A mandelic acid / glycyrrhizic acid self-assembled hydrogel and a preparation method and application thereof

Abstract

The application discloses a kind of amygdalin / liquorice acid self-assembled hydrogel and its preparation method and application, preparation method includes the following steps: amygdalin solution and liquorice acid are mixed and dispersed uniformly, stationary, obtain amygdalin / liquorice acid self-assembled hydrogel;The concentration of the amygdalin solution is 0.015~0.040g / mL, and the concentration of liquorice acid in amygdalin / liquorice acid self-assembled hydrogel is 0.015~0.040g / mL.Amygdalin / liquorice acid self-assembled hydrogel microstructure presents three-dimensional porous network structure, can be used to prepare drug for reducing or treating inflammatory encephalopathy, neuron damage encephalopathy, blood-brain barrier destruction disease, complement activation disease, the preparation of the application is simple, safe and aimed at complement activation inhibition.

Description

Technical Field

[0001] This invention relates to the field of hydrogel technology, and in particular to a self-assembled amygdalin / glycyrrhizic acid hydrogel, its preparation method, and its application. Background Technology

[0002] Traumatic brain injury (TBI) refers to a syndrome of neurological dysfunction caused by external impact, often accompanied by multiple pathological changes such as craniocerebral tissue damage and intracranial hemorrhage. Early neuroinflammation following trauma is a key factor leading to long-term, widespread secondary damage and is positively correlated with the occurrence of chronic neurodegenerative diseases. Multiple clinical and basic studies have confirmed that timely suppression of neuroinflammation is an important and modifiable key driver in reducing secondary damage after brain injury. However, existing anti-inflammatory drugs have failed to achieve the expected efficacy in current multicenter clinical trials. Current early treatment of TBI mainly focuses on surgical intervention to limit hematoma expansion and supportive care, but measures for early in-situ drug administration in the brain injury area to suppress the cytokine storm are rare.

[0003] Activation of the complement system in the area surrounding brain injury is considered an early determinant of neuroinflammatory response and functional decline after traumatic brain injury (TBI). Early inhibition of complement activation has also been shown to reduce neuroinflammation and exert neuroprotective effects. Furthermore, studies have shown that surgical injury leads to a surge in injury-related pattern molecules, thereby activating the complement system unrelated to the primary injury and triggering postoperative inflammatory responses. The complement molecule C3 has been identified as a central effector molecule regulating the complement system. Multiple preclinical studies have demonstrated that C3 inhibition can effectively reduce complement activation after brain injury, thereby alleviating neuroinflammation and improving neurological function. However, currently, there is no integrated therapeutic approach combining post-traumatic brain injury debridement with in-situ drug administration to regulate C3 expression and subsequently inhibit complement activation.

[0004] Amygdalin is the main active ingredient in the traditional Chinese medicine almond, primarily found in the seeds of plants in the Rosaceae family, with the molecular formula C. 20 H 27 NO 11 (Molecular weight 457.43). It possesses various pharmacological activities, including anti-inflammatory, anti-tumor, anti-fibrotic, immunomodulatory, and anti-atherosclerotic effects, and has already shown efficacy in treating neuroinflammation and cardiovascular diseases. However, amygdalin has good water solubility, which prevents localized administration and limits its safety and clinical application.

[0005] Glycyrrhizic acid is a triterpenoid saponin natural product and the main active ingredient of the traditional Chinese medicine licorice. Its molecular formula is C6H2O. 42 H 62 O 16(Molecular weight 822.93). Its chemical structure consists of one molecule of hydrophobic glycyrrhetinic acid and two molecules of hydrophilic glucuronic acid linked by β-glycosidic bonds, giving the glycyrrhizic acid molecule unique amphiphilic characteristics, enabling it to self-assemble into nanomaterials. It possesses a wide range of pharmacological activities, including anti-inflammatory and immunomodulatory, antioxidant and cell-protective, antiviral and antifibrotic, and antitumor effects.

[0006] Although amygdalin and glycyrrhizic acid, as single active ingredients, have demonstrated certain pharmacological activities in specific disease models, they still face significant challenges in addressing complex pathological microenvironments such as brain diseases. Firstly, single drugs are unlikely to achieve synergistic effects in complex pathological environments. Secondly, traditional systemic administration routes (such as oral or intravenous injection) suffer from significant first-pass effects, widespread distribution in non-target tissues, and shortened drug half-lives, leading to insufficient effective concentrations at the lesion site and potentially triggering systemic toxicity.

[0007] Therefore, developing in-situ drug delivery systems based on biomaterials and optimizing the spatiotemporal distribution of drugs through synergistic effects of components has become a key strategy for overcoming the bottlenecks in the treatment of complex diseases. The development of multi-component self-assembling hydrogels containing all-natural bioactive ingredients, targeting complement activation inhibition and suitable for local administration after debridement, is urgently needed. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a simple, safe, and complement activation-inhibiting self-assembled hydrogel of amygdalin / glycyrrhizic acid, its preparation method and application.

[0009] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0010] A method for preparing an amygdalin / glycyrrhizic acid self-assembled hydrogel includes the following steps: mixing and dispersing an amygdalin solution and glycyrrhizic acid evenly, and allowing it to stand to obtain an amygdalin / glycyrrhizic acid self-assembled hydrogel; wherein the concentration of the amygdalin solution is 0.015–0.040 g / mL, and the concentration of glycyrrhizic acid in the amygdalin / glycyrrhizic acid self-assembled hydrogel is 0.015–0.040 g / mL.

[0011] In some embodiments of the present invention, the concentration of the amygdalin solution can be 0.015 g / mL, 0.020 g / mL, 0.025 g / mL, 0.030 g / mL, 0.035 g / mL, or 0.040 g / mL, and the concentration of glycyrrhizic acid in the amygdalin / glycyrrhizic acid self-assembled hydrogel can be 0.015 g / mL, 0.020 g / mL, 0.025 g / mL, 0.030 g / mL, 0.035 g / mL, or 0.040 g / mL. The pH value of the amygdalin solution is 7.2-7.4, which is similar to the physiological environment of human cells.

[0012] As a further improvement to the above technical solution:

[0013] The mass ratio of amygdalin to glycyrrhizic acid is 1:1 / 2 to 1:2. In some embodiments of the present invention, the mass ratio of amygdalin to glycyrrhizic acid can be 2, 1.5, 1, or 0.5.

[0014] The process of dispersing amygdalin solution and glycyrrhizic acid evenly includes the following steps:

[0015] A1. Mix amygdalin and PBS solution, and disperse evenly by ultrasonication to obtain amygdalin solution;

[0016] A2. Mix amygdalin solution and glycyrrhizic acid, and then ultrasonically disperse the mixture at 40–70°C until it is clear and transparent. After standing, amygdalin / glycyrrhizic acid self-assembled hydrogel is obtained.

[0017] Ultrasonic dispersion at temperatures between 40 and 70°C facilitates the dissolution of glycyrrhizic acid. However, at room temperature (25°C), glycyrrhizic acid may not dissolve completely, resulting in a milky white and opaque system. In some embodiments of this invention, the temperatures may be 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, or 70°C.

[0018] In step A1 or step A2, the ultrasonic dispersion power is 70-120W, and the ultrasonic dispersion time is 1-10 minutes. In some embodiments of the present invention, for example, it can be 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, etc. When the ultrasonic dispersion temperature is 40°C, the ultrasonic dispersion time is about 6 minutes.

[0019] A self-assembled hydrogel of amygdalin / glycyrrhizic acid is prepared by the aforementioned method, and the microstructure of the self-assembled hydrogel of amygdalin / glycyrrhizic acid exhibits a three-dimensional porous network structure.

[0020] The aforementioned preparation method yields a self-assembled hydrogel of amygdalin / glycyrrhizic acid, or the aforementioned self-assembled hydrogel of amygdalin / glycyrrhizic acid is used in the preparation of a drug for alleviating or treating inflammatory encephalopathy.

[0021] The drug for inflammatory encephalopathy can inhibit the expression of pro-inflammatory factors or promote the expression of anti-inflammatory factors, wherein the pro-inflammatory factors include at least one of interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor (TNF-α), and the anti-inflammatory factors include interleukin-10 (IL-10).

[0022] The drug for the inflammatory encephalopathy can inhibit the expression of microglia marker (ionized calcium-binding adapter molecule 1, Iba-1) or astrocyte marker (glial fibrillary acidic protein, GFAP).

[0023] The inflammatory encephalopathy mentioned refers to any one or more of the following: traumatic brain injury, stroke, Alzheimer's disease, and Parkinson's disease.

[0024] The aforementioned preparation method yields a self-assembled hydrogel of amygdalin / glycyrrhizic acid, or the aforementioned self-assembled hydrogel of amygdalin / glycyrrhizic acid is used in the preparation of a drug for alleviating or treating neuronal damage encephalopathy.

[0025] The drug for neuronal damage encephalopathy can increase the number of surviving neurons after brain injury.

[0026] The aforementioned preparation method yields a self-assembled hydrogel of amygdalin / glycyrrhizic acid, or the aforementioned self-assembled hydrogel of amygdalin / glycyrrhizic acid is used in the preparation of drugs for alleviating or treating diseases involving disruption of the blood-brain barrier.

[0027] The drug for blood-brain barrier disorders can reduce the degree of IgG leakage or the level of S100β expression in serum after brain injury.

[0028] The aforementioned preparation method yields a self-assembled hydrogel of amygdalin / glycyrrhizic acid, or the aforementioned self-assembled hydrogel of amygdalin / glycyrrhizic acid is used in the preparation of drugs for alleviating or treating complement activation disorders.

[0029] The drug for complement activation disorders can inhibit the expression of complement molecules, wherein the complement molecule is C3.

[0030] The medications for complement activation disorders are locally injected formulations.

[0031] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0032] (1) The preparation method of the amygdalin / glycyrrhizic acid self-assembled hydrogel of the present invention. First, the two active ingredients (amygdalin and glycyrrhizic acid) in the raw materials are derived from traditional Chinese medicine and are natural product molecules capable of self-assembling into a hydrogel. This process, without the involvement of exogenous organic solvents, metal ions, or polymeric compounds, maximizes the biocompatibility of the hydrogel. Second, the preparation process requires no complex steps, is simple to operate, and is easily scalable for mass production. This makes the hydrogel a promising candidate for tissue engineering applications in the biomedical field.

[0033] (2) The amygdalin / glycyrrhizic acid self-assembled hydrogel of the present invention exhibits a three-dimensional porous network structure. This not only solves the problem that traditional amygdalin cannot be injected locally, but also realizes the self-assembly of amygdalin and glycyrrhizic acid, retaining the pharmacological activity of the two drug molecules. It can be administered locally by injection, avoiding the potential systemic toxicity side effects of oral administration, and synergistically enhancing the therapeutic effect of the disease.

[0034] (3) The application of the amygdalin / glycyrrhizic acid self-assembled hydrogel of the present invention in the preparation of drugs to alleviate or treat inflammatory encephalopathy, neuronal damage encephalopathy, blood-brain barrier disruption diseases, and complement activation diseases has anti-inflammatory, blood-brain barrier disruption improvement, complement activation inhibition, and neuroprotective effects. It effectively reduces the number of astrocytes and microglia in mice with traumatic brain injury, reduces the expression of pro-inflammatory factors such as interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor (TNF-α), enhances the expression of anti-inflammatory factor interleukin-10 (IL-10), reduces blood-brain barrier leakage, improves nerve function, and inhibits the expression of complement molecules. It has important practical significance for becoming a new clinical drug. Attached Figure Description

[0035] Figure 1 This is a photograph of the appearance of the amygdalin / glycyrrhizic acid self-assembled hydrogel in Example 1 of the present invention.

[0036] Figure 2 This is a scanning electron microscope (SEM) image of the amygdalin / glycyrrhizic acid self-assembled hydrogel in Example 1 of the present invention.

[0037] Figure 3 The figure shows the rheological detection results of the amygdalin / glycyrrhizic acid self-assembled hydrogel in Example 1 of the present invention.

[0038] Figure 4 This is an injectability photograph of the amygdalin / glycyrrhizic acid self-assembled hydrogel in Example 1 of the present invention.

[0039] Figure 5This is the Fourier transform infrared spectrum of the amygdalin / glycyrrhizic acid self-assembled hydrogel in Example 1 of the present invention.

[0040] Figure 6 This is the UV spectrum of the amygdalin / glycyrrhizic acid self-assembled hydrogel in Example 1 of the present invention.

[0041] Figure 7 This is the X-ray diffraction energy spectrum of the amygdalin / glycyrrhizic acid self-assembled hydrogel in Example 1 of the present invention.

[0042] Figure 8 These are photos taken before and after in situ injection of the amygdalin / glycyrrhizic acid self-assembled hydrogel in Example 2 of this invention following debridement of traumatic brain injury.

[0043] Figure 9 The images show fluorescence images and statistical results of microglia induced by traumatic brain injury in mice constructed using a controlled cortical injury instrument, including the control group, model group, amygdalin / glycyrrhizic acid self-assembled hydrogel, glycyrrhizic acid hydrogel, and amygdalin solution. (Example 2 of this invention)

[0044] Figure 10 The images show fluorescence images and statistical results of astrocytes induced by traumatic brain injury in mice constructed using a controlled cortical injury instrument, including the control group, model group, amygdalin / glycyrrhizic acid self-assembled hydrogel, glycyrrhizic acid hydrogel, and amygdalin solution. (Example 2 of this invention)

[0045] Figure 11 The figure shows the effects of the control group, model group, amygdalin / glycyrrhizic acid self-assembled hydrogel, glycyrrhizic acid hydrogel, and amygdalin solution on IL-1β and IL-6 pro-inflammatory factors induced in mice with traumatic brain injury constructed by the controlled cortical injury instrument in Example 2 of this invention.

[0046] Figure 12 The figure shows the effects of the control group, model group, amygdalin / glycyrrhizic acid self-assembled hydrogel, glycyrrhizic acid hydrogel, and amygdalin solution on TNF-α pro-inflammatory factor and IL-10 anti-inflammatory factor in traumatic brain injury mice constructed by the controlled cortical injury instrument in Example 2 of the present invention.

[0047] Figure 13 The figure shows the effects of the control group, model group, amygdalin / glycyrrhizic acid self-assembled hydrogel, glycyrrhizic acid hydrogel, and amygdalin solution on neuronal damage induced by traumatic brain injury in mice constructed using a controlled cortical injury instrument, in Example 3 of this invention.

[0048] Figure 14The figure shows the effect of the control group, model group, and amygdalin / glycyrrhizic acid self-assembled hydrogel on immunoglobulin IgG induced in traumatic brain injury mice constructed by the controlled cortical injury instrument in Example 4 of the present invention.

[0049] Figure 15 The figure shows the effect of the control group, model group, and amygdalin / glycyrrhizic acid self-assembled hydrogel on the central nervous system-specific protein S100β induced by traumatic brain injury in mice constructed using a controlled cortical injury instrument in Example 4 of this invention.

[0050] Figure 16 The figure shows the effect of the control group, model group, amygdalin solution, glycyrrhizic acid hydrogel, and amygdalin / glycyrrhizic acid self-assembled hydrogel on the complement C3 level in mice with traumatic brain injury constructed by the controlled cortical injury instrument in Example 5 of the present invention.

[0051] Figure 17 This study investigates the effects of the control group, model group, and amygdalin / glycyrrhizic acid self-assembled hydrogel on the biocompatibility of major organs in mice with traumatic brain injury constructed using a controlled cortical injury instrument, as described in Example 6 of this invention.

[0052] Figure 18 The figure shows the effects of the control group, model group, and amygdalin / glycyrrhizic acid self-assembled hydrogel on liver and kidney function indicators in mice with traumatic brain injury constructed using a controlled cortical injury instrument, as shown in Example 6 of this invention. Detailed Implementation

[0053] The present invention will be further described in detail below. Unless otherwise specified, the instruments or materials used in the present invention are commercially available.

[0054] Example 1

[0055] A self-assembled hydrogel of amygdalin / glycyrrhizic acid according to the present invention comprises an amygdalin solution and glycyrrhizic acid, and is prepared by the following method:

[0056] (1) Preparation of amygdalin solution: Take 15 mg of amygdalin, add 1 mL of PBS solution (pH = 7.4), and sonicate until fully dissolved to obtain a 15 mg / mL amygdalin solution.

[0057] (2) Self-assembly of glycyrrhizic acid and amygdalin solution: Weigh 15 mg of glycyrrhizic acid and add 1 mL of amygdalin solution from step (1) to the glycyrrhizic acid. Under conditions of 40–70°C (60°C in this example), ultrasonically disperse for 1–5 minutes (1 minute in this example) at an ultrasonic dispersion power of 70–120 W (100 W in this example). After complete dissolution, place on a table and let stand to obtain amygdalin / glycyrrhizic acid self-assembled hydrogel (abbreviated as amygdalin / glycyrrhizic acid hydrogel in the attached figure).

[0058] Appearance photos of the amygdalin / glycyrrhizic acid self-assembled hydrogel: After the amygdalin / glycyrrhizic acid self-assembled hydrogel was prepared, its appearance was photographed.

[0059] Sample preparation for scanning electron microscopy (SEM): 10 μL of hydrogel sample was pipetted onto a clean silicon wafer and immediately placed in a -20°C environment to freeze. The dried sample was then obtained using a vacuum freeze dryer and analyzed by SEM to obtain an image of the hydrogel's internal morphology.

[0060] Comparative Example 1

[0061] A comparative example of amygdalin solution was prepared by the following method: 15 mg of amygdalin was added to 1 mL of PBS solution (pH = 7.4), and the solution was sonicated until fully dissolved to obtain a 15 mg / mL amygdalin solution.

[0062] Comparative Example 2

[0063] A glycyrrhizic acid hydrogel of the present comparative example was prepared by the following method: 15 mg of glycyrrhizic acid was weighed, and 1 mL of PBS solution (pH = 7.4) was added to the glycyrrhizic acid. The mixture was ultrasonically dispersed for 1 to 5 minutes (1 minute in the present comparative example) at 40–70 °C (60 °C in the present comparative example) with an ultrasonic dispersion power of 70–120 W (100 W in the present comparative example).

[0064] Comparative Example 3

[0065] A comparative example of amygdalin / glycyrrhizic acid mobile liquid was prepared by the following method:

[0066] (1) Preparation of amygdalin solution: Take 10 mg of amygdalin, add 1 mL of PBS solution (pH = 7.4), and sonicate until fully dissolved to obtain a 10 mg / mL amygdalin solution.

[0067] (2) Mixing of glycyrrhizic acid and amygdalin solution: Weigh 10 mg of glycyrrhizic acid and add 1 mL of the aforementioned amygdalin solution to the glycyrrhizic acid. Under conditions of 40–70°C (60°C in this comparative example), ultrasonically disperse for 1–5 minutes (1 minute in this comparative example) at a power of 70–120 W. After complete dissolution, let it stand on the table to obtain a flowing liquid with a concentration of 10 mg / mL amygdalin / glycyrrhizic acid.

[0068] This is because the concentration is too low to form a hydrogel system with a stable structure.

[0069] Comparative Example 4

[0070] A comparative example of amygdalin / glycyrrhizic acid mobile liquid was prepared by the following method:

[0071] (1) Preparation of amygdalin solution: Take 8 mg of amygdalin, add 1 mL of PBS solution (pH = 7.4), and sonicate until fully dissolved to obtain an 8 mg / mL amygdalin solution.

[0072] (2) Mixing of glycyrrhizic acid and amygdalin solution: Weigh 8 mg of glycyrrhizic acid and add 1 mL of the aforementioned amygdalin solution to the glycyrrhizic acid. Under conditions of 40–70°C (60°C in this comparative example), ultrasonically disperse for 1–5 minutes (1 minute in this comparative example) at a power of 70–120 W. After complete dissolution, let it stand on the table to obtain a flowing liquid with a concentration of 8 mg / mL amygdalin / glycyrrhizic acid.

[0073] This is because at lower concentrations, it is impossible to form a hydrogel system with a stable structure.

[0074] Comparative Example 5

[0075] A comparative example of amygdalin / glycyrrhizic acid mobile liquid was prepared by the following method:

[0076] (1) Preparation of amygdalin solution: Take 4 mg of amygdalin, add 1 mL of PBS solution (pH = 7.4), and sonicate until fully dissolved to obtain a 4 mg / mL amygdalin solution.

[0077] (2) Mixing of glycyrrhizic acid and amygdalin solution: Weigh 4 mg of glycyrrhizic acid and add 1 mL of the aforementioned amygdalin solution to the glycyrrhizic acid. Under conditions of 40–70°C (60°C in this comparative example), ultrasonically disperse for 1–5 minutes (1 minute in this comparative example) at a power of 70–120 W. After complete dissolution, let it stand on the table to obtain a flowing liquid with a concentration of 4 mg / mL amygdalin / glycyrrhizic acid.

[0078] This is because the concentration is so low that it is impossible to form a hydrogel system with a stable structure.

[0079] Rheological testing: Rheological testing was performed using the amygdalin / glycyrrhizic acid self-assembled hydrogel prepared in Example 1, specifically as follows: ① Strain scan: The strain range was set to 0.01%-100%, and the frequency was set to 0.1Hz. ② Dynamic time scan: The strain force was set to 0.01%, and the duration was set to 300s. ③ Gait scan: The frequency was set to 0.1Hz. Low strain force was set to 0.01%, and the duration was set to 100s. High strain force was set to 10%, and the duration was set to 100s. The entire test process was repeated for 5 cycles. ④ Viscosity test: The shear rate was set to 0.01-100 / s, and the duration was set to 70s.

[0080] Sample preparation for Fourier transform infrared spectroscopy: The amygdalin solution, amygdalin / glycyrrhizic acid self-assembled hydrogel, and glycyrrhizic acid hydrogel prepared in Comparative Example 1, Example 1, and Comparative Example 2, respectively, were freeze-dried to obtain the powder samples to be tested. The testing process employed the potassium bromide pellet method, where the powder sample to be tested was thoroughly ground with potassium bromide and then pressed into a pellet. The testing range was 4000–400 cm⁻¹. -1 Transmittance within the wavelength range is measured using an instrument.

[0081] Sample preparation for UV spectroscopy: The amygdalin / glycyrrhizic acid self-assembled hydrogel prepared in Example 1 was diluted to different test concentrations using ultrapure water. Then, 300 μL of the sample to be tested was placed in a 10 mm quartz cuvette, and the test wavelength range was 190–400 nm before testing.

[0082] Sample preparation for X-ray diffraction energy dispersive spectroscopy (EDS): The amygdalin / glycyrrhizic acid self-assembled hydrogel prepared in Example 1 was freeze-dried to obtain the powder sample to be tested. The powder sample was thoroughly ground, and a copper target was used as the test target. The scanning speed was 10° / min, and the scanning range was 10°~90° before testing.

[0083] Figure 1 The images show the appearance of the amygdalin / glycyrrhizic acid self-assembled hydrogel prepared in Example 1 and the flowing liquids of amygdalin / glycyrrhizic acid in Comparative Examples 3, 4, and 5. Figure 1 (a) is Example 1. Figure 1 (b) is Comparative Example 3. Figure 1 (c) is Comparative Example 4. Figure 1 (d) is Comparative Example 5. For example... Figure 1 As shown in (a), the amygdalin / glycyrrhizic acid self-assembled hydrogel is a white, homogeneous, and stable gel. Figure 1 (b) Figure 1 (c) Figure 1 As shown in (d), due to the low concentration, the amygdalin / glycyrrhizic acid systems in Comparative Examples 3, 4, and 5 are all flowing liquids and cannot form a hydrogel system with a stable structure, thus lacking the ability to administer drugs in situ.

[0084] Figure 2 This is a scanning electron microscope image of the amygdalin / glycyrrhizic acid self-assembled hydrogel in Example 1. Figure 2 (b) is a magnified view of the area within the box in Figure (a). For example... Figure 2 As shown, the amygdalin / glycyrrhizic acid self-assembled hydrogel exhibits a three-dimensional porous network structure. Amygdalin / glycyrrhizic acid self-assembled hydrogels are a class of three-dimensional network materials with high water content, possessing soft tissue-like structure and soft-wet properties, local injectability, and pharmacodynamic properties. Drug molecule (small molecule traditional Chinese medicine) self-assembled hydrogels integrate drug pharmacodynamic properties (100% drug loading and drug self-delivery) and material structural characteristics (three-dimensional network support and local injectability), optimizing the traditional "drug-assisted synergy" model into a "drug-self-assisted synergy" model. This means that without additional carrier loading, drug molecules can self-assemble and act as structural carriers. This "drug-self-assisted synergy" model effectively avoids the potential toxic side effects of exogenous carriers and the complexity of system preparation. It solves the technical problems of existing technologies using traditional hydrogel systems prepared from polymeric compounds as delivery carriers for drug delivery, such as low delivery efficiency, significant toxic side effects, complex preparation procedures, and mismatch between brain tissue modulus and other factors.

[0085] Figure 3 The results are the rheological analysis of the amygdalin / glycyrrhizic acid self-assembled hydrogel in Example 1. Figure 3 (a) is a strain scan result diagram. Figure 3 (b) shows the results of a dynamic time scan. Figure 3 (c) is a gait scan result image. Figure 3 (d) shows the viscosity test results. Figure 3 As shown, strain scanning results indicate that when the strain exceeds 5%, the elastic modulus (G') is lower than the loss modulus (G”), indicating that the amygdalin / glycyrrhizic acid self-assembled hydrogel transitions from a gel state to a sol state. Dynamic time scanning results show that G' remains greater than G” throughout the test, confirming the stability of the amygdalin / glycyrrhizic acid self-assembled hydrogel. Gait scanning results show that in repeated cycles from low to high strain, the amygdalin / glycyrrhizic acid self-assembled hydrogel can recover from the sol state to the gel state when transitioning from high to low strain. Viscosity testing results show that the shear viscosity of the amygdalin / glycyrrhizic acid self-assembled hydrogel decreases with increasing shear rate, indicating good shear thinning behavior. All these results demonstrate that the amygdalin / glycyrrhizic acid self-assembled hydrogel possesses good injectability and self-healing capabilities, exhibiting significant potential for clinical applications.

[0086] Figure 4 This is an injectability photograph of the amygdalin / glycyrrhizic acid self-assembled hydrogel in Example 1. Figure 4(a) is a diagram showing the in vitro injection status of AG-gel. Figure 4 (b) is a schematic diagram of AG-gel injected into the brain tissue defect area, where AG-gel represents the amygdalin / glycyrrhizic acid self-assembled hydrogel prepared in Example 1. Figure 4 As shown, AG-gel can be smoothly dispensed from the syringe and successfully injected into the brain injury defect area. This result suggests that AG-gel can be injected into the injury area and has the potential for in-situ administration after surgical debridement.

[0087] Figure 5 The Fourier transform infrared spectrum of the amygdalin / glycyrrhizic acid self-assembled hydrogel in Example 1 is shown below. The specific groupings are as follows: Figure 5 As shown, in glycyrrhizic acid hydrogel, 3425 cm⁻¹ -1 The absorption peak for the stretching vibration of OH is 3406 cm⁻¹ in amygdalin solution. -1 The absorption peak for the stretching vibration of OH is observed, while in the amygdalin / glycyrrhizic acid self-assembled hydrogel, the absorption peak for the stretching vibration of OH shifts to 3417 cm⁻¹. -1 This indicates the presence of intermolecular hydrogen bonds in the amygdalin / glycyrrhizic acid self-assembled hydrogel.

[0088] Figure 6 This is the UV spectrum of the amygdalin / glycyrrhizic acid self-assembled hydrogel from Example 1. AG-gel represents the amygdalin / glycyrrhizic acid self-assembled hydrogel prepared in Example 1, and each curve represents AG-gel diluted to different concentrations. Figure 6 As shown, the UV spectrum exhibits a blue shift (from 219 nm to 210 nm) as the AG-gel concentration is diluted, indicating the presence of π-π stacking interactions during the assembly of AG-gel.

[0089] Figure 7 The image shows the X-ray diffraction energy distribution of the amygdalin / glycyrrhizic acid self-assembled hydrogel in Example 1. Figure 7 As shown, when the degree value is The presence of a distinct peak indicates the existence of π-π stacking interactions during the assembly of the amygdalin / glycyrrhizic acid self-assembled hydrogel.

[0090] Figure 5 , Figure 6 , Figure 7 The results confirmed that the amygdalin / glycyrrhizic acid self-assembled hydrogel in Example 1 was self-assembled through non-covalent bonding.

[0091] Example 2

[0092] The application of the amygdalin / glycyrrhizic acid self-assembled hydrogel of the present invention in the preparation of a medicament for alleviating or treating inflammatory encephalopathy, specifically the application of a medicament in treating an inflammatory response caused by traumatic brain injury, includes the following steps:

[0093] 1. Construction of a mouse model of traumatic brain injury:

[0094] C57BL / 6 mice were used as experimental animals to establish a traumatic brain injury model using a controllable cortical impact device. The specific procedure was as follows: The experimental animals were anesthetized by intraperitoneal injection of 0.3% sodium pentobarbital solution (220 μL / kg), then fixed in a prone position on a stereotaxic instrument, and the skull was adjusted to a horizontal position. After routine skin preparation and disinfection, the skin was incised along the sagittal suture to expose the skull surface. Using the anterior fontanelle as an anatomical landmark, a 4.0 mm diameter circular bone window was prepared in the corresponding area of ​​the right parietal cortex (coordinates: 2.0 mm posterior to the anterior fontanelle, 2.0 mm to the right of the midline) using a micro-bone drill. The bone flap was carefully removed with microforceps to expose the target brain region. The instrument parameters were set as follows: impact depth 1.5 mm, velocity 3.5 m / s, duration 100 ms. After the impact was completed, the surgical area was strictly disinfected, and the skin incision was sutured.

[0095] The specific experimental groups are as follows:

[0096] Control group: Bone window was opened and bone flap was removed, no traumatic impact was performed, and disinfection and suturing were performed.

[0097] Model group: Bone window was opened and bone flap was removed, while trauma was treated, disinfected and sutured.

[0098] Amygdalin solution group (Comparative Example 1): A bone window was opened and the bone flap was removed. Simultaneously, traumatic impact was performed, and amygdalin solution was injected in situ into the injured area. The area was then disinfected and sutured. The concentration of amygdalin in the amygdalin solution used in this example was 15 mg / mL.

[0099] Amygdalin / glycyrrhizic acid self-assembled hydrogel assembly (Example 1): A bone window was opened and the bone flap was removed. Simultaneously, traumatic impact was performed, and the amygdalin / glycyrrhizic acid self-assembled hydrogel was injected in situ into the injured area. The area was then disinfected and sutured. In this example, the concentrations of amygdalin and glycyrrhizic acid in the amygdalin / glycyrrhizic acid self-assembled hydrogel were 15 mg / mL and 15 mg / mL, respectively.

[0100] In the glycyrrhizic acid hydrogel group (Comparative Example 2), a bone window was opened and the bone flap was removed. Simultaneously, traumatic impact was performed, and glycyrrhizic acid hydrogel was injected in situ into the injured area. The area was then disinfected and sutured. The concentration of glycyrrhizic acid in the glycyrrhizic acid hydrogel used in this example was 15 mg / mL.

[0101] Figure 8This is a photograph of the amygdalin / glycyrrhizic acid self-assembled hydrogel used in Example 1 during intervention in a mouse model of traumatic brain injury. Figure 8 (i) is a mouse model of CCI-induced traumatic brain injury. Figure 8 (ii) is a diagram showing local drug administration after debridement. For example... Figure 8 As shown, after traumatic injury, local brain tissue in mice was damaged, resulting in bleeding. After debridement, the amygdalin / glycyrrhizic acid self-assembled hydrogel can be injected in situ to deliver medication to the brain injury area.

[0102] 2. Immunofluorescence staining was used to determine the expression of microglial cell markers (ion-calcium binding adapter molecule 1, Iba-1) and astrocyte markers (glial fibrillary acidic protein, GFAP) around the injury area of ​​brain tissue after traumatic brain injury:

[0103] (1) Brain tissue sample collection: On the third day after modeling, mice were anesthetized by intraperitoneal injection of 0.3% sodium pentobarbital, followed by cardiac perfusion with 0.9% saline. Finally, the intact brain tissue was dissected and fixed in 4% paraformaldehyde.

[0104] (2) Experimental Procedure: After fixing brain tissue samples with 4% paraformaldehyde solution for 24 hours, 3μm thick coronal brain tissue sections were prepared using standard paraffin embedding and sectioning procedures. Sections from the same coronal plane level in each group were selected, baked in a 60℃ oven for 1 hour, and then processed according to the following procedure: dewaxing with xylene solutions I and II for 10 min each (with moderate shaking during the process) → gradient ethanol hydration (100%, 100%, 95%, 75%, 5 min each) → rinsing three times (5 min each) with phosphate buffer (1×PBS, pH=7.4). Heat-induced antigen retrieval was performed using a 1× sodium citrate buffer system (pH=6.0) (continuous boiling for 20 min), followed by washing with 1×PBS and blocking with 3% bovine serum albumin at room temperature for 1 h. Primary antibody incubation: Anti-GFAP antibody (rabbit-derived, Boster Biological, BA0056, 1:400) or anti-Iba-1 antibody (rabbit-derived, CST, E404W, 1:400), incubated overnight in a humidified chamber at 4°C. After washing with 1×PBS, fluorescently labeled secondary antibody (Jackson ImmunoResearch, donkey anti-rabbit IgG, Alexa Fluor 488, 1:1000) was added and incubated at room temperature in the dark for 1 hour. After thorough washing with 1×PBS, cell nuclei were counterstained with DAPI staining solution (Solarbio, C0065, 1:100) in the dark for 10 minutes. After rinsing with 1×PBS, the cells were mounted with anti-fluorescence quenching mounting medium (Solarbio, S2110) and fluorescence signals were acquired using a Zeiss laser confocal imaging system.

[0105] Finally, representative fluorescence images were displayed, and the proportions of Iba-1 positive cells and GFAP positive cells in the cortical area surrounding brain injury were statistically analyzed. The experimental results are shown in the figures below. Figure 9 and Figure 10 .

[0106] Figure 9 The graph shows the effects of the control group, model group, amygdalin solution, amygdalin / glycyrrhizic acid self-assembled hydrogel, and glycyrrhizic acid hydrogel on Iba-1 levels induced by traumatic brain injury. The left side shows the fluorescence images of each group, and the right side shows the statistical analysis results. Figure 9 As shown, compared with the control group, the Iba-1 positive cells in the model group exhibited a swollen amoeboid morphology, while the morphology of Iba-1 positive cells in the amygdalin / glycyrrhizic acid self-assembled hydrogel group was significantly improved. Further statistical analysis indicated that the number of Iba-1 positive cells in the model group was significantly increased compared with the control group. *** p < 0.001 indicates that the traumatic brain injury model leads to a significant increase in activated Iba-1 positive cells in the cortex surrounding the injury. Compared with the model group, neither the amygdalin solution group nor the glycyrrhizic acid hydrogel group significantly reduced activated Iba-1 positive cells, indicating that a single component cannot significantly improve activated Iba-1 positive cells. However, the number of Iba-1 positive cells in the amygdalin / glycyrrhizic acid self-assembled hydrogel group was significantly lower than that in the model group. * p < 0.05 indicates that the amygdalin / glycyrrhizic acid self-assembled hydrogel can significantly reduce the activity of Iba-1 positive cells, and the effect is better than that of amygdalin solution alone and glycyrrhizic acid hydrogel alone.

[0107] Figure 10 The effects of the control group, model group, amygdalin solution, amygdalin / glycyrrhizic acid self-assembled hydrogel, and glycyrrhizic acid hydrogel on GFAP levels induced by traumatic brain injury are shown. The left side displays fluorescence images for each group, and the right side shows the statistical analysis results. Figure 10 As shown, compared with the control group, the GFAP-positive cells in the model group were larger and had thicker protrusions, and the morphology of GFAP-positive cells in the amygdalin / glycyrrhizic acid self-assembled hydrogel group was significantly improved. Further statistical analysis results indicated that the number of GFAP-positive cells in the model group was significantly increased compared with the control group. *** p < 0.001 indicates that the traumatic brain injury model leads to a significant increase in activated GFAP-positive cells in the peri-injury cortex. Compared with the model group, neither the amygdalin solution group nor the glycyrrhizic acid hydrogel group significantly reduced activated GFAP-positive cells, indicating that a single component cannot significantly improve activated GFAP-positive cells. The number of GFAP-positive cells in the amygdalin / glycyrrhizic acid self-assembled hydrogel group was significantly lower than that in the model group. ***p < 0.001 indicates that the amygdalin / glycyrrhizic acid self-assembled hydrogel can significantly reduce the number of activated GFAP-positive cells, and the effect is better than that of amygdalin solution alone and glycyrrhizic acid hydrogel alone.

[0108] 3. The expression of interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor (TNF-α), and interleukin-10 (IL-10) in serum after traumatic brain injury was determined by enzyme-linked immunosorbent assay (ELISA):

[0109] (1) Serum sample collection: On the 3rd day after modeling, mice were anesthetized by intraperitoneal injection of 0.3% sodium pentobarbital, and blood was collected by enucleation. The serum was collected after centrifugation at 4°C and 3000 rpm for 15 minutes.

[0110] (2) Experimental Procedure: The experiment was conducted according to the instructions provided with the commercially available kits. Specific information for the enzyme-linked immunosorbent assay (ELISA) kits is as follows: IL-1β (MU30369, Bioswamp), IL-6 (MU30044, Bioswamp), TNF-α (MU30030, Bioswamp), and IL-10 (MU30055, Bioswamp). Finally, the absorbance (OD value) was measured at a wavelength of 450 nm.

[0111] Finally, the expression levels of various inflammatory factors in serum were statistically compared. The experimental results are shown in the table below. Figure 11 , Figure 12 .

[0112] Figure 11 (a) This figure shows the effects of the control group, model group, amygdalin solution, amygdalin / glycyrrhizic acid self-assembled hydrogel, and glycyrrhizic acid hydrogel on the serum pro-inflammatory factor IL-1β level induced by traumatic brain injury. Figure 11 Statistical results showed that, compared with the control group, the expression of IL-1β in the model group was significantly increased ( *** p < 0.001 indicates that the traumatic brain injury model leads to a significant increase in serum pro-inflammatory factor IL-1β; compared with the model group, the expression of IL-1β in the amygdalin solution, amygdalin / glycyrrhizic acid self-assembled hydrogel, and glycyrrhizic acid hydrogel groups was significantly reduced (p < 0.001, respectively). *** p < 0.001 *** p < 0.001 *** p < 0.001 indicates that amygdalin solution, amygdalin / glycyrrhizic acid self-assembled hydrogel, and glycyrrhizic acid hydrogel can inhibit the expression level of the pro-inflammatory factor IL-1β in serum after traumatic brain injury; however, further analysis showed that compared with the amygdalin solution group, the expression of IL-1β in the amygdalin / glycyrrhizic acid self-assembled hydrogel group was significantly reduced. ***p < 0.001, indicating that the amygdalin / glycyrrhizic acid self-assembled hydrogel was more effective than the amygdalin solution alone; compared with the glycyrrhizic acid hydrogel group, the amygdalin / glycyrrhizic acid self-assembled hydrogel group showed a more significant decrease in IL-1β expression. *** p < 0.001 indicates that the amygdalin / glycyrrhizic acid self-assembled hydrogel is more effective than glycyrrhizic acid hydrogel alone.

[0113] Figure 11 (b) The effects of the control group, model group, amygdalin solution, amygdalin / glycyrrhizic acid self-assembled hydrogel, and glycyrrhizic acid hydrogel on the serum pro-inflammatory factor IL-6 level induced by traumatic brain injury. Figure 12 Statistical results showed that, compared with the control group, IL-6 expression was significantly increased in the model group. *** p < 0.001 indicates that the traumatic brain injury model leads to a significant increase in serum pro-inflammatory factor IL-6; compared with the model group, the expression of IL-6 in the amygdalin solution, amygdalin / glycyrrhizic acid self-assembled hydrogel, and glycyrrhizic acid hydrogel groups was significantly reduced (p < 0.001, respectively). *** p < 0.001 *** p < 0.001 *** p < 0.001 indicates that amygdalin solution, amygdalin / glycyrrhizic acid self-assembled hydrogel, and glycyrrhizic acid hydrogel can inhibit the expression level of the pro-inflammatory factor IL-6 in serum after traumatic brain injury; however, further analysis showed that compared with the amygdalin solution group, the amygdalin / glycyrrhizic acid self-assembled hydrogel group had a more significantly reduced IL-6 expression. *** p < 0.001, indicating that the amygdalin / glycyrrhizic acid self-assembled hydrogel was more effective than the amygdalin solution alone; compared with the glycyrrhizic acid hydrogel group, the amygdalin / glycyrrhizic acid self-assembled hydrogel group showed a more significant decrease in IL-6 expression. ** p < 0.01 indicates that the amygdalin / glycyrrhizic acid self-assembled hydrogel is more effective than glycyrrhizic acid hydrogel alone.

[0114] Figure 12 (a) The effects of the control group, model group, amygdalin solution, amygdalin / glycyrrhizic acid self-assembled hydrogel, and glycyrrhizic acid hydrogel on the serum pro-inflammatory factor TNF-α level induced by traumatic brain injury. Figure 13 Statistical results showed that, compared with the control group, the expression of TNF-α in the model group was significantly increased. ***p < 0.001 indicates that the traumatic brain injury model leads to a significant increase in serum pro-inflammatory factor TNF-α. Compared with the model group, the expression levels of TNF-α in the amygdalin solution and glycyrrhizic acid hydrogel groups showed a decreasing trend, but this was not statistically significant, indicating that amygdalin solution and glycyrrhizic acid hydrogel could not significantly inhibit the expression level of serum pro-inflammatory factor TNF-α after traumatic brain injury. However, the expression level of TNF-α in the amygdalin / glycyrrhizic acid self-assembled hydrogel group was significantly lower than that in the model group. ** p < 0.01, indicating that the amygdalin / glycyrrhizic acid self-assembled hydrogel can significantly reduce the expression level of TNF-α, and the effect is better than that of amygdalin solution alone and glycyrrhizic acid hydrogel alone.

[0115] Figure 12 (b) The effects of the control group, model group, amygdalin solution, amygdalin / glycyrrhizic acid self-assembled hydrogel, and glycyrrhizic acid hydrogel on the serum anti-inflammatory factor IL-10 level induced by traumatic brain injury. Figure 14 Statistical results showed that, compared with the control group, IL-10 expression in the model group was significantly reduced. *** p < 0.001 indicates that the traumatic brain injury model leads to a significant reduction in serum anti-inflammatory factor IL-10; compared with the model group, the expression of IL-10 in the amygdalin solution, amygdalin / glycyrrhizic acid self-assembled hydrogel, and glycyrrhizic acid hydrogel groups was significantly increased (p < 0.001, respectively). *** p < 0.001 *** p < 0.001 *** p < 0.001 indicates that amygdalin solution, amygdalin / glycyrrhizic acid self-assembled hydrogel, and glycyrrhizic acid hydrogel can promote the expression level of the anti-inflammatory factor IL-10 in serum after traumatic brain injury; however, further analysis showed that the expression of IL-10 in the amygdalin / glycyrrhizic acid self-assembled hydrogel group was significantly increased compared with that in the amygdalin solution group. *** p < 0.001, indicating that the amygdalin / glycyrrhizic acid self-assembled hydrogel was superior to the amygdalin solution alone; compared with the glycyrrhizic acid hydrogel group, the amygdalin / glycyrrhizic acid self-assembled hydrogel group showed a more significant increase in IL-10 expression. *** p < 0.001 indicates that the amygdalin / glycyrrhizic acid self-assembled hydrogel is more effective than glycyrrhizic acid hydrogel alone.

[0116] Therefore, the amygdalin / glycyrrhizic acid self-assembled hydrogel in Example 1 can be administered locally via in situ injection after debridement of traumatic brain injury, allowing for timely application to the inflammatory microenvironment caused by the injury. The synergistic effect of the two active ingredients significantly reduces the number of activated microglia and astrocytes compared to amygdalin solution or glycyrrhizic acid hydrogel alone, effectively mitigating the post-injury inflammatory cascade. Simultaneously, compared to amygdalin solution or glycyrrhizic acid hydrogel alone, the amygdalin / glycyrrhizic acid self-assembled hydrogel enhances the inhibitory effect on pro-inflammatory factors (IL-1β, IL-6, TNF-α) and the promoting effect on anti-inflammatory factor (IL-10) after injury, thereby enhancing its anti-inflammatory effect. This has positive practical significance for its development into a new clinical drug.

[0117] Example 3

[0118] The application of the amygdalin / glycyrrhizic acid self-assembled hydrogel of the present invention in the preparation of a medicament for alleviating or treating neuronal damage encephalopathy, specifically the application of the medicament in treating neuronal damage caused by traumatic brain injury, includes the following steps:

[0119] 1. Construct a mouse model of traumatic brain injury following the procedures outlined in Part 1 of Example 2. The specific experimental groups are as follows:

[0120] Control group: Bone window was opened and bone flap was removed, no traumatic impact was performed, and disinfection and suturing were performed.

[0121] Model group: Bone window was opened and bone flap was removed, while trauma was treated, disinfected and sutured.

[0122] Amygdalin solution group (Comparative Example 1): A bone window was opened and the bone flap was removed. Simultaneously, traumatic impact was performed, and amygdalin solution was injected in situ into the injured area. The area was then disinfected and sutured. The concentration of amygdalin in the amygdalin solution used in this example was 15 mg / mL.

[0123] Amygdalin / glycyrrhizic acid self-assembled hydrogel assembly (Example 1): A bone window was opened and the bone flap was removed. Simultaneously, traumatic impact was performed, and the amygdalin / glycyrrhizic acid self-assembled hydrogel was injected in situ into the injured area. The area was then disinfected and sutured. In this example, the concentrations of amygdalin and glycyrrhizic acid in the amygdalin / glycyrrhizic acid self-assembled hydrogel were 15 mg / mL and 15 mg / mL, respectively.

[0124] Glycyrrhizic acid hydrogel group (Comparative Example 2): A bone window was opened and the bone flap was removed. Simultaneously, traumatic impact was performed, and glycyrrhizic acid hydrogel was injected in situ into the injured area. The area was then disinfected and sutured. The concentration of glycyrrhizic acid in the glycyrrhizic acid hydrogel used in this example was 15 mg / mL.

[0125] 2. Nissl staining was used to detect the state of neurons in the damaged cortical area and the number of surviving neurons in different groups of mice after traumatic brain injury:

[0126] Brain tissue collection was completed according to Part 2(1) of Example 2. Experimental procedure: After fixing brain tissue samples with 4% paraformaldehyde solution for 24 hours, 3μm thick coronal brain tissue sections were prepared using standard paraffin embedding and sectioning procedures. Sections of the same coronal plane level from each group were selected, baked in a 60℃ oven for 1 hour, and then processed according to the following procedure: dewaxing with xylene solutions I and II for 10 min each (with moderate shaking during the process) → gradient ethanol hydration (100%, 100%, 95%, 75%, 5 min each) → rinsing three times with phosphate buffer (1×PBS, pH=7.4) (5 min each time). Nissl stain (G1036-100ML, Servicebio) was heated in a water bath at 60℃, and then the sections were placed in the water for staining for 10 min. After rinsing the sections clean, they were mounted with neutral resin and observed and photographed under a microscope.

[0127] Finally, Nissl staining images were collected and the number of surviving neurons in the damaged cortical area was counted and compared. The experimental results are shown in […]. Figure 13 .

[0128] Figure 13 The left side shows Nissl stained tissue sections of the control group, model group, amygdalin solution, amygdalin / glycyrrhizic acid self-assembled hydrogel, and glycyrrhizic acid hydrogel. The right side shows the statistical results of the number of surviving neurons in each group. Figure 13 As shown, in Nissl stained tissue sections, neurons in the brain cortex region of the model group exhibited dissolved / ruptured Nissl bodies and shrunken nucleoli. This abnormal neuronal morphology was significantly improved in the amygdalin / glycyrrhizic acid self-assembled hydrogel group. Further statistical results on the number of surviving neurons indicated that the number of surviving neurons in the model group was significantly reduced compared to the control group. *** p < 0.001 indicates that traumatic brain injury leads to severe neuronal damage. Compared with the model group, although the number of surviving neurons in the amygdalin solution group and the glycyrrhizic acid hydrogel group showed an increasing trend, there was no statistically significant difference, suggesting that amygdalin solution alone or glycyrrhizic acid hydrogel alone did not significantly improve neuronal damage. However, the number of surviving neurons in the amygdalin / glycyrrhizic acid self-assembled hydrogel group was significantly increased compared with the model group. *** p < 0.001, indicating that the amygdalin / glycyrrhizic acid self-assembled hydrogel can significantly improve neuronal damage after brain injury, and the effect is better than that of amygdalin solution alone and glycyrrhizic acid hydrogel alone.

[0129] Example 4

[0130] The application of the amygdalin / glycyrrhizic acid self-assembled hydrogel of the present invention in the preparation of a medicament for alleviating or treating blood-brain barrier disruption diseases, specifically in the application of a medicament for treating blood-brain barrier disruption caused by traumatic brain injury, includes the following steps:

[0131] 1. Construct a mouse model of traumatic brain injury following the procedures outlined in Part 1 of Example 2. The specific experimental groups are as follows:

[0132] Control group: Bone window was opened and bone flap was removed, no traumatic impact was performed, and disinfection and suturing were performed.

[0133] Model group: Bone window was opened and bone flap was removed, while trauma was treated, disinfected and sutured.

[0134] Amygdalin / glycyrrhizic acid self-assembled hydrogel assembly: A bone window was opened and the bone flap was removed. Simultaneously, traumatic impact was performed, and the amygdalin / glycyrrhizic acid self-assembled hydrogel was injected in situ into the injured area for drug administration. The area was then disinfected and sutured. In this example, the concentrations of amygdalin and glycyrrhizic acid in the amygdalin / glycyrrhizic acid self-assembled hydrogel were 15 mg / mL and 15 mg / mL, respectively.

[0135] 2. IgG staining was used to detect the disruption of the blood-brain barrier in mice after traumatic brain injury:

[0136] Experimental Procedure: Following the steps in Part 2 (1) and (2) of Example 2, brain tissue samples were fixed in 4% paraformaldehyde solution for 24 hours, and then 3μm thick coronal brain tissue sections were prepared using standard paraffin embedding and sectioning procedures. Sections from the same coronal plane level in each group were selected, baked in a 60℃ oven for 1 hour, and then processed according to the following procedure: dewaxing with xylene solutions I and II for 10 min each (with moderate shaking during the process) → gradient ethanol hydration (100%, 100%, 95%, and 75% for 5 min each) → rinsing three times (5 min each) with phosphate buffer (1×PBS, pH=7.4). Heat-induced antigen retrieval was performed using a 1× sodium citrate buffer system (pH=6.0) (boiling continuously for 20 min). After washing with 1×PBS, the tissue was blocked with 5% bovine serum albumin at room temperature for 2 h, IgG was added to the tissue, and it was incubated overnight at 4℃. After washing with 1×PBS, the tissue was developed with DAB chromogenic solution, and finally mounted with neutral resin and observed under a microscope.

[0137] Finally, IgG staining images were collected and the average staining intensity of IgG in brain injuries was statistically compared. Experimental results are shown below. Figure 14 .

[0138] Figure 14 Images of IgG staining in the control group, model group, and amygdalin / glycyrrhizic acid self-assembled hydrogels, along with statistical results of the average IgG staining intensity, are shown on the left. The left side displays the IgG staining image, and the right side shows the average IgG staining intensity result. Figure 14 As shown, in IgG-stained tissue sections, the area surrounding brain injury in the model group exhibited larger and deeper IgG staining, while the area surrounding brain injury in the amygdalin / glycyrrhizic acid self-assembled hydrogel group showed smaller and less intense IgG staining. Further statistical analysis of the average IgG staining intensity showed that, compared to the control group, the average IgG staining intensity in the model group was significantly increased (…). *** p < 0.001 indicates that the blood-brain barrier in the injured area was significantly disrupted after traumatic brain injury, leading to increased IgG leakage; compared with the model group, the average staining intensity of IgG in the amygdalin / glycyrrhizic acid self-assembled hydrogel group was significantly reduced ( *** p < 0.001, which indicates that amygdalin / glycyrrhizic acid hydrogel can significantly reduce the degree of IgG leakage after traumatic brain injury, suggesting that amygdalin / glycyrrhizic acid hydrogel can effectively improve blood-brain barrier damage.

[0139] 3. The expression level of S100β in serum after traumatic brain injury was determined by enzyme-linked immunosorbent assay (ELISA):

[0140] Experimental procedure: Follow the steps in Part 3 (1) and (2) of Example 2, but replace the kit with S100β (MU30367, Bioswamp).

[0141] Finally, the expression levels of S100β in serum were statistically compared. Experimental results are shown below. Figure 15 .

[0142] Figure 15 This figure shows the effects of the control group, model group, and amygdalin / glycyrrhizic acid self-assembled hydrogel on serum S100β levels induced by traumatic brain injury. Figure 15 Statistical results showed that, compared with the control group, S100β expression was significantly increased in the model group. *** p < 0.001 indicates that the traumatic brain injury model leads to a significant increase in serum S100β, suggesting that blood-brain barrier disruption causes S100β leakage; compared with the model group, the expression level of S100β in the amygdalin solution and glycyrrhizic acid hydrogel groups was significantly reduced. *** p < 0.001, indicating that the amygdalin / glycyrrhizic acid self-assembled hydrogel can significantly reduce the expression of S100β in serum after traumatic brain injury, suggesting that the amygdalin / glycyrrhizic acid hydrogel can effectively improve blood-brain barrier disruption.

[0143] Therefore, the amygdalin / glycyrrhizic acid self-assembled hydrogel can be administered locally via in situ injection after debridement for traumatic brain injury. It can reduce IgG leakage in brain tissue and S100β expression in serum, indicating that the amygdalin / glycyrrhizic acid self-assembled hydrogel effectively reduces blood-brain barrier disruption after traumatic brain injury, thereby effectively reducing the entry of peripheral substances into the brain. This can help alleviate neuroinflammation and enhance the protective effect. It has positive practical significance for becoming a new clinical drug.

[0144] Example 5

[0145] The application of the amygdalin / glycyrrhizic acid self-assembled hydrogel of the present invention in the preparation of a medicament for alleviating or treating complement activation disorders, specifically in the application of a medicament for treating abnormal increases in complement molecule C3 caused by traumatic brain injury, includes the following steps:

[0146] 1. Construct a mouse model of traumatic brain injury following the procedures outlined in Part 1 of Example 2. The specific experimental groups are as follows:

[0147] Control group: Bone window was opened and bone flap was removed, no traumatic impact was performed, and disinfection and suturing were performed.

[0148] Model group: Bone window was opened and bone flap was removed, while trauma was treated, disinfected and sutured.

[0149] Amygdalin solution group (Comparative Example 1): A bone window was opened and the bone flap was removed. Simultaneously, traumatic impact was performed, and amygdalin solution was injected in situ into the injured area. The area was then disinfected and sutured. The concentration of amygdalin in the amygdalin solution used in this example was 15 mg / mL.

[0150] Amygdalin / glycyrrhizic acid self-assembled hydrogel assembly (Example 1): A bone window was opened and the bone flap was removed. Simultaneously, traumatic impact was performed, and the amygdalin / glycyrrhizic acid self-assembled hydrogel was injected in situ into the injured area. The area was then disinfected and sutured. In this example, the concentrations of amygdalin and glycyrrhizic acid in the amygdalin / glycyrrhizic acid self-assembled hydrogel were 15 mg / mL and 15 mg / mL, respectively.

[0151] Glycyrrhizic acid hydrogel group (Comparative Example 2): A bone window was opened and the bone flap was removed. Simultaneously, traumatic impact was performed, and glycyrrhizic acid hydrogel was injected in situ into the injured area. The area was then disinfected and sutured. The concentration of glycyrrhizic acid in the glycyrrhizic acid hydrogel used in this example was 15 mg / mL.

[0152] 2. The expression level of C3 in serum after traumatic brain injury was determined by enzyme-linked immunosorbent assay (ELISA):

[0153] Experimental procedure: Follow the steps in Part 3 (1) and (2) of Example 2, but replace the reagent kit with C3 (MU30594, Bioswamp).

[0154] Finally, the expression levels of C3 in serum were statistically compared, and the experimental results are shown in [the table below]. Figure 16 .

[0155] Figure 16 The graph shows the effects of control group, model group, amygdalin solution, glycyrrhizic acid hydrogel, and amygdalin / glycyrrhizic acid self-assembled hydrogel on serum complement C3 levels induced by traumatic brain injury. Figure 16 Statistical results showed that, compared with the control group, C3 expression was significantly increased in the model group. *** p < 0.001 indicates that the traumatic brain injury model leads to a significant increase in C3 levels in the brain injury area; compared with the model group, the C3 levels in the amygdalin solution, amygdalin / glycyrrhizic acid self-assembled hydrogel, and glycyrrhizic acid hydrogel groups were significantly decreased (p < 0.001, respectively). *** p < 0.001 *** p < 0.001 *** p < 0.001 indicates that amygdalin solution, amygdalin / glycyrrhizic acid self-assembled hydrogel, and glycyrrhizic acid hydrogel can reduce C3 expression levels after traumatic brain injury; however, further analysis showed that compared with the amygdalin solution group, the amygdalin / glycyrrhizic acid self-assembled hydrogel group had a more significantly reduced C3 expression. *** p < 0.001, indicating that the amygdalin / glycyrrhizic acid self-assembled hydrogel was more effective than the amygdalin solution alone; compared with the glycyrrhizic acid hydrogel group, the amygdalin / glycyrrhizic acid self-assembled hydrogel group showed a more significant decrease in C3 expression. *** p < 0.001 indicates that the amygdalin / glycyrrhizic acid self-assembled hydrogel is more effective than glycyrrhizic acid hydrogel alone.

[0156] Therefore, the amygdalin / glycyrrhizic acid self-assembled hydrogel can be administered locally via in situ injection after debridement for traumatic brain injury. The two drug molecules synergistically enhance each other, significantly reducing C3 expression levels. Since C3 is a key central effector molecule for complement activation, this indicates that the amygdalin / glycyrrhizic acid self-assembled hydrogel effectively reduces C3 levels, thereby weakening complement system activation and alleviating the inflammatory cascade response caused by complement activation, thus enhancing the protective effect. This has positive implications for reducing postoperative complement system activation.

[0157] This invention relates to amygdalin / glycyrrhizic acid self-assembled hydrogel. When used in conjunction with debridement after traumatic brain injury, it allows for direct local injection of medication immediately after debridement, providing three-dimensional network support to the injured area. This enables early intervention and treatment of the disease, effectively reducing C3 expression levels after traumatic brain injury, thereby inhibiting complement activation, enhancing anti-inflammatory efficacy, reducing blood-brain barrier damage, avoiding systemic toxic side effects, and ultimately promoting nerve repair after injury, exerting an overall protective effect. This provides an intraoperative adjuvant drug intervention strategy for early surgical intervention of traumatic brain injury.

[0158] Example 6

[0159] Biosafety verification of the amygdalin / glycyrrhizic acid self-assembled hydrogel of the present invention in the treatment of traumatic brain injury includes the following steps:

[0160] 1. Construct a mouse model of traumatic brain injury following the procedures outlined in Part 1 of Example 2. The specific experimental groups are as follows:

[0161] Control group: Bone window was opened and bone flap was removed, no traumatic impact was performed, and disinfection and suturing were performed.

[0162] Model group: Bone window was opened and bone flap was removed, while trauma was treated, disinfected and sutured.

[0163] Amygdalin / glycyrrhizic acid self-assembled hydrogel assembly (Example 1): A bone window was opened and the bone flap was removed. Simultaneously, traumatic impact was performed, and the amygdalin / glycyrrhizic acid self-assembled hydrogel was injected in situ into the injured area. The area was then disinfected and sutured. In this example, the concentrations of amygdalin and glycyrrhizic acid in the amygdalin / glycyrrhizic acid self-assembled hydrogel were 15 mg / mL and 15 mg / mL, respectively.

[0164] 2. The biosafety of major organs in mice after traumatic brain injury was observed using hematoxylin-eosin staining:

[0165] (1) Collection of major organ tissue samples: On the third day after modeling, mice were anesthetized by intraperitoneal injection of 0.3% sodium pentobarbital, and then perfused with 0.9% saline via the heart. Finally, intact heart, liver, spleen, lung, kidney, intestine and testis tissues were dissected and fixed in 4% paraformaldehyde.

[0166] (2) Experimental Procedure: After fixing tissue samples with 4% paraformaldehyde solution for 24 hours, 3μm thick tissue sections were prepared using standard paraffin embedding and sectioning procedures. After baking in a 60℃ oven for 1 hour, the sections were processed as follows: dewaxing with xylene solutions I and II for 10 min each (with moderate shaking during the process) → gradient ethanol hydration (100%, 100%, 95%, and 75% for 5 min each) → rinsing three times (5 min each) with phosphate buffer (1×PBS, pH=7.4). The sections were then stained in hematoxylin staining solution (G1004-500ML, Servicebio) for 30–60 s, rinsed with water, differentiated in differentiation solution for 5 s, rinsed again, and then stained in eosin staining solution (G1001-500ML, Servicebio) for 10–30 s. Finally, the sections were mounted with neutral resin, observed and photographed under a microscope.

[0167] Figure 17These are images showing the pathological staining results of the control group, model group, and amygdalin / glycyrrhizic acid self-assembled hydrogel on the heart, liver, spleen, lung, kidney, intestine, and testis after traumatic brain injury. Figure 17 The results of hematoxylin-eosin staining showed that in the amygdalin / glycyrrhizic acid self-assembled hydrogel group, the tissue sections of the heart, liver, spleen, lung, kidney, intestine and testis all showed normal pathological structures and no obvious pathological changes. This indicates that the amygdalin / glycyrrhizic acid self-assembled hydrogel did not cause significant toxic damage to the organs after traumatic brain injury.

[0168] 3. Blood biochemistry was used to detect the levels of liver and kidney function indicators in mice after traumatic brain injury:

[0169] Serum samples were collected according to Part 3 (1) of Example 2, and the contents of albumin, alanine aminotransferase, aspartate aminotransferase, total bilirubin, blood urea nitrogen, creatinine, and urea nitrogen were detected using a fully automated biochemical analyzer.

[0170] Figure 18 This figure shows the expression results of liver and kidney function indicators after traumatic brain injury in the control group, model group, and amygdalin / glycyrrhizic acid self-assembled hydrogel. Figure 18 As shown, compared with the control group, the levels of albumin, alanine aminotransferase, aspartate aminotransferase, total bilirubin, blood urea nitrogen, creatinine, and urea nitrogen in the model group did not change significantly. Compared with the model group, the levels of albumin, alanine aminotransferase, aspartate aminotransferase, total bilirubin, blood urea nitrogen, creatinine, and urea nitrogen in the amygdalin / glycyrrhizic acid self-assembled hydrogel group did not change significantly. This suggests that the amygdalin / glycyrrhizic acid self-assembled hydrogel did not cause significant blood biochemical toxicity to organs after traumatic brain injury.

[0171] Therefore, the amygdalin / glycyrrhizic acid self-assembled hydrogel can be administered locally via in situ injection after debridement of traumatic brain injury. Furthermore, during treatment, it did not cause abnormal pathological changes in the organs (heart, liver, spleen, lungs, kidneys, intestines, and testes) or abnormal blood biochemical changes in liver and kidney function indicators (albumin, alanine aminotransferase, aspartate aminotransferase, total bilirubin, blood urea nitrogen, creatinine, and urea nitrogen) in mice. This demonstrates that the amygdalin / glycyrrhizic acid self-assembled hydrogel has excellent biosafety, which is of practical significance for promoting clinical translation and application.

[0172] While the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the invention. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the scope of the present invention. Therefore, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the scope of the present invention, should fall within the protection scope of the present invention.

Claims

1. The use of an amygdalin / glycyrrhizic acid self-assembled hydrogel in the preparation of a medicament for alleviating or treating traumatic brain injury, characterized in that, The drug is used to alleviate or treat traumatic brain injury by inhibiting complement activation; The preparation of the amygdalin / glycyrrhizic acid self-assembled hydrogel includes the following steps: mixing and dispersing amygdalin solution and glycyrrhizic acid evenly, and allowing it to stand to obtain the amygdalin / glycyrrhizic acid self-assembled hydrogel; the concentration of the amygdalin solution is 0.015-0.040 g / mL, and the concentration of glycyrrhizic acid in the amygdalin / glycyrrhizic acid self-assembled hydrogel is 0.015-0.040 g / mL; The steps involved in mixing and dispersing the amygdalin solution and glycyrrhizic acid evenly are as follows: A1. Mix amygdalin and PBS solution, and disperse evenly by ultrasonication to obtain amygdalin solution; A2. Mix amygdalin solution and glycyrrhizic acid, and then ultrasonically disperse the mixture at 40–70°C until it is clear and transparent. After standing, amygdalin / glycyrrhizic acid self-assembled hydrogel is obtained.

2. The application according to claim 1, characterized in that, The mass ratio of amygdalin to glycyrrhizic acid is 1:0.5 to 1:

2.

3. The application according to claim 1, characterized in that, In step A1 or step A2, the ultrasonic dispersion power is 70-120 W, and the ultrasonic dispersion time is 1-10 minutes.

4. The application according to claim 1, characterized in that: The complement molecule in complement activation is C3, the core effector molecule in the complement pathway.

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