Nasal administration rhynchophylline nano-micelle temperature-sensitive gel preparation and preparation method thereof

Abstract

The invention relates to a nasal administration rhynchophylline nano-micelle temperature-sensitive gel preparation as well as a preparation method and application thereof. The preparation is prepared by taking rhynchophylline nano-micelles as an active substance, taking poloxamer as a temperature-sensitive gel material, taking hydroxypropyl-beta-cyclodextrin as an absorption enhancer, taking polyethylene glycol 6000 and hydroxypropyl methyl cellulose as biological adhesives and taking water. Every 100 mL of the temperature-sensitive gel for nasal cavity administration contains 0.1 to 1 g of rhynchophylline nano-micelle, 18 to 21 g of poloxamer 407, 0.5 to 2.5 g of poloxamer 188, 1 to 3 g of hydroxypropyl-beta-cyclodextrin, 1 to 5 g of polyethylene glycol 6000, 0.3 to 0.8 g of hydroxypropyl methyl cellulose and the balance of water. According to the invention, the solubility of the rhynchophylline is improved, the isomerization of the rhynchophylline is inhibited, the stability of the rhynchophylline is improved, and the administration dosage of the rhynchophylline is reduced; after nasal cavity administration, the gel is converted from a liquid state to a semi-solid gel state in a short time, the residence time of the medicine in the nasal cavity is prolonged, meanwhile, the blood brain barrier is bypassed, the brain targeting property is achieved, and the bioavailability of the medicine in the brain is improved.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical preparations and relates to a thermosensitive gel formulation containing rhodopsin nanomicelles and its preparation method. Background Technology

[0002] Parkinson's disease (PD) is a common neurodegenerative disease clinically characterized by the classic triad of resting tremor, rigidity, and bradykinesia, accompanied by perceptual, cognitive, and memory impairments. Idiopathic Parkinson's disease is characterized by the progressive loss of dopaminergic neurons, but the exact cause remains unclear. The most widely studied theory is the dopamine deficiency hypothesis, which posits severe damage to dopaminergic neurons in the substantia nigra pars compacta, leading to a significant deficiency of dopamine in the striatal nerve endings. This imbalance between dopamine and acetylcholine results in motor disorders such as hypertonia. Parkinson's disease is currently incurable, but symptoms can be alleviated through medication, surgery, and rehabilitation. Drug therapy is the first-line and primary treatment for PD, commonly used drugs including anticholinergics, amantadine, levodopa / carbidopa, monoamine oxidase inhibitors, and dopamine receptor agonists. Among these, levodopa and levodopa / carbidopa are the most basic and effective drugs. However, long-term use of levodopa can cause side effects such as a significant increase in plasma homocysteine ​​levels, which manifests as a series of motor complications and limits its application. In addition, oral levodopa alone has a first-pass effect, and only 1% of the blood-brain barrier reaches the brain, which greatly affects the therapeutic effect.

[0003] Rhynchophylline (RIN) is an alkaloid extracted from the hooked branches of Uncaria rhynchophylla, a plant in the Rubiaceae family. Its molecular formula is C2. 22 H 28 N2O4, with a molecular weight of 384.46. Pharmacological studies have shown that rhynchophylline possesses various pharmacological activities, such as inhibiting peripheral vasoconstriction, reducing vascular resistance, and lowering blood pressure, and is clinically used to lower blood pressure; it also has antiplatelet aggregation and antithrombotic effects; rhynchophylline also has neuroprotective effects, by regulating glutamate and acetylcholine, regulating inflammatory factors and related pathways, inhibiting nerve cell apoptosis, and reducing intracellular calcium. 2+ The drug exerts its effects through pathways such as overload and improvement of neurodegenerative diseases, suggesting its potential development as a treatment or preventative drug for Parkinson's disease. Studies have shown that the overall clinical safety of rhynchophylline is good. However, rhynchophylline is poorly soluble in water, and when used to treat central nervous system diseases, it has difficulty crossing the blood-brain barrier to enter the central nervous system, thus failing to reach an effective therapeutic concentration in the brain. Excessive concentrations can also cause liver and nervous system toxicity, leading to hallucinations, tremors, and paroxysmal seizures, significantly limiting its clinical application and efficacy.

[0004] Therefore, developing Uncariae into a drug for treating Parkinson's disease requires addressing its solubility and blood-brain barrier issues. Summary of the Invention

[0005] The technical problems to be solved by this application are how to improve the solubility of rhynchophylline and how to overcome the blood-brain barrier.

[0006] To address the aforementioned technical problems, this invention designs a rhynchophylline / disodium glycyrrhizate nanomicelle system based on green and environmentally friendly mechanochemical technology, and provides a nasal administration rhynchophylline nanomicelle thermosensitive gel formulation, its preparation method, and its application.

[0007] The nasal administration of Uncaria rhynchophylla nanomicelle thermosensitive gel formulation of the present invention is made with poloxamer as the temperature-sensitive gel material, hydroxypropyl-β-cyclodextrin as the absorption enhancer, polyethylene glycol 6000 (PEG 6000) and hydroxypropyl methylcellulose as bioadhesives and water; wherein each 100mL of nasal administration thermosensitive gel contains 0.1-1g of Uncaria rhynchophylla nanomicelles, 18-21g of poloxamer 407, 0.5-2.5g of poloxamer 188, 1-3g of hydroxypropyl-β-cyclodextrin, 1-5g of polyethylene glycol 6000, 0.3-0.8g of hydroxypropyl methylcellulose, and the balance being water.

[0008] Preferably, each 100mL of nasal thermosensitive gel contains 0.2g of Uncaria rhynchophylla nanomicelles, 40720g of poloxamer, 1882g of poloxamer, 1g of polyethylene glycol 6000, 3g of hydroxypropyl-β-cyclodextrin, 0.8g of hydroxypropyl methylcellulose, and the remainder is water.

[0009] The applicant's research revealed that, based on the structure of disodium glycyrrhizate, glycyrrhizic acid contains a hydrophobic portion (one glycyrrhetinic acid molecule) and a hydrophilic portion (two glucuronic acid molecules). Due to its amphiphilic nature, it possesses the ability to self-assemble into micelles in aqueous media, making it suitable as a drug carrier to improve the water solubility, membrane permeability, bioavailability, and enzyme stability of drug molecules. However, under complex physiological conditions, the self-assembled micelles of glycyrrhizic acid may be unstable, leading to sudden release due to enzymatic hydrolysis in vivo. Tannic acid (TA), a natural polyphenol extracted from plants, can be added to the micelle system to form multiple hydrogen bonds with the carbonyl groups in the glycyrrhizic acid molecule, thus stabilizing the glycyrrhizic acid micelle structure and obtaining a well-stable micelle system.

[0010] Mechanochemistry is a discipline that studies chemical changes driven primarily by mechanical force. The preparation process does not require the introduction of large amounts of organic solvents; it avoids the decomposition or deterioration of thermally unstable drugs caused by high-temperature and high-heat preparation conditions; and the preparation process only requires one step to obtain the target product.

[0011] Based on the above research, the above-mentioned rhynchophylline nanomicelles were obtained by the following preparation method: rhynchophylline, disodium glycyrrhizate and tannic acid were added to a polytetrafluoroethylene ball milling jar in a mass ratio of 1:5:1 to 1:10:1, and stainless steel grinding balls were added as the grinding medium. After sealing, the ball milling jar was placed on a rolling ball mill for mechanical grinding for 2 to 16 hours to obtain rhynchophylline nanomicelles.

[0012] The preparation method of the nasal administration thermosensitive gel formulation of Uncaria rhynchophylla nanomicelles described in this invention is as follows:

[0013] First, hydroxypropyl methylcellulose was dispersed in water at 4°C for 12 hours. Then, poloxamer 407, poloxamer 188, polyethylene glycol 6000, and hydroxypropyl-β-cyclodextrin were added. The mixture was left at 4°C until a clear, transparent, lump-free, bubble-free, and uniformly dispersed thermosensitive gel solution was obtained. Then, a solution of rhynchophylline nanomicelles was added, and water was added to 100 mL. The mixture was stirred evenly to obtain a thermosensitive gel formulation for nasal administration containing rhynchophylline nanomicelles.

[0014] The above-mentioned method for obtaining the rhizomorphine nanomicelle solution is to mix rhizomorphine nanomicelles with water, place them in an air bath shaker at 37°C, and shake at 200 rpm for 3 hours to obtain the rhizomorphine nanomicelle solution.

[0015] The nasal administration thermosensitive gel containing Uncaria rhynchophylla nanomicelles of the present invention is applied to the nasal cavity in liquid form during use. It undergoes a solution-gel transition at the nasal cavity temperature, forming a semi-solid gel state and adhering to the nasal cavity.

[0016] Nasal drug delivery systems avoid the degradation processes of the gastrointestinal tract and the first-pass effect of the liver associated with oral administration, and can also bypass the blood-brain barrier. However, due to the clearance mechanisms that maintain normal nasal physiological function (such as removing dust, allergens, and bacteria), drug absorption is problematic. The continuous cleansing action of the nasal mucosa's ciliated epithelium results in a very short contact time for the drug on the nasal mucosa, and the drug is quickly cleared before it is absorbed. Thermosensitive gels have advantages such as strong affinity to the application site, especially mucosal tissue, long retention time, and good controlled drug release performance. This invention prepares a rhubarb nanomicelle thermosensitive gel, using poloxamer as the temperature-sensitive gel material, hydroxypropyl-β-cyclodextrin as an absorption enhancer, and polyethylene glycol 6000 (PEG6000) and hydroxypropyl methylcellulose (HPMC) as bioadhesives to achieve slow drug release, improve drug bioavailability, and enhance brain targeting.

[0017] The beneficial effects of this invention are:

[0018] 1. This invention designs a rhynchophylline / disodium glycyrrhizate nanomicelle system based on green and environmentally friendly mechanochemical technology, which solves the problems of rhynchophylline solubility and isomerization, and greatly reduces the dosage of rhynchophylline for administration.

[0019] 2. This invention uses rhynchophylline nanomicelles as the active ingredient, poloxamer as the temperature-sensitive gel material, hydroxypropyl-β-cyclodextrin as the absorption enhancer, and polyethylene glycol 6000 (PEG 6000) and hydroxypropyl methylcellulose (HPMC) as bioadhesives to prepare a rhynchophylline nanomicelle temperature-sensitive gel. Through nasal administration, this formulation transforms from a liquid to a semi-solid gel, increasing the drug's residence time in the nasal cavity and solving the problem of short drug contact time on the nasal mucosa. The adhesion strength and adhesion time of the temperature-sensitive gel are greatly increased, enabling slow release of rhynchophylline, improving drug bioavailability, and enhancing brain targeting. This route provides convenience and accessibility for PD patients. This invention provides a new approach for the treatment research of neurodegenerative diseases and has significant scientific value and clinical prospects. Attached Figure Description

[0020] Figure 1 The following are characterization diagrams of rhynchophylline nanomicelles from Example 1: (A) is the HPLC chromatogram of rhynchophylline in methanol and aqueous solutions, disodium glycyrrhizate in aqueous solution, tannic acid in aqueous solution, and rhynchophylline nanomicelles from Example 1; (B) is the particle size distribution of rhynchophylline nanomicelles from Example 1; (C) is the potential distribution of rhynchophylline nanomicelles from Example 1; and (D) is a physical mixture of rhynchophylline, disodium glycyrrhizate, tannic acid, and rhynchophylline / disodium glycyrrhizate / tannic acid, as well as the samples from Example 1. 1. Infrared spectrum of rhynchophylline nanomicelles, (E) is the X-ray powder diffraction pattern of rhynchophylline, disodium glycyrrhizate, tannic acid, physical mixture of rhynchophylline / disodium glycyrrhizate / tannic acid and rhynchophylline nanomicelles of Example 1, (F) is the differential scanning calorimetry pattern of rhynchophylline, disodium glycyrrhizate, tannic acid, physical mixture of rhynchophylline / disodium glycyrrhizate / tannic acid and rhynchophylline nanomicelles of Example 1, and G is the transmission electron microscope image of rhynchophylline nanomicelles of Example 1.

[0021] Figure 2 The diagram shows the molecular simulation of the three components in Example 1. (A), (B), and (C) are snapshots taken at three time points of 0, 50, and 100 ns during the molecular simulation, respectively. (D) is a kinetic evaluation diagram of the interaction energy of the three components in Example 1.

[0022] Figure 3 The diagram shows the kinetic evaluation of the three components in the molecular simulation process in Example 1, where (A) is the radius of gyration, (B) is the root mean square deviation, (C) is the solvent accessible surface area, (D) is the number of hydrogen bonds formed by glycyrrhizic acid and rhodopsin, (E) is the number of hydrogen bonds formed by glycyrrhizic acid and tannic acid, and (F) is the number of hydrogen bonds formed by rhodopsin and tannic acid.

[0023] Figure 4The following are quality evaluation diagrams for the Rhododendron nanomicelle thermosensitive gel of Example 2: (A) is a phase transition comparison diagram of the Rhododendron nanomicelle thermosensitive gel of Example 2; (B) is a diagram showing the adhesion time and adhesion strength of the Rhododendron nanomicelle thermosensitive gel of Example 2; (C) is a strain stress scan diagram of the Rhododendron nanomicelle thermosensitive gel of Example 2; and (D) is a frequency scan diagram of the Rhododendron nanomicelle thermosensitive gel of Example 2.

[0024] Figure 5 The images show the particle size stability and morphology of the Rhizoctonia solani nanomicelle thermosensitive gel in different media in Example 2. (A) No medium; (B) 10% DMEM medium; (C) 50% DMEM medium; (D) PBS medium; (E) Particle size comparison under the four conditions at the same time; (F) Particle size distribution of the Rhizoctonia solani nanomicelle thermosensitive gel in Example 2; (G) Transmission electron microscopy (TEM) image of the Rhizoctonia solani nanomicelle thermosensitive gel in Example 2; and (H) Scanning electron microscopy (SEM) image of the Rhizoctonia solani nanomicelle thermosensitive gel in Example 2.

[0025] Figure 6 In the figures, (A) shows the cytotoxicity of the rhizomorphine nanomicelle thermosensitive gel of Example 2 on SH-SY5Y and U251 cells, (B) shows the cytotoxicity of the blank gel on SH-SY5Y and U251 cells, (C) shows the effect of MPP+ on PC12 cell morphology, (D) shows the effect of different concentrations of MPP+ on PC12 cell survival, and (E) shows the in vitro anti-Parkinson's drug efficacy of the rhizomorphine nanomicelle thermosensitive gel of Example 2 (n=6, ****P<0.0001, ***P<0.001).

[0026] Figure 7 The following are in vitro drug release diagrams for the Uncaria rhynchophylla nanomicelle thermosensitive gel in Example 2: (A) Cumulative drug release rate by diffusion method, (B) Cumulative drug release rate by membrane-free method, and (C) Cumulative erosion rate by membrane-free method.

[0027] Figure 8 (A) is the metabolic kinetic curve of rhubarb in brain tissue after nasal administration of rhubarb nanomicelle thermosensitive gel of Example 2, nasal administration of rhubarb solution, tail vein injection of rhubarb solution, and gavage administration of rhubarb solution; (B) is the metabolic kinetic curve of rhubarb in blood after tail vein injection of rhubarb solution; (C) is the metabolic kinetic curve of rhubarb in blood after nasal administration of rhubarb nanomicelle thermosensitive gel of Example 2, nasal administration of rhubarb solution, and gavage administration of rhubarb solution.

[0028] Figure 9 These are pathological sections of the substantia nigra from the model group and the blank group.

[0029] Figure 10For the pharmacodynamic study of the Uncaria rhynchophylla nanomicelle thermosensitive gel in Example 2, the following data are presented: (A) Changes in mouse body weight during modeling and drug administration; (B) Time spent on the rotundus by mice in the blank control group, model group, and drug administration group; (C) Time spent on the grid by mice in the blank control group, model group, and drug administration group; (D) Center distance of the open field by mice in the blank control group, model group, and drug administration group within five minutes; (E) MDA level in the striatum of mice in the blank control group, model group, and drug administration group; (F) Total distance of the open field by mice in the blank control group, model group, and drug administration group within five minutes; (G) GPX level in the striatum of mice in the blank control group, model group, and drug administration group; (H) SOD level in the striatum of mice in the blank control group, model group, and drug administration group; and (I) Movement trajectory of mice in the mine field experiment within five minutes (n = 6, ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05).

[0030] Figure 11 Scanned images of mouse nasal mucosa sections, where (A) is the control group and (B) is the group treated with nasal administration of Uncaria rhynchophylla nanomicelle thermosensitive gel in Example 2.

[0031] Figure 12 Pathological sections of the heart, kidney, and spleen of mice in the control group and the group receiving intranasal administration of Uncaria rhynchophylla nanomicelle thermosensitive gel (Example 2). Detailed Implementation

[0032] The present invention will be further described below with reference to specific embodiments, which are carried out under preferred conditions of the present invention. Unless otherwise specified, the methods described are conventional methods, and the raw materials described are all available from publicly available commercial sources.

[0033] Example 1: Preparation and characterization of Uncariae nanomicelles

[0034] Mechanochemical techniques were used to prepare rhynchophylline nanomicelles.

[0035] 0.5g of rhizomorphine, 5.0g of disodium glycyrrhizate and 0.5g of tannic acid were placed in a 150mL polytetrafluoroethylene ball mill jar, and stainless steel grinding beads with a diameter of 15mm (total weight 340.0g) were added. Then, mechanical grinding was carried out using a rolling ball mill with a grinding acceleration of 1g for 2h, 4h, 8h and 16h respectively. The ground product was passed through a 200-mesh stainless steel sieve to obtain rhizomorphine nano micelles.

[0036] The solubility, particle size, and Zeta potential of the nanomicelles were investigated. X-ray powder diffraction, differential scanning calorimetry, infrared spectroscopy, transmission electron microscopy, molecular simulation, and molecular dynamics analysis were used to characterize the nanomicelle system. The results are as follows: Figure 1 , Figure 2and Figure 3 As shown.

[0037] Table 1 shows the solubility of the ball-milled products and their corresponding physical mixtures (PMs) of rhynchophylline, disodium glycyrrhizate, and tannic acid at different ball-milling times. The results show that the solubility of the active pharmaceutical ingredient (API) is 0.0275 g / L, indicating it is a poorly soluble drug. Furthermore, the aqueous solution of the drug is unstable. In aqueous solution, it undergoes interconversion with isorhynchophylline, forming a micelle system, which improves the stability of rhynchophylline. Figure 1 As shown in Figure A, no peak of isorhynchophylline was observed in the HPLC chromatogram of the micelle system. Furthermore, compared with the original drug powder, the ball-milled material significantly increased the solubility of the drug. This may be because the drug is dispersed in an amorphous state in the micelles formed by disodium glycyrrhizate and tannic acid. Without the lattice binding, the drug can dissolve better in aqueous solution, indicating that isorhynchophylline has been successfully encapsulated in the micelle system by disodium glycyrrhizate.

[0038] Table 1. Solubility of Uncariae and its ball-milled products at different ball-milling times

[0039]

[0040] The size distribution of micelles formed by rhizoma lucida-disodium glycyrrhizate-tannic acid micelles in aqueous solution was determined using dynamic light scattering (DLS). The particle size distribution was found to be unimodal, with the prepared nanomicelles having a particle size of approximately 150 nm, classifying them as a colloidal system. The negative zeta potential indicates that the micelles carry a negative charge on their surface. The absolute value of the zeta potential is greater than 30, demonstrating a strong electrostatic force between the micelles, which is unfavorable for aggregation and thus increases the stability of the micelles.

[0041] The rhizoid showed multiple characteristic diffraction peaks in the 2θ angle range of 0–40°, indicating that it exists in a crystalline structure. Disodium glycyrrhizate and tannic acid had only one relatively broad diffraction peak, indicating that they are amorphous compounds. The physical mixture still showed a certain number and intensity of drug crystal diffraction peaks, indicating that the drug still exists in crystalline form in this system. In the mechanically ball-milled product, the crystal diffraction peaks of the drug disappeared, indicating that the drug was highly dispersed in an amorphous state in disodium glycyrrhizate and tannic acid, indicating that under the action of mechanical force, rhizoid had been successfully encapsulated in the micelle system by disodium glycyrrhizate.

[0042] Differential scanning calorimetry (DSC) analysis, by Figure 1As shown in F, the sharp and prominent endothermic peak of rhynchophylline near 213.81℃ is its melting point. Disodium glycyrrhizate and tannic acid show no obvious melting peaks, indicating they are in an amorphous state. Three endothermic peaks appeared in the physical mixture at 140.90℃, 154.40℃, and 186.91℃, indicating that a eutectic system was formed, a phenomenon very common in physical mixtures. However, in the mechanically ball-milled product system, the melting point peak of rhynchophylline disappeared, and no eutectic system was formed, indicating that rhynchophylline was encapsulated in an amorphous state within the disodium glycyrrhizate micelle system under mechanical force.

[0043] The infrared curves of rhizoma lysine, disodium glycyrrhizate, tannic acid, physical mixture (PM), and mechanically treated product are as follows: Figure 1 As shown in D. The infrared spectrum of the rhizoma lucidae raw material shows that 2793 cm⁻¹... -1 The absorption peak for the -NH stretching vibration is located at 1707 cm⁻¹. -1 The absorption peak at this point is the C=O stretching vibration. After physical mixing, some characteristic peaks of rhynchophylline are covered by the characteristic peaks of disodium glycyrrhizate and tannic acid, becoming less obvious. However, the characteristic absorption peaks of C=O and -NH can still be observed, indicating that simple physical mixing is insufficient to form nano micelles. The infrared spectrum after mechanical ball milling is basically a simple superposition of the infrared spectra of tannic acid and disodium glycyrrhizate. However, the intensity of the characteristic absorption peak of C=O of rhynchophylline is weakened, indicating that the C=O of rhynchophylline may form micelles with the functional groups in disodium glycyrrhizate and tannic acid through hydrogen bonds.

[0044] By transmission electron microscopy (TEM) Figure 1 G) The morphology of the micelles can be clearly observed. The results show that, under electron microscopy, the micelles exhibit a spherical shape with a diameter of approximately 100–200 nm. The particle size measured using a Malvern particle size analyzer is basically consistent with the results observed under transmission electron microscopy. Simultaneously, when a laser pointer is used to illuminate a mechanically ball-milled aqueous solution on one side, a clear beam of light can be observed when viewed from a direction perpendicular to the laser irradiation. Figure 1 G) indicates that the Tyndall effect occurred, which also confirms the formation of the micelle system.

[0045] Molecular dynamics simulations of small molecule clusters were performed using the Gromacs 2019 software package. The small molecules were simulated using the GAFF2 general force field parameters, and the AM1-BCC atomic charges were calculated using the ANTECHAMBER module of Amber. The TIP3P dominant water model was selected, and periodic boundary conditions were set. The molecular dynamics simulation workflow consisted of four steps: energy minimization, heating, equilibrium, and production kinetics simulation. First, the heavy atoms were constrained, and the water molecules underwent 10,000 steps of energy minimization (including 5,000 steps of steepest descent and 5,000 steps of conjugate gradient). Then, the constraints were lifted, and the entire system underwent 10,000 steps of energy optimization. After energy minimization, the system was slowly heated to 300 K over 50 ps. After heating, the system was equilibrated under the NPT ensemble for 50 ps. Finally, the system was simulated with molecular dynamics over the NPT ensemble for 100 ns with a time step of 2 fs. Trajectory data was saved every 20 ps for correlation analysis. Five blue samples of rhizoma lysine, 40 red samples of glycyrrhizic acid, and five green samples of tannic acid were randomly mixed and placed in an 8 nm * 8 nm * 8 nm simulation box filled with water molecules. A 100 ns molecular dynamics simulation was performed to examine the system's self-assembly and cluster formation process. Figure 2 As shown in A to C, the system forms a stable micelle structure after 50 ns. Tannic acid acts as an adhesive, adsorbing glycyrrhizic acid together, while the drug molecule Uncariae is encapsulated within the micelle system. Figure 2 D represents the interaction energy between the components, where EIE represents electrostatic interaction and Vdw represents van der Waals interaction. It can be seen that the van der Waals interaction between tannic acid and glycyrrhizic acid is the strongest, reaching approximately -2200 kJ / mol, while the van der Waals interaction between glycyrrhizic acid and rhubarb alkaloid can also reach -500 kJ / mol, playing an important role in binding the drug rhubarb alkaloid. The radius of gyration (Rog) and root mean square deviation (RMSD) curves during the simulation are shown below. Figure 3 As shown in A and B, the cluster simulation exhibits a periodic mirror correction process, resulting in occasional jumps. These jumps gradually decrease and stabilize as the simulation progresses, indicating that the simulated components gradually self-aggregate to form stable clusters, and conformational changes gradually decrease. The solvent-accessible surface area gradually decreases, a trend consistent with the radius of gyration. This suggests that as the simulation continues, small molecules self-assemble to form clusters, causing water molecules to be squeezed out of the cluster interiors, reducing the solvent-accessible area, and ultimately forming a stable, solvent-inaccessible cluster system. Figure 3 As shown in D to F, glycyrrhizic acid and tannic acid can form more than 30 hydrogen bonds, which is far more than the number of hydrogen bonds between other molecules. This indicates that there is a very stable interaction between tannic acid and glycyrrhizic acid. The addition of tannic acid enables glycyrrhizic acid to form more stable self-assembled micelles.

[0046] The above results indicate that rhynchophylline has been successfully encapsulated in its micelle system by disodium glycyrrhizate, forming a new phase, and the rhynchophylline nanomicelle system has been successfully prepared.

[0047] Example 2: Preparation of Uncaria rhynchophylla nanomicelle thermosensitive gel

[0048] Add 2 ml of water to a 50 ml beaker, weigh 80 mg of HPMC-E5 and spread it on the surface. Seal the mouth of the beaker with plastic wrap and poke several air holes. Place it in a 4℃ refrigerator for 12 hours to disperse. Weigh 2 g of poloxamer 407 (P407), 0.2 g of poloxamer 188 (P188), 0.1 g of polyethylene glycol 6000 (PEG-6000), and 0.3 g of hydroxypropyl-β-cyclodextrin (HP-β-CD) and place them in a 50 mL beaker. Add the rhizoma lucidae nanomicelle solution (0.2 g of rhizoma lucidae nanomicelle dissolved in 5 mL of deionized water and shake well) and an appropriate amount of deionized water. Stir at room temperature until evenly dispersed. Store in a 4℃ refrigerator until no solids remain. Add an appropriate amount of deionized water to make up to 10 mL and store in a 4℃ refrigerator for at least 24 hours to allow all components to fully dissolve.

[0049] This invention characterized the rhynchophylline nanomicelles and conducted quality evaluation, in vitro drug release, toxicity experiments, in vitro and in vivo pharmacodynamic studies, in vivo pharmacokinetic studies, and in vivo safety evaluation on the nasal administration thermosensitive gel containing rhynchophylline nanomicelles. The results showed that forming nanomicelles with disodium glycyrrhizate and tannic acid through mechanical action not only improved the drug's solubility but also inhibited its isomerization, thus enhancing its stability. Dispersing the rhynchophylline nanomicelles within the thermosensitive gel framework allows for brain-targeting through nasal administration, and the gel can remain in the nasal cavity for a longer period, improving bioavailability. Pharmacodynamic studies, including behavioral and biochemical analyses, showed that the gel group significantly alleviated Parkinson's disease compared to the model group, and had no significant toxic effects on the nasal mucosa or vital organs.

[0050] Quality evaluation of Uncaria rhynchophylla nanomicelle thermosensitive gel

[0051] 1. Gelation temperature and gelation time

[0052] Three batches of rhubarb alkaloid thermosensitive gel were prepared in parallel according to Examples 1 and 2. For each batch, 2 mL of rhubarb alkaloid thermosensitive gel was placed in a 10 mL vial. The vial was placed in a constant temperature water bath, with the solution level in the vial always below the water bath surface. The water bath temperature was slowly increased from 20°C at a rate of 1°C / min. The vial was inverted, and the liquid flow was observed. The temperature at which the liquid stopped flowing was recorded as the gelation temperature, and the time it took for the liquid to change from a solution state to a solid state was recorded as the gelation time. The gelation temperature and gelation time were recorded. The average gelation temperature was measured to be 30.2°C, which is sufficient for phase transition at the physiological temperature of the nasal cavity without gelling at room temperature, meeting the requirements for nasal preparations.

[0053] Table 2 Gelation Temperature and Gelation Time

[0054]

[0055] 2. Visual inspection and pH value

[0056] Three batches of parallel-prepared rhynchophylline thermosensitive gels were placed in vials and observed against a black background. The pH was also measured using pH paper. The appearance of the rhynchophylline thermosensitive gels is shown in the attached image. Figure 4 A) Both pH and pH meet the relevant requirements for nasal preparations.

[0057] Table 3. Results of Appearance and pH Inspection

[0058]

[0059] 3. Determination of Adhesion Strength and Adhesion Time

[0060] 0.1 g of nasal mucosa was fixed onto a circular support with a diameter of 0.90 cm and a thickness of 0.35 cm using cyanoacrylate adhesive. The dispersion (50 mg / mL mucosa dispersion, water as solvent, dialyzed overnight in standard RC tubes with a molecular weight cutoff of 6000–8000 Da, lyophilized and stored at 2–8 °C, redispersed with pH 5.5 PBS before use) was suspended from a tension meter spring. 3 mL of each sample was then poured into a small beaker (3 cm in diameter) and gelled at 35 °C, maintained at this temperature by a water bath. The mucosa was lowered until it reached the surface of the preparation and kept in contact for 2 min without applying any force. The force required to peel the nasal mucosa from the gel represents the adhesive strength between the mucosa and the nasal gel.

[0061] Porcine nasal mucosa sections (3 cm long, 1.5 cm wide) were hydrated in PBS (pH 5.5) containing 0.05% mucin for 2 min. A gel containing 0.1% green pigment was gelled in a 34°C water bath, and 80 μL of the gel was spread onto the nasal mucosa sections. The mucosa was rinsed with a continuous flow (1 mL / min) of pH 5.5 PBS to simulate physiological conditions of the nasal cavity. The time required to remove the gel from the mucosa was measured using a stopwatch, with the disappearance of the green portion or gel detachment as indicators.

[0062] The above results are shown in Figure 4 B indicates that the adhesion time and adhesion force of the formulation increased after the addition of the bioadhesive HPMC, suggesting that this step is beneficial for improving the adhesion of the gel.

[0063] 4. Rheology

[0064] The rheological properties of the rheological gel containing rhynchophylline nanomicelles were determined using a rotational rheometer. Strain-stress scanning was employed to measure the change in viscoelastic modulus of the drug-loaded gel with strain rate at 32℃. After determining the linear viscoelastic region, the corresponding strain rate was selected, and the temperature was set at 32℃ to investigate the changes in modulus and viscosity of the drug-loaded gel.

[0065] The linear viscoelastic region refers to the phenomenon where, within a certain strain rate range, a material exhibits linear viscoelasticity, primarily characterized by a modulus that does not change with strain rate. This reflects the system's structure and can characterize sample stability. Storage modulus, denoted by G′, indicates the solid state property of the material. Loss modulus, denoted by G″, indicates the solution state property of the material. For example, after strain-stress scanning at 32℃, the linear viscoelastic region of the gel was measured to be 0.02%–0.2%. With a strain rate set at 0.05%, a frequency scan was performed at 32℃. The shear frequency f was in the range of 0.01–1 rad / s, indicating that the gel's solid properties were stronger than its liquid properties. The viscosity of the Uncaria rhynchophylla thermosensitive gel is inversely proportional to the shear frequency.

[0066] 5. Determination of gel particle size and morphology

[0067] The particle size variation under four media conditions was measured using a Malvern ZR90 particle size potentiometer. 3D waterfall plots were generated with particle size on the x-axis and intensity on the y-axis for five time periods (9:00-9:30; 10:00-10:30; 11:30-12:00; 16:00-16:30; 20:00-20:30) for comparison. Figure 5 (AE). The gel is most stable at room temperature. As time progresses, the gel particle size is mainly distributed around 20 nm, which is relatively stable and consistent with the TEM results. Figure 5G). SEM scanning results showed uniform voids on the gel surface, indicating successful preparation of the nanomicelle thermosensitive gel. Figure 5 H).

[0068] 6. In vitro toxicity

[0069] Logarithmic growth phase SH-SY5Y and U251 cells were harvested, and after adjusting the cell concentration, they were seeded into 96-well plates. After overnight cell adhesion, different drugs were added for treatment according to experimental requirements. Cells were divided into a blank group, a control group, a rhizoma sarcodile nanomicelle thermosensitive gel group, and a blank gel group without rhizoma sarcodile. Logarithmic growth phase cells were digested with trypsin, and after digestion, they were collected by centrifugation and prepared into a cell suspension. The cell concentration was adjusted to 5–10 × 10⁻⁶ cells / well. 4 After preparing the cell suspension, gently mix it. Except for the blank group, which receives an equal volume of blank culture medium, add 100 μL of cell suspension to each well of the other three groups. Fill the edge wells with sterile PBS. Place the inoculated cell culture plates in an incubator and culture until the cell monolayer covers the bottom of the wells (96-well flat-bottom plate). Discard the culture medium from all four groups. Add 100 μL of fresh culture medium to the blank and control groups. Add equal volumes of culture medium containing a concentration gradient of drugs to the rhizoma lucida nanomicelle thermosensitive gel group and the blank gel group without rhizoma lucida. The blank gel group without rhizoma lucida contains 100, 200, 400, 800, and 1600 μg / mL (calculated based on poloxamer 407), while the rhizoma lucida nanomicelle thermosensitive gel group contains 10, 20, 40, 80, and 160 μg / mL (calculated based on rhizoma lucida). Each group has 5 replicates. Incubate at 37°C with 5% CO2 for 24 h, and observe the drug effect under an inverted microscope. Add 10 μL of CCK-8 solution to each well and continue culturing for 4 hours. Observe the 96-well plate at 450 nm using a microplate reader. The formula for calculating cell viability is as follows:

[0070]

[0071] Among them, A s Absorbance of the experimental well; A b Absorbance of the blank well; A cThe absorbance of the control wells is shown in Figures 6A and 6B. The cell viability of the rhizoid micelle gel and the blank gel group are shown in Figures 6A and 6B, respectively. The cell viability of U251 and SH-SY5Y cells decreased with increasing solid gel concentration. When the rhizoid concentration reached 80 μg / ml, the cell viability of U251 and SH-SY5Y cells decreased to 56.37% and 33.26%, respectively. When the rhizoid concentration was 40 μg / ml or lower, the cell viability of U251 and SH-SY5Y cells was higher, at 91.27% and 80.74% or higher, respectively. The IC50 values ​​of U251 and SH-SY5Y cells were calculated using Prism Graphad 10.1.2, and were 86.44 μg / ml and 64.63 μg / ml, respectively. This indicates that both the rhizoid micelle gel (when the rhizoid concentration is less than 40 μg / ml) and the blank gel group had low cytotoxicity and high safety.

[0072] 7. In vitro efficacy

[0073] Logarithmic growth phase PC12 cells were harvested, and after adjusting the cell concentration, they were seeded into 96-well plates. After the cells adhered overnight, they were injected with 0, 1, 2, 3, 4, 5, 6, 7, and 8 mM MPP. + After incubating at 37℃ and 5% CO2 for 24 hours, the drug's effect was observed under an inverted microscope. 10 μL of CCK-8 solution was added to each well, and incubation continued for another 4 hours. The 96-well plate was then observed at 450 nm using a microplate reader, and cell viability was calculated.

[0074] via MPP + To establish a Parkinson's disease model, PC12 cells were damaged. As MPP+ concentration increased, the cells became rounder, and cell viability gradually decreased. The optimal MPP+ concentration for establishing a Parkinson's disease model was the one corresponding to a cell viability of 50%–60%. + Effects on cell morphology and different concentrations of MPP + The effects on cell viability are as follows: Figure 6 As shown in C and D, the MPP corresponding to a cell viability of 50-60% + The concentration is 2 mM, therefore MPP with a concentration of 2 mM is used. + PC12 cells were induced for subsequent Parkinson's disease modeling studies.

[0075] Logarithmic growth phase PC12 cells were harvested, and after adjusting the cell concentration, they were seeded into 96-well plates. After overnight cell adhesion, different drugs were added for treatment according to experimental requirements. Cells were divided into a blank group, a control group, a model group, and a rutin-based nanomicelle thermosensitive gel group. Logarithmic growth phase cells were digested with trypsin, and after digestion, they were collected by centrifugation and prepared into a cell suspension. The cell concentration was adjusted to 5 × 10⁻⁶ cells / well. 4After preparing the cell suspension, mix gently and divide the PC12 cells in logarithmic growth phase into a control group, a model group, and a RIN-GEL treatment group. The control group received 100 μL of culture medium; the model group received 100 μL of 2 mM MPP. + The RIN-GEL treatment group was pre-treated with 100 μL of RIN-GEL solution at serial concentrations (10, 20 μg / mL) for 4 hours, followed by the addition of 2 mM MPP. + After incubating at 37℃ and 5% CO2 for 24 hours, the drug's effect was observed under an inverted microscope. 10 μL of CCK-8 solution was added to each well, and incubation continued for another 4 hours. The 96-well plate was then observed at 450 nm using a microplate reader. The formula for calculating cell viability is as follows:

[0076]

[0077] Among them, A s Absorbance of the experimental well; A b Absorbance of the blank well; A c The absorbance of the control well is shown in Figure 6E. The in vitro anti-Parkinson's disease efficacy results are shown in Figure 6E. Compared with the control group, the survival rate of the model group was 51.32%, showing a significant change, indicating that the Parkinson's disease model was successfully established. Compared with the model group, the RIN-GEL group showed a significant increase in PC12 cell survival, indicating that the rhizome alkaloid thermosensitive gel has an anti-Parkinson's disease effect.

[0078] In vitro drug release of Uncaria rhynchophylla nanomicelle thermosensitive gel

[0079] 1. Diffusion method

[0080] One mL each of 20 mg / mL rhynchophylline thermosensitive gel and rhynchophylline solution were placed in two identical dialysis bags. A pH 5.8 phosphate buffer solution was used as the release medium, with a volume of 300 mL. The temperature was (37 ± 0.5) °C, and the rotation speed was 100 r / min. One mL of the outer medium was collected at 0, 0.5, 1, 2, 4, 8, 12, and 24 h, and an equal volume of release medium was immediately added at the same temperature. The drug concentration in the release medium was determined by HPLC, and the cumulative release rate at different time points was calculated. The calculation formula is as follows:

[0081]

[0082] Among them, Q n Cumulative release rate (%); V is the volume of the release medium (mL); C n Ci is the concentration (μg / mL) measured at the nth sampling point; Ci is the concentration (μg / mL) measured at the ith sampling point; V0 is the sampling volume (mL); and A is the mass of drug added to the gel (μg).

[0083] 2. Membrane-free method

[0084] Take 1 mL of Uncaria rhynchophylla thermosensitive gel and place it in a 5 mL centrifuge tube. Incubate at 37°C to form a gel. After gelation, accurately weigh the gel and add 1 mL of physiological saline. Place the centrifuge tube in a constant-temperature shaker and shake at a frequency of 100 rpm. At 15, 30, 60, 90, and 180 min, pour out all the solution from the centrifuge tube, wipe the surface of the centrifuge tube dry with filter paper, accurately weigh it, and add the same volume of physiological saline to continue shaking at the constant temperature. Filter the solutions poured out at different time points, and analyze the filtrate using a high-performance liquid chromatograph. Calculate the cumulative dissolution rate X and the cumulative release rate Q. n The formula is as follows:

[0085]

[0086] Where X is the cumulative dissolution rate (%); Q n W0 represents the cumulative release rate (%); W0 represents the mass (g) of the gel and centrifuge tube before the addition of physiological saline; W i The mass (g) of the centrifuge tube; W n Mass (g) of gel to centrifuge tube at each time point; m i denoted as mi, where mi is the mass of drug released in the i-th release (g); m0 is the total mass of drug in the entire gel system (g).

[0087] Results of in vitro drug release: Figure 7 As shown, the release rate of the rhizoma lucida solution is relatively fast, reaching 90% within 1 hour and complete release within 2 hours. In contrast, the gel achieves a cumulative release rate of 80% within 8 hours, indicating that the rhizoma lucida thermosensitive gel exhibits sustained-release properties compared to the rhizoma lucida solution. Furthermore, the cumulative dissolution rate and cumulative release rate of the rhizoma lucida thermosensitive gel are approximately 60% after 180 minutes.

[0088] Pharmacokinetic Study of Uncariae Nanomicelle Thermosensitive Gel

[0089] 1. Liquid chromatography-mass spectrometry conditions

[0090] Mobile phase: Phase A: 5 mmol / L ammonium formate aqueous solution containing 0.1% formic acid; Phase B: methanol:acetonitrile (v / v) = 1:1 solution, with a ratio of Phase A to Phase B of 60:40. Elution was performed using isocratic elution. Column: ODS-SP (4.6×150mm, 5μm), pre-column C18 guard column (BHZXC18), flow rate 0.9mL / min, column temperature 35℃, injection volume 10μL.

[0091] The mass spectrometer employed positive ionization detection mode, with the ionization voltage (IS) controlled at 5500 V, the curtain gas (CUR) at 30 psi, the spray gas (GS1) at 55 psi, the auxiliary heating gas (GS2) at 55 psi, and the temperature at 550 °C. Multiple reaction monitoring (MRM) was used. The quantitative analysis ion reaction for rhynchophylline was m / z 385.2 → m / z 160.0, with the declustering voltage controlled at 120 V and the collision energy at 39 V. The quantitative analysis ion reaction for dehydrorhynchophylline was m / z 383.2 → m / z 159.8, with the declustering voltage controlled at 130 V and the collision energy at 44 V.

[0092] 2. Grouping and Dosing

[0093] Twenty-four male SD rats, ranging in weight from 190 to 230 g, were divided into four groups and administered the drug at a dose of 10 mg / kg. These groups included a nasal administration of Uncaria rhynchophylla nanomicelle gel group, a nasal administration of Uncaria rhynchophylla solution group, a tail vein injection of Uncaria rhynchophylla solution group, and a gavage administration of Uncaria rhynchophylla solution group. Blood samples were collected at 5 min, 10 min, 15 min, 30 min, 45 min, 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h after drug administration. Approximately 0.3 mL of blood was collected each time, centrifuged in anticoagulant tubes to obtain plasma, and stored at -80°C.

[0094] One hundred and forty-four male KM mice, weighing 14–17 g, were divided into four groups and administered the drug at a dose of 10 mg / kg: intranasal administration of Uncaria rhynchophylla nanomicelle gel, intranasal administration of Uncaria rhynchophylla solution, intravenous injection of Uncaria rhynchophylla solution, and gavage administration of Uncaria rhynchophylla solution. After administration, the mice were euthanized by cervical dislocation at 15 min, 30 min, 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h, and the brain tissue was harvested. The brain tissue was rinsed thoroughly with physiological saline, and after removing excess water, it was weighed. Physiological saline was added at a ratio of 3:1 (volume to tissue mass), and the mixture was homogenized to obtain a brain tissue homogenate, which was then stored at -80℃.

[0095] 3. Methods for processing plasma and brain tissue

[0096] Place 50 μL of plasma sample (supernatant) in an Eppendorf tube, add 100 μL of 80 ng / mL internal standard dehydrouncitonin solution (dissolved in acetonitrile), then add 50 μL of acetonitrile, vortex for 1 min to mix, centrifuge for 10 min (4℃, 16000 rpm), and take the supernatant for LC-MS / MS quantitative analysis.

[0097] Take 50 μL of the supernatant brain tissue sample and place it in an Eppendorf tube. Add 100 μL of acetonitrile solution containing the internal standard dehydrorheumatine (80 ng / mL), then add 50 μL of acetonitrile. Vortex to mix the parts evenly. Centrifuge at 16000 rpm at 4℃ for 10 min. Take the supernatant for LC-MS / MS quantitative analysis.

[0098] 4. Data Processing and Analysis

[0099] With the help of The 8.3.5 version (Pharsight Corporation, CA) software processes the blood drug concentration-time data and plots the blood drug concentration-time curve. Figure 8 Peak concentration Cmax, half-life t 1 / 2 The pharmacokinetic parameters were calculated using the non-compartmental model method (NCA method), and the absolute bioavailability was also calculated.

[0100] Table 4. Pharmacokinetic parameters of rhodopsin in plasma and brain tissue of rats after administration.

[0101]

[0102] Pharmacokinetic results showed that nasal administration had higher bioavailability compared to oral administration, demonstrating that nasal delivery was more effective at reaching the brain. Furthermore, the bioavailability of the nasal gel group was higher than that of the nasal solution group. Calculations of the relative bioavailability of the rutin solution and gel showed that in brain tissue, the nasal gel was 2.24 times more bioavailable than the nasal solution and 5.73 times more bioavailable than the gavage group; in blood, the nasal gel was 1.49 times more bioavailable than the nasal solution and 2.48 times more bioavailable than the gavage group, demonstrating that nasal gel can increase drug concentration in the brain. Half-life t 1 / 2 The nasal administration of the gel can last for 13.12 hours, which is 6.05 times that of the nasal solution, indicating that the Uncaria rhynchophylla gel has a sustained-release effect in vivo.

[0103] Pharmacodynamic study of rhizoside nanomicelle thermosensitive gel

[0104] 1. Grouping and Modeling

[0105] Balb / c mice (18–22 g) were randomly divided into 5 groups: a blank control group, a model group (MPTP 30 mg / kg), an MPTP + gavage administration of Uncaria rhynchophylla solution group, an MPTP + intranasal administration of Uncaria rhynchophylla solution group, and an MPTP + intranasal administration of Uncaria rhynchophylla gel group, with 6 mice in each group. The blank control group received intraperitoneal injection of saline, while the other groups received intraperitoneal injection of MPTP 30 mg / kg for 5 consecutive days. Mice were observed to exhibit tail stiffness and erection, involuntary tremors, and reduced spontaneous activity. The body weight of each group was recorded during model establishment and drug administration. At the time of model establishment, a decrease in animal body weight was observed. Figure 10 A) and H&E staining of the substantia nigra in both the model group and the control group, such as Figure 9 As shown, obvious lesions were found in the model group, indicating that the Parkinson's disease model was successful.

[0106] 2. Drug administration and testing

[0107] After successful model establishment, the gavage group received 10 mg / kg of rutin solution via gavage, while the nasal administration groups (10 mg / kg and nasal administration of rutin gel, respectively) received rutin solution and thermosensitive gel via nasal administration. The blank control group and the model group received physiological saline. Administration continued for 10 days. Rotary bar and string hanging tests were performed on days 15 and 16, and the open field test was performed on day 17. Biochemical indicators were tested after the behavioral experiments.

[0108] Mice were pre-trained to rotarod before using MPTP to establish a Parkinson's disease model. After drug treatment, mice were also pre-trained to rotarod, and then observed on the rotarod, with the time spent on the rod recorded. Figure 10 B). The results showed that compared with the control group, the time spent on the rotundus in the model group was significantly shortened. After drug treatment, the time spent on the rotundus in mice increased, with the most significant difference in the intranasal administration group. Among them, the intranasal administration of Uncaria rhynchophylla nanomicelle gel group had the longest extension time, indicating that the gel preparation had a better therapeutic effect.

[0109] Mice used in the wire-hanging experiment should be acclimatized to the laboratory one hour before the test. They are placed on a wire mesh so they can grip it with their limbs. The mesh is then flipped so the mice are hanging upside down. A timer is started, and the duration the mouse can remain on the mesh is measured. Figure 10C). The maximum test length is 2 minutes and 30 seconds. If the animal does not escape the grid after reaching the maximum test length, it should be removed from the apparatus and returned to its cage. The test should be conducted three times daily. Mice should be given a rest period between each test. The test can be repeated at intervals appropriate to the strain used. With a cutoff time of 150 seconds, most mice in the control group were able to reach this point. Compared to the control group, the fall time in the model group was significantly shorter. After drug treatment, the descent latency of mice increased, with the most significant difference observed in the intranasal administration group. The intranasal administration of Uncaria rhynchophylla nanomicelle gel group showed the best recovery, indicating that the gel formulation had a better therapeutic effect.

[0110] The open field test is mainly used to observe the spontaneous movement ability, exploratory behavior in novel environments, stress, mania, anxiety, and depressive behavior of experimental animals. An open field device with a length of 50 cm, a width of 50 cm, and a height of 40 cm is used. Four virtual squares of equal area are marked at the bottom of the device, and a suitable scale is selected. Before the formal experiment, mice are placed in the open field device for 5-10 minutes to allow them to adapt to the environment. Then, with the help of an autonomous photography system, the spontaneous activities of the mice are recorded continuously for 300 seconds. The results are shown in Figures 10D, F, and I. Compared with the control group, the total distance and central movement distance of the model group were significantly reduced, while those of the drug-treated groups increased. The group receiving intranasal administration of Uncaria rhynchophylla nanomicelle gel showed the best recovery, indicating that the gel preparation had a better therapeutic effect.

[0111] Bilateral striatum samples were collected, and an appropriate amount of homogenate was added. After centrifugation, the supernatant was extracted, and malondialdehyde (MDA) activity was determined using a BCA protein assay kit. Similarly, glutathione peroxidase (GPX) and superoxide dismutase (SOD) activities were measured. The results are shown in Figures 10E, G, and H. Compared to the control group, the MDA level in the model group was significantly increased, but after ten days of administration of Uncaria rhynchophylla solution and gel, the MDA level decreased. Compared to the control group, the glutathione peroxidase activity in the model group was significantly decreased. After drug treatment, the enzyme activities of the nasally administered Uncaria rhynchophylla solution and nasally administered Uncaria rhynchophylla nanomicelle gel groups increased, showing a significant difference from the model group. Compared to the control group, the superoxide dismutase activity in the model group was significantly decreased. After drug treatment, the enzyme activities of the nasally administered Uncaria rhynchophylla solution and nasally administered Uncaria rhynchophylla nanomicelle gel groups increased, showing a significant difference from the model group.

[0112] Safety evaluation of Uncaria rhynchophylla nanomicelle thermosensitive gel

[0113] 1. Nasal mucosal toxicity evaluation

[0114] After the behavioral experiment was completed, the nasal cavity of the mice was dissected, and nasal mucosal tissue sections were prepared and H&E stained to observe morphological changes in the nasal mucosa. Results are shown below. Figure 11 The results, observed under a 40x light microscope, showed that in the control group, the nasal mucosa was intact, the pseudostratified ciliated columnar epithelial cells were neatly arranged and structurally complete, with a small number of scattered goblet cells, and the submucosal connective tissue and glands were evenly distributed, with no obvious vasodilation or congestion. Compared with the control group, the nasal gel group showed no obvious pathological behavior, indicating that the Uncaria rhynchophylla nanomicelle gel caused less damage to the nasal mucosa.

[0115] 2. Complete blood count and organ safety evaluation

[0116] Balb / c mice weighing 18–22 g were randomly divided into two groups according to body weight: a control group and a nasal administration group of Uncaria rhynchophylla thermosensitive gel, with 6 mice in each group. The dosage for both groups was 10 mg / kg. Whole blood (50–100 μL) was collected from each animal in blood collection tubes containing EDTA anticoagulant for routine blood analysis. Freshly dissected organs were fixed with 4% paraformaldehyde and prepared into pathological sections using H&E staining. Pathological changes in each tissue were observed. Compared with the normal group, there were no significant differences in white blood cell count, red blood cell count, hemoglobin, platelet count, lymphocyte count, monocyte count, and neutrophil count in the nasal gel group, and all were within the normal range, as shown in Table 5. Pathological sections of important organs such as the heart, liver, spleen, lungs, and kidneys were prepared. Figure 12 As shown, no obvious pathological changes were found. This indicates that the rhizoma sarcodile nanomicelle gel is non-toxic to mice.

[0117] Table 5 Important parameters of complete blood count

[0118]

Claims

1. A rhynchophylline nano-micelle thermosensitive gel preparation for nasal administration, characterized in that: The invention is prepared with rhynchophylline nano-micelles as active substance, poloxamer as temperature-sensitive gel material, hydroxypropyl-β-cyclodextrin as absorption promoter, polyethylene glycol 6000 (PEG 6000) and hydroxypropyl methylcellulose as bioadhesives and water; each 100 mL of the thermosensitive gel for nasal administration contains 0.1-1 g of rhynchophylline nano-micelles, 18-21 g of poloxamer 407, 0.5-2.5 g of poloxamer 188, 1-3 g of hydroxypropyl-β-cyclodextrin, 1-5 g of polyethylene glycol 6000, 0.3-0.8 g of hydroxypropyl methylcellulose, and the balance is water.

2. The thermosensitive gel preparation for nasal administration of rhynchophylline nano-micelles according to claim 1, characterized in that: Each 100 mL of the thermosensitive gel for nasal administration contains 0.2 g of rhynchophylline nano-micelles, 20 g of poloxamer 407, 2 g of poloxamer 188, 1 g of polyethylene glycol 6000, 3 g of hydroxypropyl-β-cyclodextrin, 0.8 g of hydroxypropyl methylcellulose, and the balance is water.

3. The thermosensitive gel preparation for nasal administration of rhynchophylline nano-micelles according to claim 1, characterized in that: The rhynchophylline nano-micelles are obtained by the following preparation method: rhynchophylline, disodium glycyrrhizinate and tannic acid are added into a ball mill according to a mass ratio of 1:5:1 to 1:10:1, and then ball mill beads are added as ball milling media. After sealing, the ball mill is placed on a rolling ball mill for mechanical grinding for 2 to 16 hours to obtain rhynchophylline nano-micelles.

4. The thermosensitive gel preparation for nasal administration of rhynchophylline nano-micelles according to claim 3, characterized in that: The ball milling jar is a polytetrafluoroethylene ball milling jar, and the ball milling beads are stainless steel ball milling beads.

5. The method for preparing the thermosensitive gel preparation of rhynchophylline nano-micelles for nasal administration according to any one of claims 1 to 4, characterized in that: First, hydroxypropyl methylcellulose was dispersed in water at 4°C for 12 h, and then poloxamer 407, poloxamer 188, polyethylene glycol 6000, and hydroxypropyl-β-cyclodextrin were added, and the mixture was allowed to stand at 4°C until a clear, transparent, lump-free, bubble-free, and uniformly dispersed thermosensitive gel solution was obtained. The rhynchophylline nanomicelle solution was added, and the mixture was made up to 100 mL with water and stirred evenly to obtain a thermosensitive gel preparation for nasal administration containing rhynchophylline nanomicelles.

6. The method for preparing the rhynchophylline nano-micelle solution according to claim 4, characterized in that: The method for obtaining the rhynchophylline nano-micelle solution is to mix the rhynchophylline nano-micelles with water, place the mixture in an air bath shaker at 37° C., and shake at 200 rpm for 3 hours to obtain the rhynchophylline nano-micelle solution.

7. Use of the thermosensitive gel preparation for nasal administration of rhynchophylline nano-micelles according to claim 1 in the preparation of drugs for treating neurodegenerative diseases.

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