Amygdalin / glycyrrhizic acid self-assembled hydrogel as well as preparation method and application thereof

Abstract

The invention discloses an amygdalin / glycyrrhizic acid self-assembled hydrogel and a preparation method and application thereof, and the preparation method comprises the following steps: mixing an amygdalin solution and glycyrrhizic acid, uniformly dispersing, and standing to obtain the amygdalin / glycyrrhizic acid self-assembled hydrogel; the concentration of the amygdalin solution is 0.015 to 0.040 g / mL, and the concentration of the glycyrrhizic acid in the amygdalin / glycyrrhizic acid self-assembled hydrogel is 0.015 to 0.040 g / mL. The micro-morphology of the amygdalin / glycyrrhizic acid self-assembled hydrogel is of a three-dimensional porous network structure, the amygdalin / glycyrrhizic acid self-assembled hydrogel can be used for preparing drugs for relieving or treating inflammatory encephalopathy, neuronal damage encephalopathy, blood-brain barrier damage diseases and complement activation diseases, and the amygdalin / glycyrrhizic acid self-assembled hydrogel is simple and safe to prepare and aims at complement activation inhibition.

Description

Technical Field

[0001] The present invention relates to the technical field of hydrogels, in particular to a laetrile / glycyrrhizic acid self-assembly hydrogel and a preparation method and application thereof. Background Art

[0002] Traumatic brain injury refers to a syndrome of neurological impairment caused by external impact, often accompanied by multiple pathological changes such as cranial brain tissue damage and intracranial hemorrhage. Neuroinflammation in the early stage of trauma is a key factor leading to long-term and extensive secondary damage, and is positively correlated with the occurrence of long-term chronic neurodegenerative diseases. A number of clinical and basic studies have confirmed that timely inhibition of neuroinflammation is an important and controllable key driving factor in reducing secondary damage after brain injury. However, existing anti-inflammatory drugs have failed to achieve the expected therapeutic effect in current multicenter clinical trials. At present, the early treatment of traumatic brain injury mainly focuses on surgical intervention to limit hematoma expansion and supportive treatment, but there are few measures to administer drugs in situ in the brain injury area in the early stage to suppress the inflammatory storm.

[0003] Activation of the complement system in the peri-injured area of the brain is considered an early determinant of neuroinflammatory responses and functional decline after traumatic brain injury. Early inhibition of complement activation has also been shown to reduce neuroinflammation and exert neuroprotective effects. In addition, studies have shown that surgical injury can cause a surge in injury-related pattern molecules, which in turn activates the complement system unrelated to the primary injury and triggers a postoperative inflammatory response. The complement molecule C3 has been pointed out as a central effector molecule regulating the complement system. Multiple preclinical experiments have confirmed that C3 inhibition can effectively reduce complement activation after brain injury, thereby reducing neuroinflammation and improving neurological function. However, there is currently no integrated therapeutic drug that integrates in situ administration after debridement of traumatic brain injury to regulate C3 expression and then inhibit complement activation.

[0004] Amygdalin is the main active ingredient of traditional Chinese medicine apricot kernel, mainly found in the seeds of Rosaceae plants, with the molecular formula C 20 H 27 NO 11 (Molecular weight 457.43). It exhibits multiple pharmacological activities, including anti-inflammatory, anti-tumor, anti-fibrotic, immune-modulating, and anti-atherosclerotic activities, and has demonstrated efficacy in neuroinflammatory, cardiovascular, and cerebrovascular diseases. However, amygdalin's high water solubility precludes localized delivery, limiting its safety and clinical application.

[0005] Glycyrrhizic acid is a natural triterpenoid saponin product and the main active ingredient of the traditional Chinese medicine licorice. Its molecular formula is C 42 H 62 O 16(Molecular weight 822.93). Its chemical structure consists of one molecule of hydrophobic glycyrrhetinic acid linked to two molecules of hydrophilic glucuronic acid via a β-glycosidic bond. This gives glycyrrhizic acid its unique amphiphilic properties, enabling it to self-assemble into nanomaterials. It exhibits a wide range of pharmacological activities, including anti-inflammatory and immunomodulatory, antioxidant and cytoprotective, antiviral and anti-fibrotic, and anti-tumor activities.

[0006] Although amygdalin and glycyrrhizic acid, as single active ingredients, have demonstrated a degree of pharmacological activity in specific disease models, they still face significant challenges in addressing complex pathological microenvironments such as brain diseases. First, single drugs struggle to achieve synergistic effects in complex pathological environments. Second, traditional systemic routes of administration (such as oral or intravenous injection) are limited by significant first-pass effects, widespread non-target tissue distribution, and a shortened drug half-life, resulting in insufficient local effective concentrations and potential systemic toxicity.

[0007] Therefore, developing in situ drug delivery systems based on biomaterials and optimizing the spatiotemporal distribution of drugs through component synergy has become a key strategy to overcome the bottleneck in the treatment of complex diseases. Furthermore, the development of multi-component self-assembled hydrogels containing all-natural bioactive ingredients that can inhibit complement activation and be used for local delivery after debridement is urgently needed. Summary of the Invention

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

[0009] In order to solve the above technical problems, the present invention adopts the following technical solutions:

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

[0011] In certain 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 close 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 certain 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 uniformly dispersing the amygdalin solution and glycyrrhizic acid comprises the following steps:

[0015] A1, mixing amygdalin and PBS solution, and uniformly dispersing them by ultrasonication to obtain amygdalin solution;

[0016] A2, mixing the amygdalin solution and glycyrrhizic acid, ultrasonically dispersing the mixture at a temperature of 40-70° C. until the mixture becomes clear and transparent, and then allowing the mixture to stand to obtain amygdalin / glycyrrhizic acid self-assembled hydrogel.

[0017] Ultrasonic dispersion at a temperature of 40-70°C is beneficial for the dissolution of glycyrrhizic acid. If the temperature is at room temperature (25°C), glycyrrhizic acid will not dissolve fully, and the system will appear milky white and opaque. In certain embodiments of the present invention, for example, the temperature 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 to 120 W, and the ultrasonic dispersion time is 1 to 10 minutes. In certain embodiments of the present invention, for example, the ultrasonic dispersion time may 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 laetrile / glycyrrhizic acid self-assembled hydrogel is prepared by the above-mentioned preparation method. The microscopic morphology of the laetrile / glycyrrhizic acid self-assembled hydrogel presents a three-dimensional porous network structure.

[0020] A method for preparing amygdalin / glycyrrhizic acid self-assembled hydrogel or the use of the amygdalin / glycyrrhizic acid self-assembled hydrogel in preparing a drug for alleviating or treating inflammatory encephalopathy.

[0021] The drug for inflammatory encephalopathy can inhibit the expression of proinflammatory factors or promote the expression of anti-inflammatory factors, wherein the proinflammatory 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 inflammatory encephalopathy can inhibit the expression of microglial cell markers (ionized calcium binding adaptor molecule 1, Iba-1) or astrocyte markers (glial fibrillary acidic protein, GFAP).

[0023] The inflammatory encephalopathy is any one or more of brain trauma, cerebral stroke, Alzheimer's disease, and Parkinson's disease.

[0024] A method for preparing amygdalin / glycyrrhizic acid self-assembled hydrogel or the use of the amygdalin / glycyrrhizic acid self-assembled hydrogel in preparing a drug for alleviating or treating neuronal damage encephalopathy.

[0025] The drug for neuron-damaging encephalopathy can increase the number of surviving neurons after brain injury.

[0026] A self-assembled amygdalin / glycyrrhizic acid hydrogel prepared by the aforementioned preparation method or the use of the aforementioned self-assembled amygdalin / glycyrrhizic acid hydrogel in the preparation of a drug for alleviating or treating diseases of blood-brain barrier destruction.

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

[0028] A self-assembled amygdalin / glycyrrhizic acid hydrogel prepared by the aforementioned preparation method or the use of the aforementioned self-assembled amygdalin / glycyrrhizic acid hydrogel in the preparation of a drug for alleviating or treating complement activation diseases.

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

[0030] The medicine for complement activation diseases is a local injection preparation.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[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 preparation raw materials are derived from Chinese herbal medicines and are natural product molecules that can self-assemble into hydrogels. This method, without the participation of exogenous organic solvents, metal ions, or polymer compounds, maximizes the biosafety of the hydrogel. Secondly, the preparation process does not require complex steps, is simple to operate, and is easy to scale up for production. This makes the hydrogel have broad prospects in tissue engineering applications in the biomedical field.

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

[0034] (3) The application of the amygdalin / glycyrrhizic acid self-assembled hydrogel of the present invention in the preparation of drugs for alleviating or treating inflammatory encephalopathy, neuronal damage encephalopathy, blood-brain barrier damage diseases, and complement activation diseases has anti-inflammatory, blood-brain barrier damage 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 the anti-inflammatory factor interleukin 10 (IL-10), reduces blood-brain barrier leakage, improves neurological function, and inhibits the expression of complement molecules. It has important practical significance for becoming a new clinical drug. BRIEF DESCRIPTION OF THE DRAWINGS

[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 This is a graph showing the rheological test results of the amygdalin / glycyrrhizic acid self-assembled hydrogel in Example 1 of the present invention.

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

[0039] Figure 5This is a 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 ultraviolet 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 of the amygdalin / glycyrrhizic acid self-assembled hydrogel in Example 2 of the present invention before and after in situ injection after debridement of traumatic brain injury.

[0043] Figure 9 These are the fluorescence images and statistical results of microglia induced by the control group, model group, amygdalin / glycyrrhizic acid self-assembled hydrogel, glycyrrhizic acid hydrogel, and amygdalin solution in Example 2 of the present invention on mice with traumatic brain injury constructed using a controlled cortical injury apparatus.

[0044] Figure 10 These are the fluorescence images and statistical results of astrocytes induced by the control group, model group, amygdalin / glycyrrhizic acid self-assembled hydrogel, glycyrrhizic acid hydrogel, and amygdalin solution in Example 2 of the present invention in mice with traumatic brain injury constructed using a controlled cortical injury apparatus.

[0045] Figure 11 This is a graph showing the effects of the control group, model group, amygdalin / glycyrrhizic acid self-assembled hydrogel, glycyrrhizic acid hydrogel, and amygdalin solution in Example 2 of the present invention on IL-1β pro-inflammatory factors and IL-6 pro-inflammatory factors in mice with traumatic brain injury constructed in a controlled cortical injury apparatus.

[0046] Figure 12 This is a graph showing the effects of the control group, model group, amygdalin / glycyrrhizic acid self-assembled hydrogel, glycyrrhizic acid hydrogel, and amygdalin solution in Example 2 of the present invention on TNF-α pro-inflammatory factors and IL-10 anti-inflammatory factors in mice with traumatic brain injury in a controlled cortical injury apparatus.

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

[0048] Figure 14This is a graph showing the effects of the control group, model group, and amygdalin / glycyrrhizic acid self-assembled hydrogel on immunoglobulin IgG in mice with traumatic brain injury using a controlled cortical injury apparatus in Example 4 of the present invention.

[0049] Figure 15 This is a graph showing the effects of the control group, model group, and amygdalin / glycyrrhizic acid self-assembled hydrogel on the central nervous system-specific protein S100β in mice with traumatic brain injury caused by a controlled cortical injury apparatus in Example 4 of the present invention.

[0050] Figure 16 This figure shows the effects 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 using a controlled cortical injury apparatus in Example 5 of the present invention.

[0051] Figure 17 This is the effect of the control group, model group, and amygdalin / glycyrrhizic acid self-assembled hydrogel in Example 6 of the present invention on the biosafety of major organs in mice with traumatic brain injury constructed using a controlled cortical injury apparatus.

[0052] Figure 18 This is a graph showing 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 apparatus in Example 6 of the present invention. DETAILED DESCRIPTION

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

[0054] Example 1

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

[0056] (1) Preparation of amygdalin solution: 15 mg of amygdalin was added to 1 mL of PBS solution (pH = 7.4), and ultrasonicated 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 the amygdalin solution from step (1) to the glycyrrhizic acid. Ultrasonic dispersion is performed at 40-70°C (60°C in this embodiment) with an ultrasonic dispersion power of 70-120 W (100 W in this embodiment) for 1-5 minutes (1 minute in this embodiment). After complete dissolution, the solution is placed on a table and allowed to stand to obtain amygdalin / glycyrrhizic acid self-assembled hydrogel (referred to as amygdalin / glycyrrhizic acid hydrogel in the accompanying drawings).

[0058] Appearance photo of amygdalin / glycyrrhizic acid self-assembled hydrogel: After the preparation of the amygdalin / glycyrrhizic acid self-assembled hydrogel is completed, its appearance is photographed using a camera.

[0059] SEM sample preparation: Pipette 10 μL of the hydrogel sample onto a clean silicon wafer and immediately freeze it at -20°C. Then, use a vacuum freeze dryer to dry the sample and examine it with a SEM to obtain the internal morphology of the hydrogel.

[0060] Comparative Example 1

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

[0062] Comparative Example 2

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

[0064] Comparative Example 3

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

[0066] (1) Preparation of amygdalin solution: 10 mg of amygdalin was added to 1 mL of PBS solution (pH = 7.4), and ultrasonicated 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. Ultrasonic dispersion was performed at 40-70°C (60°C in this comparative example) with a power of 70-120 W (100 W in this comparative example) for 1-5 minutes (1 minute in this comparative example). After complete dissolution, the mixture was placed on a table and allowed to stand to obtain a mobile 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 laetrile / glycyrrhizic acid mobile liquid of this comparative example was prepared by the following method:

[0071] (1) Preparation of amygdalin solution: 8 mg of amygdalin was added to 1 mL of PBS solution (pH = 7.4), and ultrasonicated 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. Ultrasonic dispersion was performed at 40-70°C (60°C in this comparative example) with a power of 70-120 W (100 W in this comparative example) for 1-5 minutes (1 minute in this comparative example). After complete dissolution, the mixture was placed on a table and allowed to stand to obtain a mobile liquid with a concentration of 8 mg / mL amygdalin / glycyrrhizic acid.

[0073] This is because the concentration is lower and a hydrogel system with a stable structure cannot be formed.

[0074] Comparative Example 5

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

[0076] (1) Preparation of amygdalin solution: 4 mg of amygdalin was added to 1 mL of PBS solution (pH = 7.4), and ultrasonicated 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. Ultrasonic dispersion is performed at 40-70°C (60°C in this comparative example) with a power of 70-120 W (100 W in this comparative example) for 1-5 minutes (1 minute in this comparative example). After complete dissolution, place the mixture on a table and let it stand to obtain a liquid with a concentration of 4 mg / mL amygdalin / glycyrrhizic acid.

[0078] This is because the concentration is extremely low and a hydrogel system with a stable structure cannot be formed.

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

[0080] Fourier transform infrared spectroscopy sample preparation: 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 powder samples to be tested. The test process adopted the potassium bromide tableting method, in which the powder sample to be tested was fully ground with potassium bromide and then tableted. The test range was 4000-400 cm -1 The transmittance within the wavelength range is tested on the machine.

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

[0082] Sample preparation for X-ray diffraction spectroscopy: The amygdalin / glycyrrhizic acid self-assembled hydrogel prepared in Example 1 was freeze-dried to obtain a powder sample. The powder sample was thoroughly ground and tested using a copper target at a scanning speed of 10° / min and a scanning range of 10° to 90°.

[0083] Figure 1 These are photos of the appearance of the amygdalin / glycyrrhizic acid self-assembled hydrogel prepared in Example 1 and the amygdalin / glycyrrhizic acid flowing liquids 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. Figure 1 As shown in (a), the amygdalin / glycyrrhizic acid self-assembled hydrogel is a white, uniform 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, and do not have the ability to deliver 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 partial enlarged view of the box in Figure (a). Figure 2 As shown, the amygdalin / glycyrrhizic acid self-assembled hydrogel exhibits a microscopic morphology of a three-dimensional porous network structure. Amygdalin / glycyrrhizic acid self-assembled hydrogel is a type of three-dimensional mesh material with a high water content, which has a structure similar to soft tissue and soft and wet properties, local injectability, and pharmacodynamic properties. Drug molecule (traditional Chinese medicine small molecule) self-assembled hydrogel integrates the pharmacodynamic properties of drugs (100% drug loading and drug self-delivery) and material structural characteristics (three-dimensional network support and local injectability), and can optimize the traditional "drug-assisted synergy" model to a "drug-self-assisted synergy" model, that is, without the need for additional carrier loading, the drug molecules self-assemble and can 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 various traditional hydrogel systems prepared with polymer compounds as raw materials in the existing technology to achieve drug delivery as delivery carriers, but with low delivery efficiency, large toxic side effects, complex preparation procedures, and mismatch with brain tissue modulus.

[0085] Figure 3 The rheological test results of the amygdalin / glycyrrhizic acid self-assembled hydrogel in Example 1 are as follows: Figure 3 (a) is the strain scanning result diagram, Figure 3 (b) is the dynamic time scanning result, Figure 3 (c) is the gait scanning result diagram, Figure 3 (d) is the viscosity test result diagram. Figure 3 As shown, the strain sweep results show 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 transforms from a gel state to a sol state. The dynamic time sweep results show that G' always remains greater than G" throughout the test process, and its value remains relatively unchanged, which confirms the stability of the amygdalin / glycyrrhizic acid self-assembled hydrogel. The gait sweep results show that in repeated cycles from low strain to high strain, the amygdalin / glycyrrhizic acid self-assembled hydrogel can recover from the sol state to the gel state when transitioning from high strain to low strain. The viscosity test results show that the shear viscosity of the amygdalin / glycyrrhizic acid self-assembled hydrogel decreases with increasing shear rate, indicating that it has good shear thinning behavior. All these results indicate that the amygdalin / glycyrrhizic acid self-assembled hydrogel has good injectability and self-healing ability, and has great potential for clinical application.

[0086] Figure 4 This is a photo of the injectability of the amygdalin / glycyrrhizic acid self-assembled hydrogel in Example 1. Figure 4(a) is the in vitro injection state diagram of AG-gel, Figure 4 (b) is a schematic diagram of AG-gel injected into the brain tissue defect area, AG-gel represents the amygdalin / glycyrrhizic acid self-assembled hydrogel prepared in Example 1. Figure 4 As shown in the figure, AG-gel can be smoothly ejected 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 clinical application in situ drug delivery after surgical debridement.

[0087] Figure 5 This is the Fourier transform infrared spectrum of the amygdalin / glycyrrhizic acid self-assembled hydrogel in Example 1. The specific groups are as follows: Figure 5 As shown, the glycyrrhizic acid hydrogel has a 3425 cm -1 is the stretching vibration absorption peak of OH, 3406 cm in amygdalin solution -1 The stretching vibration absorption peak of OH shifts to 3417 cm in the amygdalin / glycyrrhizic acid self-assembled hydrogel. -1 , which indicates that there are intermolecular hydrogen bonds in the amygdalin / glycyrrhizic acid self-assembled hydrogel.

[0088] Figure 6 The UV spectrum of the amygdalin / glycyrrhizic acid self-assembled hydrogel in 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 in Figure 3, as the concentration of AG-gel was diluted, the UV spectrum showed a blue shift (from 219 nm to 210 nm), which indicated the existence of π-π stacking interactions during the assembly process of AG-gel.

[0089] Figure 7 This is the X-ray diffraction spectrum of the amygdalin / glycyrrhizic acid self-assembled hydrogel in Example 1. Figure 7 As shown, when the degree value is There is an obvious peak at , which indicates that there is a π-π stacking interaction during the assembly process of 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 invention discloses a method for preparing a drug for alleviating or treating inflammatory encephalopathy using the amygdalin / glycyrrhizic acid self-assembled hydrogel, specifically for treating inflammatory reactions caused by traumatic brain injury, comprising the following steps:

[0093] 1. Construction of Traumatic Brain Injury Mouse Model:

[0094] C57BL / 6 mice were used as experimental animals, and a traumatic brain injury model was established using a controlled cortical impact device. The specific procedure was as follows: the animals were anesthetized with an intraperitoneal injection of 0.3% sodium pentobarbital solution (220 μL / kg) and then positioned prone on a stereotaxic apparatus with the skull adjusted to a horizontal position. After routine skin preparation and disinfection, the surgical area was incised along the sagittal suture to expose the skull surface. Using the bregma as an anatomical landmark, a 4.0 mm diameter circular bone window was created using a microbone drill in the corresponding region of the right parietal cortex (coordinates: 2.0 mm posterior to the bregma and 2.0 mm right of the midline). Microtweezers were used to carefully remove the bone flap to expose the target brain region. Instrument parameters were set as follows: impact depth 1.5 mm, velocity 3.5 m / s, and duration 100 ms. After the impact, the surgical area was rigorously 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 without trauma, and the wound was disinfected and sutured.

[0097] Model group: bone window was opened and bone flap was removed, trauma was performed, and disinfection and suture were performed at the same time.

[0098] Amygdalin solution group (Comparative Example 1): A bone window was opened and the bone flap was removed. Traumatic impact was performed simultaneously. Amygdalin solution was injected in situ into the injured area, followed by disinfection and suture. The concentration of amygdalin in the amygdalin solution used in this example was 15 mg / mL.

[0099] Amygdalin / glycyrrhizic acid self-assembling hydrogel group (Example 1): A bone window was created and the bone flap was removed. Simultaneously, trauma was applied. The injured area was in situ injected with amygdalin / glycyrrhizic acid self-assembling hydrogel, followed by disinfection and suture. The concentrations of amygdalin and glycyrrhizic acid in the amygdalin / glycyrrhizic acid self-assembling hydrogel used in this example 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, trauma was performed, and the injured area was in situ injected with glycyrrhizic acid hydrogel, followed by disinfection and suture. The concentration of glycyrrhizic acid in the glycyrrhizic acid hydrogel used in this example was 15 mg / mL.

[0101] Figure 8This is a photo of the intervention of the amygdalin / glycyrrhizic acid self-assembled hydrogel in the traumatic brain injury mouse model in Example 1. Figure 8 (i) is a diagram of a mouse model of traumatic brain injury induced by CCI. Figure 8 (ii) is a diagram of local drug administration after debridement. Figure 8 As shown in the figure, after traumatic injury, the mouse brain tissue was damaged, resulting in injury and bleeding. After debridement, the amygdalin / glycyrrhizic acid self-assembled hydrogel was injected in situ to achieve in situ drug delivery to the brain injury area.

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

[0103] (1) Brain tissue sample collection: On day 3 after modeling, mice were anesthetized with 0.3% sodium pentobarbital via intraperitoneal injection and then perfused transcardially with 0.9% saline. Finally, the intact brain tissue was dissected and fixed in 4% paraformaldehyde.

[0104] (2) Experimental procedure: Brain tissue samples were fixed with 4% paraformaldehyde solution for 24 hours, and then 3 μm thick coronal brain tissue sections were prepared by standard paraffin embedding and sectioning procedures. Sections at the same coronal level in each group were selected and dried in a 60°C oven for 1 hour. Then, they were processed according to the following process: dewaxing in xylene solution I and II for 10 minutes each (moderate shaking during the process) → hydration in graded ethanol (100%, 100%, 95%, 75% for 5 minutes each) → rinsing in phosphate buffer (1×PBS, pH=7.4) 3 times (5 minutes / time). Heat-induced antigen retrieval was performed using 1× sodium citrate buffer system (pH=6.0) (continuous boiling for 20 minutes), and after washing with 1×PBS, the sections were blocked with 3% bovine serum albumin at room temperature for 1 hour. Incubation was performed with primary antibodies (rabbit, Boster Biological, BA0056, 1:400) or anti-Iba-1 (rabbit, CST, E404W, 1:400) at 4°C in a humidified chamber overnight. After rinsing with 1× PBS, fluorescently labeled secondary antibodies (Jackson ImmunoResearch, donkey anti-rabbit IgG, Alexa Fluor 488, 1:1000) were added and incubated for 1 hour at room temperature in the dark. After extensive washing with 1× PBS, nuclei were counterstained with DAPI staining solution (Solarbio, C0065, 1:100) for 10 minutes in the dark. After rinsing with 1× PBS, the sections were mounted with anti-fading 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 proportion of Iba-1 positive cells and GFAP positive cells in the cortical area around the brain injury were counted. The experimental results are shown in Figure 2. Figure 9 and Figure 10 .

[0106] Figure 9 The results of the effects of the control group, model group, amygdalin solution, amygdalin / glycyrrhizic acid self-assembled hydrogel, and glycyrrhizic acid hydrogel on Iba-1 levels caused by traumatic brain injury are shown in the figure below. The left side shows the fluorescence images of each group, and the right side shows the statistical analysis results. Figure 9 As shown in the figure, compared with the control group, the Iba-1 positive cells in the model group showed a swollen amoeboid morphology, and the Iba-1 positive cells in the amygdalin / glycyrrhizic acid self-assembled hydrogel group showed a significant improvement in morphology. Further statistical analysis results showed that compared with the control group, the number of Iba-1 positive cells in the model group increased significantly ( *** p<0.001), indicating that the traumatic brain injury model can lead to a significant increase in the activated Iba-1 positive cells in the cortex around the injury; compared with the model group, the amygdalin solution group and the glycyrrhizic acid hydrogel group failed to significantly reduce the activated Iba-1 positive cells, indicating that a single component cannot significantly improve the activated Iba-1 positive cells; and 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), indicating that amygdalin / glycyrrhizic acid self-assembled hydrogel can significantly reduce the activation 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 caused by traumatic brain injury are shown on the left, and the statistical analysis results are shown on the right. Figure 10 As shown in the figure, compared with the control group, the GFAP-positive cells in the model group became larger and the processes became thicker, and the GFAP-positive cells in the amygdalin / glycyrrhizic acid self-assembled hydrogel group showed a significant improvement in morphology. Further statistical analysis results showed that compared with the control group, the number of GFAP-positive cells in the model group increased significantly ( *** p<0.001), indicating that the traumatic brain injury model can lead to a significant increase in activated GFAP-positive cells in the cortex around the injury; compared with the model group, the amygdalin solution group and the glycyrrhizic acid hydrogel group failed to significantly reduce the number of activated GFAP-positive cells, indicating that a single component cannot significantly improve the number of activated GFAP-positive cells; and 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), indicating that amygdalin / glycyrrhizic acid self-assembled hydrogel can significantly reduce the activation of GFAP-positive cells, and the effect is better than that of amygdalin solution alone and glycyrrhizic acid hydrogel alone.

[0108] 3. Enzyme-linked immunosorbent assay was used to determine 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.

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

[0110] (2) Experimental Procedure: The experiments were performed according to the instructions provided with the commercially available kits. The specific enzyme-linked immunosorbent assay kits are as follows: IL-1β (MU30369, Bioswamp), IL-6 (MU30044, Bioswamp), TNF-α (MU30030, Bioswamp), and IL-10 (MU30055, Bioswamp). Finally, the absorbance (i.e., OD value) was measured at a wavelength of 450 nm.

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

[0112] Figure 11 (a) is the effect of the control group, model group, amygdalin solution, amygdalin / glycyrrhizic acid self-assembled hydrogel, and glycyrrhizic acid hydrogel on the level of pro-inflammatory factor IL-1β in serum caused by traumatic brain injury. Figure 11 The statistical results showed that compared with the control group, the expression of IL-1β in the model group increased significantly ( *** p<0.001), indicating that the traumatic brain injury model can lead to a significant increase in the pro-inflammatory factor IL-1β in serum; 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), indicating that amygdalin solution, amygdalin / glycyrrhizic acid self-assembled hydrogel and glycyrrhizic acid hydrogel can inhibit the expression level of pro-inflammatory factor IL-1β in serum after traumatic brain injury; however, further analysis showed that compared with the amygdalin solution group, the IL-1β expression in the amygdalin / glycyrrhizic acid self-assembled hydrogel group was more significantly reduced ( ***p<0.001), indicating that the effect of amygdalin / glycyrrhizic acid self-assembled hydrogel is better than that of amygdalin solution alone; compared with the glycyrrhizic acid hydrogel group, the expression of IL-1β in the amygdalin / glycyrrhizic acid self-assembled hydrogel group was significantly reduced ( *** p<0.001), indicating that the effect of amygdalin / glycyrrhizic acid self-assembled hydrogel is better than that of glycyrrhizic acid hydrogel alone.

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

[0114] Figure 12 (a) Effects of the control group, model group, amygdalin solution, amygdalin / glycyrrhizic acid self-assembled hydrogel, and glycyrrhizic acid hydrogel on the level of pro-inflammatory factor TNF-α in serum 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 increased significantly ( ***p<0.001), indicating that the traumatic brain injury model will lead to a significant increase in the pro-inflammatory factor TNF-α in the serum; compared with the model group, the expression levels of TNF-α in the amygdalin solution and glycyrrhizic acid hydrogel groups showed a downward trend, but there was no statistical significance, indicating that the amygdalin solution and glycyrrhizic acid hydrogel could not significantly inhibit the expression level of the pro-inflammatory factor TNF-α in the serum 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), which shows that 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) Effects of the control group, model group, amygdalin solution, amygdalin / glycyrrhizic acid self-assembled hydrogel, and glycyrrhizic acid hydrogel on the level of anti-inflammatory factor IL-10 in serum induced by traumatic brain injury. Figure 14 The statistical results showed that compared with the control group, the expression of IL-10 in the model group was significantly decreased ( *** p<0.001), indicating that the traumatic brain injury model can lead to a significant decrease in the anti-inflammatory factor IL-10 in serum; 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), indicating that amygdalin solution, amygdalin / glycyrrhizic acid self-assembled hydrogel and glycyrrhizic acid hydrogel can promote the expression level of anti-inflammatory factor IL-10 in serum after traumatic brain injury; however, further analysis showed that compared with the amygdalin solution group, the IL-10 expression in the amygdalin / glycyrrhizic acid self-assembled hydrogel group was more significantly increased ( *** p<0.001), indicating that the effect of amygdalin / glycyrrhizic acid self-assembled hydrogel is better than that of amygdalin solution alone; compared with the glycyrrhizic acid hydrogel group, the IL-10 expression in the amygdalin / glycyrrhizic acid self-assembled hydrogel group was significantly increased ( *** p<0.001), indicating that the effect of amygdalin / glycyrrhizic acid self-assembled hydrogel is better than that of glycyrrhizic acid hydrogel alone.

[0116] Therefore, in Example 1, amygdalin / glycyrrhizic acid self-assembly hydrogel can be injected in situ to achieve local administration after traumatic brain injury debridement, and is promptly applied to the inflammatory microenvironment caused by injury. The two active ingredients of the medicine work together to enhance the effect, and can well reduce the number of activated microglia and astrocytes compared with amygdalin solution and glycyrrhizic acid hydrogel, which effectively reduces the post-injury inflammatory cascade. Meanwhile, compared with amygdalin solution and glycyrrhizic acid hydrogel, amygdalin / glycyrrhizic acid self-assembly hydrogel can enhance the inhibitory effect of proinflammatory factors (IL-1β, IL-6, TNF-α) after injury and the promoting effect of anti-inflammatory factors (IL-10), so as to play an enhanced anti-inflammatory effect. It has positive practical significance to becoming a new clinical drug.

[0117] Example 3

[0118] The invention discloses a method for preparing a drug for alleviating or treating neuronal damage encephalopathy using the amygdalin / glycyrrhizic acid self-assembled hydrogel, specifically for treating neuronal damage caused by traumatic brain injury, comprising the following steps:

[0119] 1. Complete the construction of a traumatic brain injury mouse model according to the procedures 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 without trauma, and the wound was disinfected and sutured.

[0121] Model group: bone window was opened and bone flap was removed, trauma was performed, and disinfection and suture were performed at the same time.

[0122] Amygdalin solution group (Comparative Example 1): A bone window was opened and the bone flap was removed. Traumatic impact was performed simultaneously. Amygdalin solution was injected in situ into the injured area, followed by disinfection and suture. The concentration of amygdalin in the amygdalin solution used in this example was 15 mg / mL.

[0123] Amygdalin / glycyrrhizic acid self-assembling hydrogel group (Example 1): A bone window was created and the bone flap was removed. Simultaneously, trauma was applied. The injured area was in situ injected with amygdalin / glycyrrhizic acid self-assembling hydrogel, followed by disinfection and suture. The concentrations of amygdalin and glycyrrhizic acid in the amygdalin / glycyrrhizic acid self-assembling hydrogel used in this example 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, and trauma was performed simultaneously. The injured area was injected with glycyrrhizic acid hydrogel in situ, and 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 status of neurons in the damaged cortical area and the number of surviving neurons in each group of mice after traumatic brain injury:

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

[0127] Finally, Nissl-stained images were collected and the number of surviving neurons in the cortical area of brain injury 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, and 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 cortical region of the brain injury in the model group showed dissolved / ruptured Nissl bodies and shrunken nucleoli. This abnormal neuronal morphology was significantly improved in the amygdalin / glycyrrhizic acid self-assembled hydrogel group. The statistical results of the number of surviving neurons further showed that compared with the control group, the number of surviving neurons in the model group was significantly reduced ( *** p<0.001), indicating that traumatic brain injury can cause serious damage to neurons; compared with the model group, the number of surviving neurons in the amygdalin solution group and the glycyrrhizic acid hydrogel group showed an increasing trend, but there was no statistical difference, which suggests that amygdalin solution or glycyrrhizic acid hydrogel alone did not significantly improve neuronal damage; while 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), which shows that amygdalin / glycyrrhizic acid self-assembled hydrogel can significantly improve the damage of neurons after brain injury, and the effect is better than that of amygdalin solution alone and glycyrrhizic acid hydrogel alone.

[0129] Example 4

[0130] The invention discloses a method for preparing a drug for alleviating or treating blood-brain barrier damage, and specifically for treating blood-brain barrier damage caused by traumatic brain injury, comprising the following steps:

[0131] 1. Complete the construction of a traumatic brain injury mouse model according to the procedures 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 without trauma, and the wound was disinfected and sutured.

[0133] Model group: bone window was opened and bone flap was removed, trauma was performed, and disinfection and suture were performed at the same time.

[0134] In the Amygdalin / Glycyrrhizic Acid Self-Assembling Hydrogel Group, a bone window was created and the bone flap removed. Traumatic impact was then performed, and the injured area was in situ injected with the amygdalin / Glycyrrhizic Acid Self-Assembling Hydrogel. The injured area was disinfected and sutured. The concentrations of amygdalin and glycyrrhizic acid in the amygdalin / Glycyrrhizic Acid Self-Assembling Hydrogel used in this example were 15 mg / mL and 15 mg / mL, respectively.

[0135] 2. IgG staining to detect blood-brain barrier damage in each group of mice after traumatic brain injury:

[0136] Experimental operation steps: According to the steps of part 2 (1) (2) in Example 2, brain tissue samples were fixed with 4% paraformaldehyde solution for 24 hours, and then 3 μm thick coronal brain tissue sections were prepared by standard paraffin embedding and sectioning procedures. Slices at the same coronal level in each group were selected, and after being baked in a 60℃ oven for 1 hour, they were processed according to the following process: dewaxing in xylene solution I and II for 10 minutes each (moderate shaking during the period) → hydration with gradient ethanol (100%, 100%, 95%, 75% each for 5 minutes) → rinsing with phosphate buffer (1×PBS, pH=7.4) 3 times (5 minutes / time). Heat-induced antigen retrieval was performed using 1× sodium citrate buffer system (pH=6.0) (continuous boiling for 20 minutes). After washing with 1×PBS, the sections were blocked with 5% bovine serum albumin at room temperature for 2 hours. IgG was added to the tissue and incubated at 4℃ overnight. After washing with 1×PBS, the sections were stained with DAB colorimetric solution, and finally sealed with neutral gum and observed under a microscope.

[0137] Finally, IgG staining images were collected and statistically compared with the average IgG staining intensity in brain injury. Figure 14 .

[0138] Figure 14 The IgG staining images and statistical results of the average IgG staining intensity of the control group, model group, and amygdalin / glycyrrhizic acid self-assembled hydrogel are shown on the left, and the average IgG staining intensity is shown on the right. Figure 14 As shown in the IgG staining tissue sections, the area around the brain injury in the model group showed a larger area and darker staining of IgG staining results, while the area around the brain injury in the amygdalin / glycyrrhizic acid self-assembled hydrogel group showed a smaller area and earlier staining of IgG staining results. Further statistical results of the average IgG staining intensity showed that: compared with the control group, the average IgG staining intensity of the model group increased significantly ( *** p<0.001), indicating that the blood-brain barrier in the injured area was significantly damaged after traumatic brain injury, resulting in increased IgG leakage; compared with the model group, the average IgG staining intensity in the amygdalin / glycyrrhizic acid self-assembled hydrogel group was significantly reduced ( *** p<0.001), which shows 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. ELISA was used to determine the expression level of S100β in serum after traumatic brain injury:

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

[0141] Finally, the expression levels of S100β in serum were statistically compared. Figure 15 .

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

[0143] Therefore, the amygdalin / glycyrrhizic acid self-assembled hydrogel can be locally administered via in situ injection after debridement of traumatic brain injury, reducing IgG leakage from brain tissue and S100β expression in serum. This suggests that the amygdalin / glycyrrhizic acid self-assembled hydrogel effectively mitigates 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 protective effects, which has positive practical significance for becoming a new clinical drug.

[0144] Example 5

[0145] The invention discloses a method for preparing a drug for alleviating or treating complement activation diseases, and specifically for treating abnormal increase of complement molecule C3 caused by traumatic brain injury, comprising the following steps:

[0146] 1. Complete the construction of a traumatic brain injury mouse model according to the procedures 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 without trauma, and the wound was disinfected and sutured.

[0148] Model group: bone window was opened and bone flap was removed, trauma was performed, and disinfection and suture were performed at the same time.

[0149] Amygdalin solution group (Comparative Example 1): A bone window was opened and the bone flap was removed. Traumatic impact was performed simultaneously. Amygdalin solution was injected in situ into the injured area, followed by disinfection and suture. The concentration of amygdalin in the amygdalin solution used in this example was 15 mg / mL.

[0150] Amygdalin / glycyrrhizic acid self-assembling hydrogel group (Example 1): A bone window was created and the bone flap was removed. Simultaneously, trauma was applied. The injured area was in situ injected with amygdalin / glycyrrhizic acid self-assembling hydrogel, followed by disinfection and suture. The concentrations of amygdalin and glycyrrhizic acid in the amygdalin / glycyrrhizic acid self-assembling hydrogel used in this example 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, and trauma was performed simultaneously. The injured area was injected with glycyrrhizic acid hydrogel in situ, and then disinfected and sutured. The concentration of glycyrrhizic acid in the glycyrrhizic acid hydrogel used in this example was 15 mg / mL.

[0152] 2. ELISA was used to determine the expression level of C3 in serum after traumatic brain injury:

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

[0154] The final statistical comparison of C3 expression levels in serum, the experimental results are shown in Figure 16 .

[0155] Figure 16 The results of the control group, model group, amygdalin solution, glycyrrhizic acid hydrogel, and amygdalin / glycyrrhizic acid self-assembled hydrogel on the serum complement C3 level caused by traumatic brain injury are shown in the figure. Figure 16 Statistical results showed that compared with the control group, the expression of C3 in the model group increased significantly ( *** p<0.001), indicating that the traumatic brain injury model can lead to a significant increase in the C3 level 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 reduced (p<0.001, respectively). *** p<0.001, *** p<0.001, *** p<0.001), indicating that amygdalin solution, amygdalin / glycyrrhizic acid self-assembled hydrogel and glycyrrhizic acid hydrogel can reduce the expression level of C3 after traumatic brain injury; however, further analysis showed that compared with the amygdalin solution group, the C3 expression in the amygdalin / glycyrrhizic acid self-assembled hydrogel group was more significantly reduced ( *** p<0.001), indicating that the effect of amygdalin / glycyrrhizic acid self-assembled hydrogel is better than that of amygdalin solution alone; compared with the glycyrrhizic acid hydrogel group, the C3 expression in the amygdalin / glycyrrhizic acid self-assembled hydrogel group was significantly reduced ( *** p<0.001), indicating that the effect of amygdalin / glycyrrhizic acid self-assembled hydrogel is better than that of glycyrrhizic acid hydrogel alone.

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

[0157] The amygdalin / glycyrrhizic acid self-assembled hydrogel of the present invention can be directly injected locally after debridement of traumatic brain injury, providing three-dimensional network support for the injured area, achieving early intervention treatment of the disease, and effectively reducing the C3 expression level 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 post-injury neural repair, exerting an overall protective effect, and providing an intraoperative auxiliary drug intervention strategy for early surgical intervention of traumatic brain injury.

[0158] Example 6

[0159] A biosafety verification method for the amygdalin / glycyrrhizic acid self-assembled hydrogel of the present invention in treating traumatic brain injury comprises the following steps:

[0160] 1. Complete the construction of a traumatic brain injury mouse model according to the procedures 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 without trauma, and the wound was disinfected and sutured.

[0162] Model group: bone window was opened and bone flap was removed, trauma was performed, and disinfection and suture were performed at the same time.

[0163] Amygdalin / glycyrrhizic acid self-assembling hydrogel group (Example 1): A bone window was created and the bone flap was removed. Simultaneously, trauma was applied. The injured area was in situ injected with amygdalin / glycyrrhizic acid self-assembling hydrogel, followed by disinfection and suture. The concentrations of amygdalin and glycyrrhizic acid in the amygdalin / glycyrrhizic acid self-assembling hydrogel used in this example were 15 mg / mL and 15 mg / mL, respectively.

[0164] 2. Hematoxylin-eosin staining was used to observe the biosafety of the main organs in each group of mice after traumatic brain injury:

[0165] (1) Collection of major organ tissue samples: On day 3 after modeling, mice were anesthetized with 0.3% sodium pentobarbital via intraperitoneal injection and then perfused transcardially with 0.9% saline. Finally, the intact heart, liver, spleen, lung, kidney, intestine, and testis tissues were dissected and fixed in 4% paraformaldehyde.

[0166] (2) Experimental procedure: After the tissue samples were fixed with 4% paraformaldehyde solution for 24 hours, 3 μm thick tissue sections were prepared by standard paraffin embedding and sectioning procedures. After baking in a 60°C oven for 1 hour, the sections were processed according to the following process: dewaxing in xylene solutions I and II for 10 minutes each (moderate shaking during the process) → hydration with graded ethanol (100%, 100%, 95%, 75% for 5 minutes each) → rinsing in phosphate buffer (1×PBS, pH=7.4) three times (5 minutes each). The sections were placed in hematoxylin staining solution (G1004-500ML, Servicebio) for 30-60 seconds, rinsed with water, and then differentiated in differentiation solution for 5 seconds. After rinsing again, the sections were placed in eosin staining solution (G1001-500ML, Servicebio) for 10-30 seconds. Finally, the sections were mounted with neutral gum and observed under a microscope and photographed.

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

[0168] 3. Blood biochemistry was used to detect the liver and kidney function levels of mice in each group 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 measured using a fully automatic biochemical analyzer.

[0170] Figure 18 The graph shows the expression results of liver and kidney function indexes after traumatic brain injury in the control group, model group, and amygdalin / glycyrrhizic acid self-assembled hydrogel. Figure 18 As shown in the data, 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, indicating that the amygdalin / glycyrrhizic acid self-assembled hydrogel did not cause obvious blood biochemical toxicity to organs after traumatic brain injury.

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

[0172] Although the present invention has been disclosed above with reference to preferred embodiments, this is not intended to limit the present invention. Any person skilled in the art can, without departing from the scope of the technical solution of the present invention, utilize the technical content disclosed above to make many possible changes and modifications to the technical solution of the present invention, or modify it into an equivalent embodiment with equivalent changes. Therefore, any simple modification, equivalent change, and modification made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention shall fall within the scope of protection of the technical solution of the present invention.

Claims

1. A method for preparing amygdalin / glycyrrhizic acid self-assembled hydrogel, characterized in that: The following steps are involved: The amygdalin solution and glycyrrhizic acid are mixed and dispersed evenly, and allowed to stand to obtain amygdalin / glycyrrhizic acid self-assembled hydrogel; the concentration of the amygdalin solution is 0.015 to 0.040 g / mL, and the concentration of the glycyrrhizic acid in the amygdalin / glycyrrhizic acid self-assembled hydrogel is 0.015 to 0.040 g / mL.

2. The preparation method according to claim 1, characterized in that The mass ratio of the amygdalin to the glycyrrhizic acid is 1:1 / 2 to 1:

2.

3. The preparation method according to claim 1 or 2, characterized in that The process of uniformly dispersing the amygdalin solution and glycyrrhizic acid comprises the following steps: A1, mixing amygdalin and PBS solution, and uniformly dispersing them by ultrasonication to obtain amygdalin solution; A2, mixing the amygdalin solution and glycyrrhizic acid, ultrasonically dispersing the mixture at a temperature of 40-70° C. until the mixture becomes clear and transparent, and then allowing the mixture to stand to obtain amygdalin / glycyrrhizic acid self-assembled hydrogel.

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

5. A laetrile / glycyrrhizic acid self-assembled hydrogel, characterized in that: The amygdalin / glycyrrhizic acid self-assembled hydrogel is prepared according to the preparation method according to any one of claims 1 to 4, and the microscopic morphology of the amygdalin / glycyrrhizic acid self-assembled hydrogel presents a three-dimensional porous network structure.

6. Use of the amygdalin / glycyrrhizic acid self-assembled hydrogel prepared according to the preparation method according to any one of claims 1 to 4, or the amygdalin / glycyrrhizic acid self-assembled hydrogel according to claim 5, in the preparation of a medicament for alleviating or treating inflammatory encephalopathy.

7. Use of the amygdalin / glycyrrhizic acid self-assembled hydrogel prepared according to the preparation method of any one of claims 1 to 4, or the amygdalin / glycyrrhizic acid self-assembled hydrogel according to claim 5, in the preparation of a medicament for alleviating or treating neuronal encephalopathy.

8. Use of the amygdalin / glycyrrhizic acid self-assembled hydrogel prepared according to the preparation method of any one of claims 1 to 4, or the amygdalin / glycyrrhizic acid self-assembled hydrogel according to claim 5, in the preparation of a medicament for alleviating or treating diseases involving blood-brain barrier destruction.

9. Use of the amygdalin / glycyrrhizic acid self-assembled hydrogel prepared according to the preparation method of any one of claims 1 to 4, or the amygdalin / glycyrrhizic acid self-assembled hydrogel according to claim 5, in the preparation of a medicament for alleviating or treating complement activation diseases.

10. The use according to claim 9, characterized in that: The complement molecule in the complement activation diseases is the core effector molecule C3 in the complement pathway.

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