutes to form a uniform viscoelastic composite carrier solution.

[0060] S3: Pretreatment and addition of active ingredients: Weigh out astragaloside A (2.0 mg / mL), salvianolic acid B (1.2 mg / mL), ferulic acid (0.8 mg / mL), and notoginsenoside R1 (0.6 mg / mL), and dissolve them separately in phosphate buffer at pH 7.3 to form single-component solutions.

[0061] According to the mass ratio of astragaloside A: salvianolic acid B: ferulic acid: notoginsenoside R1 = 1:1.0:0.4:0.3, notoginsenoside R1 and ferulic acid were added to the base solution in step S2 and stirred for 15 minutes. Then salvianolic acid B and astragaloside A were added, and stirring was continued for 45 minutes to ensure that the active components were fully integrated and there was no visible precipitate.

[0062] S4: Cross-linking reaction treatment: Maleimide-modified hyaluronic acid and L-cysteine-modified chitosan were dissolved separately in buffer solution, and the pH was adjusted to 6.9. They were then mixed in a 1:1 volume ratio to form a cross-linking agent solution. This solution was added slowly dropwise to the mixture obtained in step S3, and a Michael addition reaction was carried out at 25°C with stirring for 25 minutes to generate a stable C-S bond cross-linked structure.

[0063] Comparative Example 1 (C1):

[0064] The formulation used in this comparative example is similar to that in Example 3, but salvianolic acid B has been removed from the active ingredient combination. The formulation composition is as follows (by mass percentage): 3.0% chitosan, 1.0% sodium hyaluronate, 0.6% astragaloside A, 0.2% ferulic acid, 0.1% notoginsenoside R1, 0.3% lactate buffer (pH 6.6), 0.5% cross-linking agent, and the balance is water for injection to bring the total to 100%.

[0065] The preparation method is basically the same as in Example 3, including:

[0066] Chitosan was dissolved in lactate buffer at a mass concentration of 1.2% and stirred at room temperature (23°C) for 5 hours to form a homogeneous solution.

[0067] Sodium hyaluronate was slowly added dropwise to the chitosan solution and stirred for 45 minutes.

[0068] The active ingredients include only astragaloside A (2.0 mg / mL), ferulic acid (0.8 mg / mL) and notoginsenoside R1 (0.6 mg / mL), which are compounded in a mass ratio of astragaloside A: ferulic acid: notoginsenoside R1 = 1:0.4:0.3 and added to the carrier solution in the same manner as in Example 3.

[0069] Prepare and add maleimide-cysteine ​​crosslinking agent, and react for 25 minutes;

[0070] Comparative Example 2 (C2):

[0071] The formulation of this comparative example is basically the same as that of Example 3, except that the molecular weight of the chitosan used is 50kDa, and its role in the formation and stability of the hydrogel structure is investigated.

[0072] The formulation consists of the following components (by weight percentage): 3.0% chitosan (molecular weight approximately 50 kDa, degree of deacetylation > 85%), 1.0% sodium hyaluronate (molecular weight 150 kDa), 0.3% astragaloside A, 0.3% salvianolic acid B, 0.2% ferulic acid, 0.1% notoginsenoside R1, 0.3% lactate buffer (pH 6.6), 0.5% cross-linking agent, and the balance is water for injection to bring the total to 100%.

[0073] The preparation method is as described in Example 3, and is as follows:

[0074] Chitosan was added to lactate buffer (pH 6.6) at a mass concentration of 1.2% and the solution was prepared by magnetic stirring at 23°C for 5 hours; the sodium hyaluronate was added dropwise in the same manner as in Example 3, and the mixture was stirred for 45 minutes.

[0075] The active ingredients were compounded in the ratio of astragaloside A: salvianolic acid B: ferulic acid: notoginsenosides = 1:1.0:0.4:0.3, and the concentrations were 2.0, 1.2, 0.8 and 0.6 mg / mL, respectively. After being dissolved in pH 7.3 buffer, they were added to the carrier solution in sequence and stirred for 60 minutes.

[0076] Prepare a 1:1 volume ratio maleimide-thiol crosslinking agent solution, add it to the system and react for 25 minutes.

[0077] Comparative Example 3 (C3):

[0078] This comparative example uses the same hydrogel-based formulation and process as Example 3, the difference being the absence of astragaloside A. The formulation is as follows (by mass percentage): 3.0% chitosan (molecular weight 250 kDa, degree of deacetylation > 85%), 1.0% sodium hyaluronate (molecular weight 150 kDa), 0.6% salvianolic acid B, 0.2% ferulic acid, 0.1% notoginsenoside R1, 0.3% lactate buffer (pH 6.6), 0.5% crosslinking agent, with the balance being water for injection to bring the total to 100%.

[0079] The preparation process is as follows:

[0080] Chitosan was dissolved in lactate buffer at a mass concentration of 1.2% and magnetically stirred at room temperature (23°C) for 5 hours to form a transparent and homogeneous polymer solution.

[0081] Add 1.0% sodium hyaluronate solution dropwise at a rate of 2 mL per minute, and stir for 45 minutes;

[0082] The active ingredients consist only of salvianolic acid B (1.2 mg / mL), ferulic acid (0.8 mg / mL), and notoginsenoside R1 (0.6 mg / mL), which are dissolved separately in phosphate buffer at pH 7.3 and then mixed. The mixture is added to the base solution at a mass ratio of salvianolic acid B: ferulic acid: notoginsenoside R1 = 1.0:0.4:0.3 and stirred for 60 minutes.

[0083] Add the pre-prepared maleimide-modified hyaluronic acid and L-cysteine-modified chitosan crosslinking agent, maintain pH 6.9, and stir at 25°C for 25 minutes.

[0084] Comparative Example 4 (C4):

[0085] This comparative example uses a chitosan-hyaluronic acid physically mixed hydrogel system, without any pharmaceutical ingredients or functional crosslinking agents. The chitosan-hyaluronic acid gel is formed only under weakly acidic conditions to evaluate its basic gelation and material properties.

[0086] The formulation consists of the following components (by mass percentage): 2.5% chitosan (molecular weight 150kDa, degree of deacetylation > 85%), 1.5% sodium hyaluronate (molecular weight 100kDa), 0.3% lactate buffer (pH 6.5), and the remainder is water for injection, to be made up to 100%.

[0087] The preparation method is as follows:

[0088] Chitosan was added to lactate buffer (pH 6.5) at a mass concentration of 2.5% and magnetically stirred at room temperature (23°C) for 5 hours to form a homogeneous solution in a flowable state.

[0089] Sodium hyaluronate was dissolved in deionized water to form a 1.5% solution, which was then slowly added dropwise to the chitosan solution and stirred for 30 minutes.

[0090] The solution is injected directly into the mold or PBS buffer and allowed to stand naturally at room temperature to form a gel.

[0091] Comparative experiment:

[0092] Experimental objective: To verify the film-forming integrity, biodegradability, pharmacological activity, and anti-adhesion effect of the composite hydrogels described in Examples T1, T2, and T3 in postoperative wounds of the uterine cavity, and to compare them with comparative examples C1-C4 to clarify the contribution of key components (such as astragaloside A and salvianolic acid B) and structural parameters (such as chitosan molecular weight and cross-linking mode) to the overall therapeutic effect.

[0093] Animal model selection: 8 female rats per group;

[0094] Model establishment method: After anesthesia, a standardized uterine cavity wound was created by mechanical curettage; immediately after trauma, hydrogel of each group was injected into the uterine cavity (about 0.3-0.5 mL), and then the vaginal opening was sutured to prevent leakage; each group maintained a 14-day observation period after the operation.

[0095] To comprehensively evaluate the effectiveness of each composition in preventing postoperative adhesions in the uterine cavity, the following three core evaluation indicators were set:

[0096] 1. Re-adhesion degree rating:

[0097] Objective: To evaluate the physical barrier effect and adhesion inhibition ability of each group of hydrogels on the postoperative uterine cavity wound.

[0098] Methods: Animals were euthanized on the 14th day after surgery, and the uterus was completely removed. The AFS scoring system was used for evaluation, examining the following three dimensions (0-4 points for each item, 0-12 points in total): extent of intrauterine adhesions; adhesion strength (separability); endometrial regeneration; the higher the score, the more severe the adhesions.

[0099] 2. Gel degradation behavior:

[0100] Objective: To evaluate the degradation rate, structural integrity maintenance, and space occupation effect of different compositions in the uterine cavity.

[0101] Methods: On postoperative days 3, 7, 10 and 14, two animals from each group were randomly sacrificed, the uterus was dissected, and the amount of gel residue was observed; the degree of adhesion was scored (0: no adhesion, 1: partial adhesion, 2: complete adhesion).

[0102] 3. Pharmacological effects analysis:

[0103] Objective: To evaluate the bioregulatory function of the composition from two aspects: inflammation suppression and fibrosis factor expression.

[0104] Methods: Detection of inflammatory factors in uterine cavity irrigation fluid (day 7); Uterine cavity irrigation fluid was collected from each group on day 7 postoperatively (0.5 mL of sterile PBS was instilled and aspirated), and the supernatant was collected after centrifugation;

[0105] Detection indicators: TNF-α, IL-6, IL-1β;

[0106] Detection method: ELISA kit, follow the instructions;

[0107] Unit: pg / mL, repeated 3 times, and the average value is taken.

[0108] The experimental results are shown in Tables 1 to 3:

[0109] Table 1 Comparison of experimental results (adhesion score)

[0110]

[0111] Table 2 Comparison of experimental results (gel degradation residue rate)

[0112]

[0113] Table 3 Comparison of experimental results (inflammatory factor levels, postoperative day 7, pg / mL)

[0114]

[0115] Data Analysis:

[0116] 1. Analysis of adhesion scoring results:

[0117] From Table 1 and Figure 1 It can be seen that the adhesion scores of the T1, T2 and T3 groups were 3.8, 2.2 and 1.5, respectively, which were much lower than those of the comparative examples (C1: 5.5, C2: 6.2, C3: 5.8, C4: 8.7) and the blank control group N (9.5). Among them, T3 had the lowest score, indicating that its anti-adhesion effect was the most significant.

[0118] Analysis of the causes reveals that T3's formulation utilizes a moderate concentration of chitosan (3.0%), sodium hyaluronate (1.0%), and a well-balanced combination of active ingredients, achieving a balance between gelling properties and bioactivity. In contrast, C1 and C3 respectively omits salvianolic acid B or astragaloside A, resulting in decreased synergistic anti-inflammatory and anti-adhesion capabilities; C2 uses low molecular weight chitosan (50kDa), significantly reducing the material's adhesive strength and steric barrier capacity; C4 completely lacks active pharmaceutical ingredients, remaining merely a physical mixed gel, thus exhibiting the worst effect in inhibiting tissue adhesion.

[0119] 2. Analysis of gel degradation residue rate:

[0120] Table 2 and Figure 2 This reflects the residual hydrogel degradation rate in different groups on postoperative days 3, 7, 10, and 14. In the T group, T2 showed the highest residual rate (91.5% and 75.3%) on days 3 and 7, respectively, with the slowest degradation. T1 and T3 were next. Figure 2 The curve shows a sharp rise. By day 14, the residual rates of T1, T2, and T3 decreased to 3.6%, 7.1%, and 4.8%, respectively, indicating that the degradation in vivo was basically completed.

[0121] The difference is closely related to the molecular weight and content of chitosan and hyaluronic acid: T2 uses high molecular weight chitosan (300kDa) and high concentration of hyaluronic acid (2.0%) to form a denser, more cross-linked three-dimensional network structure, which slows down the hydrolysis rate; T3 achieves a balance between structural stability and biodegradability, ensuring sustained-release performance while avoiding the impact of slow degradation on subsequent tissue repair.

[0122] In the comparative group, although C1-C3 used similar formulations, incomplete or excessive degradation occurred due to missing components or insufficient chitosan molecular weight, with residual rates still as high as 15.9% (C1), 12.7% (C2), and 18.4% (C3) on day 14. C4 degraded rapidly from day 3, with only 6.3% remaining on day 14. Although the residual rate was close to that of group T, its adhesion score and inflammatory indicators were extremely high, indicating that its degradation process was not accompanied by effective drug release and barrier protection.

[0123] 3. Analysis of inflammatory factor levels:

[0124] Table 3 and Figure 3 Data showed that the T group significantly suppressed the levels of inflammatory factors such as TNF-α, IL-6, and IL-1β after surgery, with T2 showing the best performance (64.7, 45.9, and 55.1 pg / mL, respectively), followed by T3 and T1; while the levels of inflammatory factors in the C and N groups were significantly increased, especially the TNF-α concentrations in the C4 and N groups, which were as high as 129.5 and 137.8 pg / mL, respectively.

[0125] The comparison revealed that T2 had higher levels of astragaloside A and salvianolic acid B (0.5%), and with its highly cross-linked matrix, it effectively controlled drug release and local inflammatory response. Although T3 had slightly lower drug content, its balanced formulation and good viscoelasticity resulted in an anti-inflammatory effect close to that of T2. T1 had the lowest concentration of active components, thus its control of inflammatory factors was relatively weaker, but still superior to group C.

[0126] C1 and C3, by removing salvianolic acid B and astragaloside A respectively, resulted in incomplete anti-inflammatory pathways and significantly increased inflammation levels; C2, although complete in its components, had low molecular weight chitosan that accelerated drug release and reduced residence time, making it unable to maintain effective inflammation control; C4 lacked active ingredients and had the worst performance in all inflammatory indicators.

[0127] 4. Attribution Analysis and Structure-Function Relationship:

[0128] 4.1 Synergistic Effect of Core Active Ingredients: The combination of astragaloside A and salvianolic acid B is key to inhibiting inflammation, regulating immunity, and promoting tissue repair. Results from both T3 and T2 tests validated this synergistic advantage. Removal of either ingredient (C1 or C3) significantly weakened the effect.

[0129] 4.2 Chitosan molecular weight regulation of structural mechanics: Chitosan with a molecular weight of 250-300 kDa provides good adhesion and degradation rate control. T3 exhibits excellent balance, while C2 uses 50 kDa chitosan, resulting in a loose gel structure and insufficient stability.

[0130] 4.3 Cross-linking method improves stability and targeted release: In the example, maleimide and L-cysteine ​​are used to modify C-S covalent cross-linking method, which not only enhances structural stability, but also ensures the controllable release of drug active ingredients. C4 is structurally fragile due to the lack of cross-linking agent, resulting in poor clinical efficacy.

[0131] In summary, Examples T1-T3 significantly outperformed the comparative examples in inhibiting adhesion formation, regulating degradation cycles, and controlling postoperative inflammation, with T3 showing the best overall performance, demonstrating its excellent balance and biocompatibility in formulation design. In contrast, Examples C1-C4 suffered from defects such as missing key components, insufficient raw material quality, or coarse structural design, resulting in suboptimal overall efficacy. This experiment fully demonstrates the importance of synergistic strategies for composite active ingredients and precise cross-linking mechanisms in the design of bio-hydrogels.

[0132] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A chitosan-hyaluronic acid hydrogel composition based on astragaloside A and salvianolic acid B, characterized in that it comprises the following components in weight percentage: 1.0–5.0% chitosan; 0.2–2.0% sodium hyaluronate; 0.05–0.5% astragaloside A; 0.05–0.5% salvianolic acid B; 0.01–0.3% ferulic acid; 0.01–0.2% notoginsenoside R1; 0.1–0.5% lactate buffer, pH 6.2–6.8; 0.2–1.0% cross-linking agent; balance water, make up to 100%. The chitosan is a chitosan with a degree of deacetylation greater than 85% and a molecular weight of 150-300 kDa; The hyaluronic acid is a sodium salt type hyaluronic acid with a molecular weight of 100-200 kDa; Astragaloside A, salvianolic acid B, ferulic acid and notoginsenoside R1 are added in a predetermined mass ratio; The crosslinking agent is a mixture of maleimide-modified hyaluronic acid and L-cysteine-modified chitosan, dissolved separately and then mixed in a 1:1 volume ratio. The crosslinking reaction is achieved by Michael addition reaction, with the reaction temperature controlled at 20-30°C and the reaction time at 15-30 minutes. The composition is stirred evenly to form an injectable pregel liquid, which is then cross-linked to form a semi-solid three-dimensional hydrogel structure. The chitosan-hyaluronic acid hydrogel composition based on astragaloside A and salvianolic acid B was prepared by the following steps: S1. Dissolve chitosan in lactate buffer to form a chitosan solution with a mass concentration of 1.2%, and stir at room temperature for 5 hours to fully swell and form a viscous homogeneous solution; S2. Add sodium hyaluronate to the chitosan solution dropwise, while stirring and keeping the pH of the buffer solution between 6.4 and 6.

6. Continue mixing for 30 minutes to obtain the composite carrier base solution. S3. Weigh out astragaloside A, salvianolic acid B, ferulic acid and notoginsenoside R1, dissolve them separately in phosphate buffer solution with pH 7.2-7.4, mix them and add them to the solution obtained in step S2 in proportion, and stir for 60 minutes. The order of addition is as follows: first, add Panax notoginseng saponin R1 and ferulic acid to the basic carrier solution and stir for 15 minutes; then add salvianolic acid B and astragaloside A in sequence and stir for 45 minutes; during the reaction, a magnetic stirrer is used to control the speed at 300~500 rpm. S4. Prepare a crosslinking agent solution separately, including glutaraldehyde solution or a multifunctional polymer solution containing reactive groups. Adjust the pH to 6.8-7.0 and add it to the mixture in step S3. Continue the reaction for 20-30 minutes to obtain the pre-formed hydrogel composition.

2. The chitosan-hyaluronic acid hydrogel composition based on astragaloside A and salvianolic acid B according to claim 1, characterized in that: The addition of astragaloside A, salvianolic acid B, ferulic acid, and notoginsenoside R1 in a predetermined mass ratio specifically refers to: The following single-component solutions were prepared by dissolving the following ingredients in phosphate buffer solution at pH 7.2–7.4: astragaloside A: 0.5–2.0 mg / mL, salvianolic acid B: 0.4–1.6 mg / mL, ferulic acid: 0.2–1.0 mg / mL, and notoginsenoside R1: 0.2–0.8 mg / mL. The mixture was prepared according to the mass ratio of astragaloside A: salvianolic acid B: ferulic acid: notoginsenosides = 1:(0.8~1.2):(0.3~0.6):(0.2~0.4), and continuously stirred at room temperature (20~25℃) for 60 minutes under magnetic stirring to form a composite active solution. The composite active solution was slowly added dropwise to the chitosan-hyaluronic acid-based carrier liquid in a lactate buffer stable environment, and stirring was continued for 30 minutes.

3. The chitosan-hyaluronic acid hydrogel composition based on astragaloside A and salvianolic acid B according to claim 1, characterized in that: The chitosan was first dissolved in a lactic acid buffer solution with a mass concentration of 1.0-1.5% by stirring at room temperature (20-25°C) for 4-6 hours. The hyaluronic acid was added to the chitosan solution by slow dropwise addition, and the mixture was stirred for 30 to 60 minutes.

4. The chitosan-hyaluronic acid hydrogel composition based on astragaloside A and salvianolic acid B according to claim 1, characterized in that: The hydrogel formed by the composition is a non-self-flowing gel with a storage modulus of 200–600 Pa at a frequency of 1 Hz. Its degradation rate in PBS body fluid simulation solution at pH 7.4 is 15-30% reduction in dry weight every 3 days, and complete degradation within 10-14 days; Its hydration swelling rate remains stable in the range of 500% to 800% at a body temperature of 37°C.

5. The use of the chitosan-hyaluronic acid hydrogel composition based on astragaloside A and salvianolic acid B according to any one of claims 1-4 in the preparation of a medicament for preventing postoperative re-adhesion of the uterine cavity.

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