Chitosan-hyaluronic acid hydrogel composition based on astragaloside and salvianolic acid B as well as preparation method and application of chitosan-hyaluronic acid hydrogel composition

Abstract

The invention relates to the technical field of biomedical materials, in particular to a chitosan-hyaluronic acid hydrogel composition based on astragaloside and salvianolic acid B as well as a preparation method and application of the chitosan-hyaluronic acid hydrogel composition. According to the hydrogel, a three-dimensional network formed by synergistic crosslinking of chitosan and hyaluronic acid is constructed, various natural active ingredients including astragaloside, salvianolic acid B, ferulic acid and notoginsenoside R1 are loaded, and the composite hydrogel with good biocompatibility, controllable degradability and anti-inflammatory and anti-adhesion functions is formed. C-S covalent crosslinking is realized by adopting a maleimide-cysteine system, so that the structural stability and the component release efficiency are improved. Animal experiment results show that the hydrogel can significantly reduce postoperative adhesion score and inflammatory factor level, and has wide postoperative anti-adhesion application potential.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical materials, and particularly relates to a chitosan-hyaluronic acid hydrogel composition based on astragaloside IV and salvianolic acid B, a preparation method and application. BACKGROUND

[0002] Hysteroscopic surgery (such as hysteroscopic adhesion release, endometrial polyp resection, etc.) is widely used in the treatment of female infertility and abnormal uterine bleeding, etc., but postoperative re-adhesion often occurs due to the damage of the endometrial basal layer, which significantly affects the repair ability of the endometrium and the reproductive prognosis. Even if traditional physical isolation means such as balloons, contraceptive rings or hyaluronic acid gel is used, it is still difficult to block the pathological cascade process of inflammation-fibrosis-adhesion. The main difficulty of current postoperative intervention in the uterine cavity is: how to form a barrier system with good biocompatibility, degradability and anti-scarring function locally, and continuously provide biological active support for promoting angiogenesis and tissue regeneration.

[0003] Traditional active ingredients of Chinese medicine have been gradually introduced into the design of tissue engineering materials: astragaloside IV is the main effective monomer component in astragalus, which has been proved to have the effects of inhibiting endometrial cell apoptosis and resisting oxidative stress; salvianolic acid B is a water-soluble phenolic acid component in Danshen, which has the biological functions of anti-fibrosis, free radical scavenging, inhibition of TGF-beta1 / Smad signaling pathway, etc. However, as small molecule natural products, astragaloside IV and salvianolic acid B have the limitations of short local retention time, low bioavailability and uncontrollable release rate when used alone, which restricts their efficacy stability and clinical feasibility in postoperative repair.

[0004] Therefore, it is urgent to construct a drug delivery system with controllable sustained release, strong tissue adhesion, anti-fibrosis and double functions of promoting regeneration, so as to realize the precise delivery and long-term effect of the above active ingredients of traditional Chinese medicine in the postoperative repair environment of the uterine cavity. SUMMARY

[0005] In view of the above problems, the present application aims to provide a chitosan-hyaluronic acid hydrogel composition based on astragaloside IV and salvianolic acid B, which comprises the following components in mass percentage:

[0006] 1.0-5.0% of chitosan; 0.2-2.0% of sodium hyaluronate; 0.05-0.5% of astragaloside IV; 0.05-0.5% of salvianolic acid B; 0.01-0.3% of ferulic acid; 0.01-0.2% of notoginseng saponin R1; 0.1-0.5% of lactic acid buffer, pH 6.2-6.8; 0.2-1.0% of crosslinking agent; and the balance is water, supplemented 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] The astragaloside, salvianolic acid B, ferulic acid and panax notoginseng saponin R1 are compounded and added in a preset 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 uniformly to form an injectable pre-gel liquid, and a semi-solid three-dimensional hydrogel structure is formed through a crosslinking reaction.

[0012] As a preferred technical solution, the astragaloside, salvianolic acid B, ferulic acid and panax notoginseng saponin R1 are compounded and added in a preset mass ratio, specifically:

[0013] According to the concentrations of astragaloside 0.5-2.0 mg / mL, salvianolic acid B 0.4-1.6 mg / mL, ferulic acid 0.2-1.0 mg / mL and panax notoginseng saponin R1 0.2-0.8 mg / mL, respectively, dissolved in a phosphate buffer solution with a pH of 7.2-7.4 to form a single-component solution;

[0014] According to the mass ratio of astragaloside: salvianolic acid B: ferulic acid: panax notoginseng saponin = 1: (0.8-1.2): (0.3-0.6): (0.2-0.4), compounded and continuously stirred at room temperature 20-25°C for 60 minutes under magnetic stirring to form a composite active liquid;

[0015] The composite active liquid is slowly added to the chitosan-hyaluronic acid base carrier liquid in a lactic acid buffer stable environment, and continues to stir for 30 minutes to make the four components uniformly distributed at the molecular level, forming a stable solution state to avoid precipitation, crystallization or pH-induced decomposition reaction.

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

[0017] The hyaluronic acid is added to the chitosan solution by slow dripping, and continues to mix uniformly under stirring for 30-60 minutes to fully entangle the composite network molecular chains.

[0018] As a preferred technical solution, the cross-linking agent is hyaluronic acid modified by maleimide and L-cysteine modified chitosan respectively dissolved and mixed in a volume ratio of 1:1, the cross-linking reaction is realized by Michael addition reaction, the reaction temperature is controlled at 20-30 DEG C, the reaction time is 15-30 minutes, and a stable irreversible carbon-sulfur bond cross-linking structure is formed.

[0019] As a preferred technical solution, the hydrogel formed by the composition is a non-self-flowing gel, and the storage modulus thereof is 200-600 Pa under the condition of a frequency of 1 Hz, and the hydrogel has a moderate form retention capacity.

[0020] The degradation rate of the hydrogel in a PBS body fluid simulation solution at pH 7.4 is 15%-30% of dry weight reduction per 3 days, and the hydrogel is degraded completely within 10-14 days; and the hydration expansion rate of the hydrogel is stable in the range of 500%-800% at a body temperature of 37 DEG C.

[0021] The application further provides a preparation method of the chitosan-hyaluronic acid hydrogel composition based on astragaloside and salvianolic acid B.

[0022] S1, chitosan is dissolved in a lactic acid buffer to form a chitosan solution with a mass concentration of 1.2%, and the chitosan solution is stirred at room temperature for 5 hours to be fully swollen and form a viscous and uniform solution;

[0023] S2, sodium hyaluronate is added dropwise to the chitosan solution, the buffer pH is maintained at 6.4-6.6 while stirring, and the mixture is uniformly mixed for 30 minutes to obtain a composite carrier base solution;

[0024] S3, astragaloside, salvianolic acid B, ferulic acid and notoginsenoside R1 are weighed and dissolved in a phosphate buffer with pH 7.2-7.4, and then added to the solution obtained in step S2 in a proportion, and stirred for 60 minutes;

[0025] S4, a cross-linking agent solution is prepared, including a glutaraldehyde solution or a multi-functional polymer solution containing a reaction group, and the pH is adjusted to 6.8-7.0, then the mixed solution of step S3 is added, and the reaction is continued for 20-30 minutes to obtain a preformed hydrogel composition.

[0026] As a preferred technical solution, the active ingredients are added in the following order: notoginsenoside R1 and ferulic acid are first added to the base carrier solution and stirred for 15 minutes; then salvianolic acid B and astragaloside are added in sequence, and the stirring time is 45 minutes to prevent crystallization and precipitation between the components with similar structures; and a magnetic stirrer is used to control the rotation speed to be 300-500 rpm during the reaction.

[0027] The application also provides application of the astragaloside IV and salvianolic acid B based chitosan-hyaluronic acid hydrogel composition in preparation of a medicine for preventing post-hysteroscopy re-adhesion.

[0028] Beneficial effects:

[0029] The astragaloside IV and salvianolic acid B compound chitosan-hyaluronic acid hydrogel composition provided by the application has good physical and chemical properties and biological adaptability, and by optimizing the proportion and molecular weight range of chitosan and hyaluronic acid, the prepared hydrogel has stable structure, uniform gel formation, moderate shape maintaining capacity and controllable degradation rate. By proportionally compounding astragaloside IV, salvianolic acid B, ferulic acid and notoginsenoside R1 in a pH stable system, a compound active liquid is formed, so that uniform distribution of the components is ensured, crystallization and precipitation are avoided, and the synergistic effect of the components is improved. The stable carbon-sulfur bond is constructed by using Michael addition reaction to realize a mild crosslinking process, and the biological active ingredients are effectively reserved. The obtained hydrogel is injectable and forms a gel in situ at body temperature, and is degraded within 10-14 days, and is suitable for filling and sealing the wound site after hysteroscopy, reducing the risk of tissue re-adhesion, and improving the postoperative healing quality. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 A schematic diagram of the comparative experimental results (AFS adhesion score) of the application is shown in the following figure:

[0031] Figure 2 A schematic diagram of the comparative experimental results (gel degradation residual rate) of the application is shown in the following figure:

[0032] Figure 3 A schematic diagram of the comparative experimental results (inflammatory factor level) of the application is shown in the following figure. DETAILED DESCRIPTION

[0033] In order to deepen the understanding of the application, the application will be further described in combination with examples, and the examples are only used to explain the application and do not constitute a limitation on the protection scope of the application.

[0034] Example one (T1):

[0035] The example provides an astragaloside IV and salvianolic acid B based chitosan-hyaluronic acid hydrogel composition, and the specific formula and preparation steps are as follows:

[0036] The composition includes the following components in mass percentage: 1.0% of chitosan, 0.2% of sodium hyaluronate, 0.05% of astragaloside IV, 0.05% of salvianolic acid B, 0.01% of ferulic acid, 0.01% of notoginsenoside R1, 0.1% of lactic acid buffer (pH 6.2), 0.2% of crosslinking agent, and the balance of injection water to 100%.

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

[0038] The preparation steps are as follows:

[0039] S1, weigh the chitosan, dissolve it in lactic acid buffer (pH 6.2) at a mass concentration of 1.0%, stir at room temperature (20°C) for 5 hours, and fully swell to form a viscous transparent uniform solution.

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

[0041] S3, respectively weigh 0.05% astragaloside, 0.05% salvianolic acid B, 0.01% ferulic acid and 0.01% notoginseng saponin R1, and configure them at concentrations of 0.5 mg / mL, 0.4 mg / mL, 0.2 mg / mL and 0.2 mg / mL respectively, all dissolved in pH 7.2 phosphate buffer, to form single component solutions. According to the mass ratio of astragaloside: salvianolic acid B: ferulic acid: notoginseng saponin R1 = 1:0.8:0.3:0.2, mix the four solutions in turn, and stir at 20°C under magnetic stirring (400 rpm) for 60 minutes, to form a complex active liquid.

[0042] Slowly add the above complex active liquid to the carrier liquid prepared in S2, continue to stir for 30 minutes to ensure uniform dispersion and avoid local precipitation and component decomposition.

[0043] S4, respectively prepare the maleimide-modified hyaluronic acid and L-cysteine-modified chitosan solutions, control the pH to be 6.9, mix them at a volume ratio of 1:1, immediately add them to the S3 mixed solution, maintain the reaction temperature at 25°C, and magnetically stir for 20 minutes, so that they form stable cross-linking through Michael addition reaction.

[0044] Example Two (T2):

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

[0046] The composition includes, by mass percentage, 5.0% chitosan, 2.0% sodium hyaluronate, 0.5% astragaloside, 0.5% salvianolic acid B, 0.3% ferulic acid, 0.2% notoginseng saponin R1, 0.5% lactic acid buffer (pH 6.8), 1.0% cross-linking agent, and the balance is water for injection, supplemented to 100%.

[0047] The chitosan used is medium viscosity chitosan with a deacetylation degree of >85% and a molecular weight of about 300 kDa, the sodium hyaluronate has a molecular weight of about 200 kDa, and the active ingredients are added in a complex manner with a fixed ratio as follows: Astragaloside B: Salvianolic acid B: Ferulic acid: Notoginseng saponin = 1:1.2:0.6:0.4.

[0048] The specific preparation steps are as follows:

[0049] S1, chitosan dissolution: chitosan is weighed and added to a lactic acid buffer solution with a mass concentration of 1.5% and a pH of 6.8, stirred at room temperature (25°C) for 6 hours to form a transparent viscous chitosan solution.

[0050] S2, hyaluronic acid mixing: a 2.0% concentration of sodium hyaluronate solution is slowly added to the chitosan solution while maintaining a pH of 6.6, and a magnetic stirrer is used to stir at 500 rpm for 60 minutes to obtain a high molecular entanglement complex carrier base solution.

[0051] S3, active ingredient dissolution and compounding: Astragaloside B, Salvianolic acid B, Ferulic acid and Notoginseng saponin R1 are respectively dissolved in a phosphate buffer solution with a pH of 7.4 at concentrations of 2.0, 1.6, 1.0 and 0.8 mg / mL to form four single-component solutions. According to the above mass ratio (1:1.2:0.6:0.4), the four solutions are sequentially mixed, and the mixture is stirred at 25°C for 60 minutes to form a uniform complex active solution. Complex active liquid mixing: first, the mixture of Notoginseng saponin R1 and Ferulic acid is added to the carrier liquid prepared in step S2 and stirred for 15 minutes; then the mixture of Salvianolic acid B and Astragaloside B is added sequentially, and stirred for 45 minutes to ensure that no precipitation or crystallization occurs; the whole mixing process is controlled at a magnetic stirring speed of 500 rpm.

[0052] S4, crosslinking agent addition and gel formation: 1.0% maleimide-modified hyaluronic acid and 1.0% L-cysteine-modified chitosan solutions are respectively prepared and dissolved in a buffer solution, and the pH is adjusted to 6.9. Then, the two solutions are mixed in a volume ratio of 1:1, immediately added to the mixed solution obtained in S3, and stirred at 25°C for 30 minutes to complete the Michael addition reaction and form a stable C—S covalent crosslinked structure.

[0053] Example Three (T3):

[0054] This example provides a chitosan-hyaluronic acid hydrogel composition based on Astragaloside B and Salvianolic acid B, the component ratio, raw material processing and preparation method are as follows:

[0055] The composition consists of the following components by mass percentage: 3.0% of chitosan, 1.0% of sodium hyaluronate, 0.3% of astragaloside IV, 0.3% of salvianolic acid B, 0.2% of ferulic acid, 0.1% of notoginseng saponin R1, 0.3% of lactic acid buffer (pH 6.6), 0.5% of crosslinking agent, and the balance of water for injection, supplemented to 100%.

[0056] The chitosan used is a medium viscosity product with a degree of deacetylation > 85% and a molecular weight of about 250 kDa; the sodium hyaluronate has a molecular weight of about 150 kDa; 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: chitosan is weighed and dissolved in a lactic acid buffer with a pH of 6.6 at a mass concentration of 1.2%, and magnetically stirred at room temperature (23°C) for 5 hours to form a transparent and uniform polymer solution.

[0059] S2: Sodium hyaluronate compounding: a 1.0% concentration of sodium hyaluronate solution is slowly added to the chitosan solution at a speed of 2 mL per minute, the stirring process is controlled at a speed of 400 rpm, and the temperature is maintained at 23°C, and the mixing is continued for 45 minutes to form a uniform viscoelastic composite carrier liquid.

[0060] S3: Active ingredient pretreatment and addition: astragaloside IV (2.0 mg / mL), salvianolic acid B (1.2 mg / mL), ferulic acid (0.8 mg / mL), and notoginseng saponin R1 (0.6 mg / mL) are weighed respectively and dissolved in a phosphate buffer with a pH of 7.3 to form a single component solution.

[0061] According to the mass ratio of astragaloside IV: salvianolic acid B: ferulic acid: notoginseng saponin = 1:1.0:0.4:0.3, notoginseng saponin R1 and ferulic acid are sequentially added to the base liquid of step S2 and stirred for 15 minutes, then salvianolic acid B and astragaloside IV are added and continue to stir for 45 minutes to ensure that the active components are fully integrated and there is no visible precipitation.

[0062] S4: Crosslinking reaction treatment: maleimide-modified hyaluronic acid and L-cysteine-modified chitosan are dissolved in buffer respectively, the pH is adjusted to 6.9, and a crosslinking agent liquid is formed by mixing at a volume ratio of 1:1. The liquid is added to the mixture obtained in step S3 in a slow dripping manner, and a Michael addition reaction is carried out at 25°C, stirring for 25 minutes to generate a stable C—S bond crosslinking structure.

[0063] Comparative Example One (C1):

[0064] The formula of this comparative example is similar to that of Example 3, but the active ingredient combination excludes Danshensuan B. The formula composition is as follows (in mass percentage): 3.0% chitosan, 1.0% sodium hyaluronate, 0.6% astragaloside IV, 0.2% ferulic acid, 0.1% notoginsenoside R1, 0.3% lactic acid buffer (pH 6.6), 0.5% crosslinking agent, and the balance is made up to 100% with water for injection.

[0065] The preparation method is basically consistent with that of Example 3, comprising:

[0066] Chitosan is dissolved in lactic acid buffer at a mass concentration of 1.2%, stirred at room temperature (23°C) for 5 hours to form a uniform solution;

[0067] Sodium hyaluronate is slowly added to the chitosan solution and stirred for 45 minutes;

[0068] The active ingredients only include astragaloside IV (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 IV: ferulic acid: notoginsenoside R1 = 1:0.4:0.3 and added to the carrier liquid in the same way as in Example 3;

[0069] The maleimide-cysteine crosslinking agent is prepared and added, and the reaction is carried out for 25 minutes;

[0070] Comparative Example 2 (C2):

[0071] This comparative example is basically consistent with the formula of Example 3, except that the molecular weight of the chitosan used is 50 kDa, and its role in the formation of hydrogel structure and stability is investigated.

[0072] The formula composition is as follows (in mass percentage): 3.0% chitosan (molecular weight about 50 kDa, degree of deacetylation > 85%), 1.0% sodium hyaluronate (molecular weight 150 kDa), 0.3% astragaloside IV, 0.3% Danshensuan B, 0.2% ferulic acid, 0.1% notoginsenoside R1, 0.3% lactic acid buffer (pH 6.6), 0.5% crosslinking agent, and the balance is made up to 100% with water for injection.

[0073] The preparation method is referred to Example 3, and the details are as follows:

[0074] Chitosan is added to lactic acid buffer (pH 6.6) at a mass concentration of 1.2%, and a solution is prepared by magnetic stirring at 23°C for 5 hours; the sodium hyaluronate addition process is the same as in Example 3, and stirring is carried out for 45 minutes;

[0075] The active ingredients were compounded in the ratio of Astragaloside IV: Salvianolic acid B: Ferulic acid: Notoginseng saponins = 1:1.0:0.4:0.3, and the solubility concentrations were 2.0, 1.2, 0.8, and 0.6 mg / mL respectively. The active ingredients were dissolved in a pH 7.3 buffer solution, and then the carrier liquid was added in sequence, and stirred for 60 minutes.

[0076] A 1:1 volume ratio of maleimide-thiol crosslinking agent solution was prepared and added to the system and reacted for 25 minutes.

[0077] Comparative Example Three (C3):

[0078] This comparative example used the same hydrogel base formula and process as Example Three, except that Astragaloside IV was not added. The formula is as follows (in 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% Notoginseng saponins R1, 0.3% lactic acid buffer (pH 6.6), 0.5% crosslinking agent, and the balance made up to 100% with water for injection.

[0079] The preparation process is as follows:

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

[0081] The 1.0% sodium hyaluronate solution was added dropwise at a rate of 2 mL per minute, and stirred for 45 minutes;

[0082] The active ingredients included only Salvianolic acid B (1.2 mg / mL), Ferulic acid (0.8 mg / mL), and Notoginseng saponins R1 (0.6 mg / mL), which were mixed after being dissolved in a pH 7.3 phosphate buffer solution. The active ingredients were added to the base liquid in the mass ratio of Salvianolic acid B: Ferulic acid: Notoginseng saponins = 1.0:0.4:0.3, and stirred for 60 minutes.

[0083] The pre-prepared maleimide-modified hyaluronic acid and L-cysteine-modified chitosan crosslinking agent was added, the pH was maintained at 6.9, and the mixture was stirred at 25°C for 25 minutes.

[0084] Comparative Example Four (C4):

[0085] This comparative example selected a chitosan-hyaluronic acid physical mixing type hydrogel system, which did not contain any medicinal ingredients or functional crosslinking agents, but only used chitosan-hyaluronic acid gel formed under weak acid conditions to evaluate the basic gel formation and material performance.

[0086] The formulation composition is as follows (in mass percentage): 2.5% chitosan (molecular weight 150 kDa, degree of deacetylation > 85%), 1.5% sodium hyaluronate (molecular weight 100 kDa), 0.3% lactic acid buffer (pH 6.5), and the balance is water for injection, supplemented to 100%.

[0087] The preparation method is as follows:

[0088] Chitosan is added to the lactic acid buffer (pH 6.5) at a mass concentration of 2.5%, and stirred magnetically at room temperature (23°C) for 5 hours to form a uniform solution in a flowable state;

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

[0090] The solution is directly injected into the mold or PBS buffer and left to set naturally at room temperature.

[0091] Comparative experiment:

[0092] Purpose of the experiment: To verify the film-forming integrity, biodegradability, pharmacological activity, and anti-adhesion effect of the composite hydrogel described in Examples T1, T2, and T3 in the postoperative wound of the uterine cavity, and to control the comparative examples C1-C4, to determine the contribution of key ingredients (such as astragaloside A and salvianolic acid B) and structural parameters (such as chitosan molecular weight and cross-linking method) to the overall therapeutic effect.

[0093] Selection of animal models: 8 female rats per group;

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

[0095] To comprehensively evaluate the effectiveness of each group's composition in preventing postoperative re-adhesion of the uterine cavity, the following three core evaluation indicators are set:

[0096] 1. Re-adhesion degree score:

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

[0098] Method: The animals are sacrificed on the 14th day after surgery, and the uterus is completely dissected; the AFS scoring system is used for evaluation, and the following three dimensions are investigated (each item scored 0-4, total score 0-12): uterine cavity adhesion range; adhesion strength (separability); endometrial regeneration; the higher the score, the more severe the adhesion.

[0099] 2. Gel degradation behavior:

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

[0101] Method: On the 3rd day, 7th day, 10th day and 14th day after operation, 2 animals in each group were randomly killed, and the uterus was dissected to observe the gel residue; the degree of adhesion score (0: no adhesion, 1: partial adhesion, 2: complete adhesion);

[0102] 3. Pharmacological effect analysis:

[0103] Objective: To evaluate the biological regulation function of the composition from the aspects of inflammation inhibition and fibrosis factor expression.

[0104] Method: Inflammation factor detection of uterine cavity irrigation fluid (7th day); uterine cavity irrigation fluid was collected (0.5 mL of sterile PBS was poured in and sucked out) on the 7th day after operation in each group, and the supernatant was taken after centrifugation;

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

[0106] Detection method: ELISA kit, operation according to the instruction;

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

[0108] The experimental result data is 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 (inflammation factor level, 7th day after operation, pg / mL)

[0114]

[0115] Data analysis:

[0116] 1. Analysis of adhesion score results:

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

[0118] The cause of the analysis, T3 in the formula using moderate chitosan concentration (3.0%), sodium hyaluronate concentration (1.0%) and reasonable proportion of active ingredient combination, both the gel performance and biological activity. In contrast, C1 and C3 respectively eliminated salvianolic acid B or astragaloside, leading to the ability to reduce the synergistic anti-inflammatory and anti-adhesion; C2 using low molecular weight chitosan (50kDa), significantly reduced the adhesion strength and space barrier ability of the material; C4 is completely missing active pharmaceutical ingredients, only physical mixed gel, so the effect is the worst in inhibiting tissue adhesion.

[0119] 2, gel degradation residual rate analysis:

[0120] Table 2 and Figure 2 Reflects the different groups of hydrogel degradation residual rate on the 3rd, 7th, 10th, 14th day. In the T group, T2 has the highest residual rate on the 3rd and 7th day (91.5%, 75.3%), the degradation is the slowest, T1 and T3 are second, in Figure 2 The curve is protruding. To the 14th day, T1, T2, T3 residual rate decreased to 3.6%, 7.1% and 4.8% respectively, basically complete in vivo degradation.

[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%), forming a more dense, higher cross-linking three-dimensional network structure, delaying the hydrolysis rate; T3 balances the structural stability and biodegradability, while ensuring the slow-release performance, avoiding the influence of subsequent tissue repair due to too slow degradation.

[0122] And the comparative example, C1-C3 although using similar formula, but due to the lack of ingredients or chitosan molecular weight, leading to incomplete degradation or too fast, residual rate on the 14th day is still as high as 15.9% (C1), 12.7% (C2), 18.4% (C3); C4 degradation is very fast from the 3rd day, only 6.3% on the 14th day, although the residual rate is close to T group, but its adhesion score and inflammation index is very high, indicating that its degradation process is not accompanied by effective drug release and barrier protection.

[0123] 3, inflammation factor level analysis:

[0124] Table 3 and Figure 3 The data shows that the T group significantly inhibits the level of TNF-α, IL-6, IL-1β and other inflammatory factors, T2 shows the best (64.7, 45.9, 55.1 pg / mL respectively), T3 and T1 are second; and the inflammatory factor level of C group and N group is significantly increased, especially the TNF-α concentration of C4 and N group is 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 above shows and describes the basic principles, main features and advantages of the present application. Those skilled in the art should understand that the present application is not limited to the above-mentioned embodiments, and the above-mentioned embodiments and descriptions in the specification are only to illustrate the principles of the present application. Without departing from the spirit and scope of the present application, various changes and improvements can be made to the present application, and these changes and improvements all fall within the scope of the claimed present application. The scope of protection of the present application is defined by the appended claims and their equivalents.

Claims

1. A chitosan-hyaluronic acid hydrogel composition based on astragaloside IV and salvianolic acid B, characterized in that, Comprise the following components by mass percentage: 1.0%~5.0% of chitosan; 0.2%~2.0% of sodium hyaluronate; 0.05%~0.5% of astragaloside IV; 0.05%~0.5% of salvianolic acid B; 0.01%~0.3% of ferulic acid; 0.01%~0.2% of notoginseng saponin R1; 0.1%~0.5% of lactic acid buffer, pH 6.2~6.8; 0.2%~1.0% of crosslinking agent; the balance is water, supplemented to 100%; The chitosan is medium viscosity chitosan with a degree of deacetylation greater than 85% and a molecular weight of 150~300kDa; The hyaluronic acid is sodium salt type hyaluronic acid with a molecular weight of 100~200kDa; The astragaloside IV, salvianolic acid B, ferulic acid and notoginseng saponin R1 are compounded and added in a preset mass ratio; The crosslinking agent is a reaction mixture of glutaraldehyde or aldehyde-modified hyaluronic acid and thiolated chitosan; The composition forms an injectable pre-gel liquid after uniform stirring and forms a semi-solid three-dimensional hydrogel structure after crosslinking reaction.

2. The chitosan-hyaluronic acid hydrogel composition based on astragaloside IV and salvianolic acid B according to claim 1, characterized in that: The astragaloside IV, salvianolic acid B, ferulic acid and notoginseng saponin R1 are compounded and added in a preset mass ratio specifically refers to: According to the concentrations of astragaloside IV: 0.5~2.0mg / mL, salvianolic acid B: 0.4~1.6mg / mL, ferulic acid: 0.2~1.0mg / mL, notoginseng saponin R1: 0.2~0.8mg / mL, respectively dissolved in phosphate buffer with pH 7.2~7.4 to form single-component solutions; According to the mass ratio of astragaloside IV: salvianolic acid B: ferulic acid: notoginseng saponin = 1: (0.8~1.2): (0.3~0.6): (0.2~0.4), compound and under the condition of magnetic stirring, continuously stirring for 60 minutes at room temperature 20~25℃ to form a complex active liquid; The complex active liquid is slowly added to the chitosan-hyaluronic acid base carrier liquid in the lactic acid buffer stable environment, continue to stir for 30 minutes, make the four components uniformly distributed at the molecular level, form a stable solution state, avoid precipitation, crystallization or pH-induced decomposition reaction.

3. The chitosan-hyaluronic acid hydrogel composition based on astragaloside IV and salvianolic acid B according to claim 1, characterized in that: The chitosan is first dissolved in lactic acid buffer with a mass concentration of 1.0~1.5% at room temperature 20~25℃ for 4~6 hours to ensure its full swelling and form a uniform high molecular solution; The hyaluronic acid is added to the chitosan solution by slow dropwise addition and continue to mix for 30~60 minutes under stirring conditions to make the complex network molecular chains fully entangled.

4. The chitosan-hyaluronic acid hydrogel composition based on astragaloside IV and salvianolic acid B according to claim 1, characterized in that: The cross-linking agent is prepared by mixing hyaluronic acid modified by maleimide and chitosan modified by L-cysteine at a volume ratio of 1:1, and the cross-linking reaction is realized by Michael addition reaction, the reaction temperature is controlled at 20-30 DEG C, and the reaction time is 15-30 minutes, to form a stable irreversible carbon-sulfur bond cross-linking structure. 5.The chitosan-hyaluronic acid hydrogel composition based on astragaloside and salvianolic acid B according to claim 1, characterized in that: The hydrogel formed by the composition is a non-self-flowing gel, the storage modulus thereof is 200-600 Pa at a frequency of 1 Hz, and the hydrogel has a moderate form retention capacity; The degradation rate of the hydrogel in a PBS body fluid simulation solution at pH 7.4 is 15%-30% of the dry weight per 3 days, and the hydrogel is degraded completely within 10-14 days; The hydration expansion rate of the hydrogel is stable in the range of 500%-800% at a body temperature of 37 DEG C.

6. A process for the preparation of a chitosan-hyaluronic acid hydrogel composition based on astragaloside IV and salvianolic acid B as claimed in any one of claims 1 to 5, characterized in that, The method comprises the following steps: S1, dissolving chitosan in a lactic acid buffer to form a chitosan solution with a mass concentration of 1.2%, and stirring at room temperature for 5 hours to fully swell and form a viscous and uniform solution; S2, adding sodium hyaluronate to the chitosan solution in a dropwise manner, stirring while maintaining the buffer pH at 6.4-6.6, and continuing to mix for 30 minutes to obtain a composite carrier base solution; S3, weighing astragaloside, salvianolic acid B, ferulic acid and notoginsenoside R1, respectively dissolving them in a phosphate buffer solution at pH 7.2-7.4, mixing, and then adding the mixture to the solution obtained in step S2 in a proportion, and stirring for 60 minutes; S4, preparing a cross-linking agent solution including a glutaraldehyde solution or a multi-functional polymer solution containing a reaction group, adjusting the pH to 6.8-7.0, and then adding the solution to the mixture in step S3, and continuing to react for 20-30 minutes to obtain a preformed hydrogel composition. 7.The preparation method according to claim 6, characterized in that: The active ingredients are added in the following order: first, notoginsenoside R1 and ferulic acid are added to the base carrier solution and stirred for 15 minutes; then, salvianolic acid B and astragaloside are added in sequence, and the stirring time is 45 minutes to prevent crystallization and precipitation between the ingredients with similar structures; and a magnetic stirrer is used to control the rotation speed to be 300-500 rpm during the reaction. 8.Use of the chitosan-hyaluronic acid hydrogel composition based on astragaloside and salvianolic acid B according to any one of claims 1-5 in the preparation of a medicine for preventing postoperative adhesion of the uterine cavity.

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