Traditional Chinese medicine supramolecular nanoparticle, preparation method and application thereof

Abstract

The application belongs to the field of traditional Chinese medicines, and discloses a traditional Chinese medicine supramolecular nanoparticle, which is prepared from Uncaria as a raw medicinal material, is extracted by heating reflux with 68-72% ethanol, is filtered, and is dried after being dialyzed after being dispersed in pure water, centrifuged, resuspended, and centrifuged again. The traditional Chinese medicine supramolecular nanoparticle has uniform particle size, a narrow particle size distribution, and high stability. The nanoparticle has obvious anti-nerve oxidation and obvious nerve protection effects according to in-vitro cell activity detection. The nanoparticle has good in-vivo nerve protection effect, can improve memory impairment and cognitive impairment, and can prevent hippocampal neuron reduction. Compared with Uncaria effective parts, the effective administration dose of the traditional Chinese medicine supramolecular nanoparticle is obviously reduced. The application also discloses a use of the traditional Chinese medicine supramolecular nanoparticle in preparation of a medicine for treating nervous system diseases.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine, and relates to a supramolecular nanoparticle of traditional Chinese medicine, its preparation method and uses. Background Technology

[0002] Neurodegenerative diseases are characterized by dysfunction of neurons in the central or peripheral nervous system, and are often difficult to treat once diagnosed. Currently, the main treatments for neurodegenerative diseases are small molecule drug therapy and immunotherapy, but these can only alleviate symptoms and cannot stop the progression of the disease. Furthermore, immunotherapy is expensive and exhibits significant individual variability.

[0003] Traditional Chinese medicine, including formulas like Gou Teng San and Tianma Gou Teng Yin, has a long history of using it to treat nervous system diseases. Uncaria rhynchophylla (Miq.) Miq. ex Havil., the principal ingredient in Gou Teng compound formulas, is derived from plants in the Rubiaceae family, specifically the hooked stems and branches. The effects of indole alkaloids in Gou Teng, such as rhynchophylline and isorhynchophylline, on the nervous system have been widely reported. Both the active components of Gou Teng and its combination with other drugs exhibit protective effects on the degenerative nervous system, including anti-neuroinflammatory, anti-oxidative stress, and neuroprotective effects.

[0004] Traditional Chinese medicine (TCM) extracts often exhibit complex pharmacodynamic substances, poor solubility, and low bioavailability. Supramolecular technology for TCM offers the advantages of clearly defining the pharmacodynamic material basis of TCM and explaining compatibility theories. During the decoction process, small molecules of TCM self-assemble to form supramolecular structures. Different conformations of monomeric components can lead to different supramolecular morphologies induced by non-chemical bonds through intermolecular interactions, thereby affecting biological activity. Summary of the Invention

[0005] The inventors used Uncaria rhynchophylla as a raw material and conducted in vitro neuroprotective activity tests on total extracts from different ethanol-water extracts through dual characterization and activity targeting. They screened out ethanol extracts that could improve the cell viability of oxidatively damaged nerve cells to a greater extent, and further extracted nanoparticles with neuroprotective effects from these ethanol extracts.

[0006] The purpose of this invention is to provide a supramolecular nanoparticle of traditional Chinese medicine. It is made from Uncaria rhynchophylla as raw material. First, it is extracted by heating and reflux with 68-72% ethanol, filtered, and the solvent is removed from the extract to obtain a total extract. Then, the total extract is ultrasonically dispersed in pure water, centrifuged at high speed, the precipitate is resuspended in pure water, and centrifuged at low speed to remove large particles. The supernatant is then placed in a dialysis bag, dialyzed, and dried to obtain a lyophilized powder.

[0007] Another objective of this invention is to provide a method for preparing the aforementioned supramolecular nanoparticles, comprising: using Uncaria rhynchophylla as the raw material, using 68-72% ethanol (by volume) as the extraction solvent, heating and reflux extraction, filtering, removing the solvent from the extract to obtain a total extract, ultrasonically dispersing the total extract in pure water, centrifuging at high speed, discarding the supernatant, resuspending the precipitate in pure water, centrifuging at low speed, then placing the supernatant into a dialysis bag, dialyzing in pure water, taking the solution from the dialysis bag, freeze-drying, and obtaining traditional Chinese medicine supramolecular nanoparticles (i.e., Uncaria rhynchophylla NPs).

[0008] The ratio of Uncaria rhynchophylla to the extraction solvent is 1:10-1:25 g / mL, preferably 1:20 g / mL.

[0009] Preferably, the extraction solvent is 70% ethanol by volume.

[0010] The number of heating and reflux extractions is 1-3 times, preferably 2 times; the time for each heating and reflux extraction is 0.5-2 hours, preferably 1 hour.

[0011] The extract can be first subjected to vacuum distillation to remove most of the solvent, and then dried at room temperature to obtain the total extract; the vacuum concentration temperature is 50-70℃.

[0012] The ratio of total extract to pure water is 1:50 g / mL. The ultrasonic dispersion is performed at a frequency of 20-30 kHz for 20-30 min.

[0013] The ratio of the precipitate (based on total extract) to pure water is 1:50 g / mL.

[0014] The high-speed centrifugation process involves centrifuging at a temperature of 2-8℃ and a rotation speed of 13000-15000 rpm for 15-20 minutes.

[0015] Preferably, the high-speed centrifugation process is performed at a temperature of 4°C and a rotation speed of 14,000 rpm for 20 minutes.

[0016] The low-speed centrifugation process is carried out at a temperature of 2-8℃ and a rotation speed of 4000-6000 rpm for 5-20 minutes.

[0017] Preferably, the low-speed centrifugation process is performed at a temperature of 4°C and a rotation speed of 5000 rpm for 5 minutes.

[0018] The dialysis bag has a molecular weight cutoff of 2000-5000 Da, preferably 3500 Da; the dialysis time is 20-28 hours, preferably 24 hours, and the pure water is changed twice during the dialysis.

[0019] The main components of the aforementioned traditional Chinese medicine supramolecular nanoparticles are acetyl catalol, chlorogenic acid, and rhynchophylline.

[0020] The aforementioned supramolecular nanoparticles of traditional Chinese medicine have a particle size of 152.1 ± 1.8 nm, a polydispersity index (PDI) of 0.171 ± 0.054, and a zeta potential of -24.9 ± 3.1 mV. The inventors adjusted different pH values ​​to simulate the in vivo environment to test the stability of the supramolecular nanoparticles of traditional Chinese medicine in vivo. In simulated in vivo metabolism, the nanoparticles were stable in the stomach for the first 2 hours and remained stable in the small intestine and colon, exhibiting high digestive stability. This indicates that the supramolecular nanoparticles of traditional Chinese medicine of the present invention have uniform particle size, narrow particle size distribution, and high stability.

[0021] Another objective of this invention is to provide an effective component of Uncaria rhynchophylla, which is obtained by extracting Uncaria rhynchophylla from raw material by heating and refluxing with 68-72% ethanol, filtering, removing the solvent from the extract to obtain a total extract, or by drying the total extract to obtain a powder.

[0022] Preferably, the effective part of Uncaria rhynchophylla is obtained by extracting Uncaria rhynchophylla from raw material by heating and refluxing with 70% ethanol, filtering, removing the solvent from the extract to obtain a total extract, or by drying the total extract to obtain a powder.

[0023] In vitro cell viability tests showed that the supramolecular nanoparticles of the traditional Chinese medicine and the effective parts of Uncaria rhynchophylla had significant anti-neurooxidative effects and significant neuroprotective effects. The supramolecular nanoparticles of the traditional Chinese medicine and the effective parts of Uncaria rhynchophylla had good in vivo neuroprotective effects, which could improve memory and cognitive impairment and prevent the reduction of hippocampal neurons. Moreover, compared with the effective parts of Uncaria rhynchophylla, the effective dosage of the supramolecular nanoparticles of the traditional Chinese medicine was significantly reduced.

[0024] Another object of the present invention is to provide the use of the aforementioned supramolecular nanoparticles of traditional Chinese medicine or the effective parts of the aforementioned Uncaria rhynchophylla in the preparation of drugs for treating nervous system diseases.

[0025] The neurological diseases mentioned include Alzheimer's disease, Parkinson's disease, cerebral hemorrhage, and epilepsy, especially Alzheimer's disease, Parkinson's disease, cerebral hemorrhage, and epilepsy related to nerve damage.

[0026] Another object of the present invention is to provide the use of the aforementioned supramolecular nanoparticles of traditional Chinese medicine or the effective parts of the aforementioned Uncaria rhynchophylla in the preparation of anti-nerve damage drugs.

[0027] The nerve damage described is oxidative nerve damage.

[0028] A pharmaceutical composition comprising the traditional Chinese medicine supramolecular nanoparticles described in this invention and a pharmaceutically acceptable carrier.

[0029] The dosage form of the pharmaceutical composition is an oral preparation.

[0030] Compared with the prior art, the present invention has the following advantages:

[0031] (1) This invention, guided by both efficacy and characterization, uses enrichment and purification methods to obtain supramolecular nanoparticles of traditional Chinese medicine from Uncaria rhynchophylla, clarifying the pharmacodynamic material basis of Uncaria rhynchophylla's neuroprotective effect and improving the scientific basis of traditional Chinese medicine in treating nervous system diseases. Compared with 70% total extract of Uncaria rhynchophylla, the effective dosage of nanoparticles is significantly reduced.

[0032] (2) The preparation method of the traditional Chinese medicine supramolecular nanoparticles of the present invention is simple and easy to operate. The obtained particle size has good uniformity, high storage stability, and is soluble in water.

[0033] (3) The supramolecular nanoparticles of traditional Chinese medicine in this invention have good digestive stability under physiological conditions and can meet the absorption conditions of oral preparations.

[0034] (4) The supramolecular nanoparticles of traditional Chinese medicine in this invention have good in vivo safety and will not cause damage to tissues and organs.

[0035] (5) The supramolecular nanoparticles of traditional Chinese medicine in this invention have good neuroprotective effects and can significantly inhibit oxidative stress in nerve cells.

[0036] (6) The supramolecular nanoparticles of traditional Chinese medicine in this invention have good in vivo neuroprotective effects, which can improve memory and cognitive impairment and prevent the reduction of hippocampal neurons. Attached Figure Description

[0037] Figure 1 Effects of different concentrations of H2O2 on cell viability; where, compared with the control group, ns indicates no significant difference, ****P<0.0001.

[0038] Figure 2 Tyndall effect diagram of supramolecular nanoparticles of traditional Chinese medicine.

[0039] Figure 3 Dynamic light scattering columnar distribution of supramolecular nanoparticles of traditional Chinese medicine.

[0040] Figure 4 Transmission electron microscopy (TEM) images of supramolecular nanoparticles of traditional Chinese medicine; the left image shows the TEM distribution of Uncaria rhynchophylla NPs aqueous solution (concentration 1 mg / mL) at a 200 nm scale bar, and the right image shows the TEM distribution of Uncaria rhynchophylla NPs aqueous solution at a 100 nm scale bar.

[0041] Figure 5 UV spectra of supramolecular nanoparticles of traditional Chinese medicine and total extract of 70% ethanol.

[0042] Figure 6 Infrared-Fourier spectra of supramolecular nanoparticles of traditional Chinese medicine and total extract of 70% ethanol.

[0043] Figure 7 X-ray diffraction patterns of supramolecular nanoparticles of traditional Chinese medicine and total extract of 70% ethanol.

[0044] Figure 8 Results of in vitro detection of the effect of supramolecular nanoparticles of traditional Chinese medicine on enhancing the viability of cells damaged by oxidation; among them, compared with the control group, ****P<0.0001; compared with the model group, ns represents no significant difference. #### P < 0.0001.

[0045] Figure 9 Liquid chromatogram of supramolecular nanoparticles of traditional Chinese medicine.

[0046] Figure 10 Total ion flow chromatogram of supramolecular nanoparticles of traditional Chinese medicine in positive and negative ion modes.

[0047] Figure 11 Results of digestibility testing of supramolecular nanoparticles of traditional Chinese medicine; where A: particle size, B: PDI.

[0048] Figure 12 Results of storage stability testing of supramolecular nanoparticles of traditional Chinese medicine.

[0049] Figure 13 Results of water maze experiments using model-based drug delivery methods; where A: water maze trajectory diagram, B: percentage of time spent in the target quadrant, and C: escape latency.

[0050] Figure 14 : Results of H&E staining safety test in histopathology.

[0051] Figure 15 : Detection of the neuroprotective efficacy of supramolecular nanoparticles of traditional Chinese medicine in vivo - Water maze test results; where A: Water maze trajectory diagram, B: Percentage of time spent in the target quadrant, C: Number of times the platform was crossed, D: Escape latency; Compared with the control group, *P<0.5, **P<0.01, ***P<0.001; Compared with the model group, ### P < 0.001.

[0052] Figure 16 : Detection of neuroprotective efficacy of supramolecular nanoparticles of traditional Chinese medicine in vivo - Y-maze experiment results; where A: Y-maze trajectory diagram, B: spontaneous alternation rate; compared with the model group, *P<0.05, **P<0.01.

[0053] Figure 17Results of in vivo neuroprotective efficacy detection of supramolecular nanoparticles of traditional Chinese medicine - novel object recognition experiment; where A: novel object recognition trajectory map, B: novelty preference index, C: relative discrimination index; compared with the control group, *P<0.05.

[0054] Figure 18 In vivo neuroprotective efficacy detection of supramolecular nanoparticles of traditional Chinese medicine - Nissl staining analysis of hippocampal neuronal damage; where A: scan images of CA1, CA3, and DG regions of the hippocampus, B: quantitative map of hippocampal neurons; where, compared with the control group, **P<0.01, ***P<0.001, ****P<0.0001; compared with the model group, ## P < 0.01, ### P < 0.001, #### P < 0.0001; *The underlined part indicates a significant difference between the two. Detailed Implementation

[0055] Example 1

[0056] Preparation of ethanol extracts of different concentrations from the traditional Chinese medicine Uncaria rhynchophylla

[0057] Take 50g of dried Uncaria rhynchophylla (i.e., stems and branches with hooks), add 1L of 60% ethanol, soak at room temperature for 30min, reflux for 1h, filter with gauze, add 1L of 60% ethanol to the residue again and reflux for 1h, combine the two extracts, concentrate under reduced pressure at 60℃ until most of the solvent is evaporated, and then evaporate naturally at room temperature to obtain 4.62g of total extract, freeze-dry to obtain 60% ethanol total extract.

[0058] Take 50g of dried Uncaria rhynchophylla, add 1L of 70% ethanol, soak at room temperature for 30min, reflux for 1h, filter with gauze, add 1L of 70% ethanol to the residue again and reflux for 1h, combine the two extracts, concentrate under reduced pressure at 60℃ until most of the solvent is evaporated, and evaporate naturally at room temperature to obtain 8.38g of total extract, freeze-dry to obtain 70% ethanol total extract.

[0059] Take 50g of dried Uncaria rhynchophylla, add 1L of 80% ethanol, soak at room temperature for 30min, reflux for 1h, filter with gauze, add 1L of 80% ethanol to the residue again and reflux for 1h, combine the two extracts, concentrate under reduced pressure at 60℃ until most of the solvent is evaporated, and then evaporate naturally at room temperature to obtain 7.26g of total extract, freeze-dry to obtain 80% ethanol total extract.

[0060] Take 50g of dried Uncaria rhynchophylla, add 1L of 95% ethanol, soak at room temperature for 30min, reflux for 1h, filter with gauze, add 1L of 95% ethanol to the residue again and reflux for 1h, combine the two extracts, concentrate under reduced pressure at 60℃ until most of the solvent is evaporated, and then evaporate naturally at room temperature to obtain 12.67g of total extract, freeze-dry to obtain 95% ethanol total extract.

[0061] Characterization of total extract

[0062] Weigh 2 mg of each lyophilized powder of ethanol extracts at different concentrations and add 1 mL of pure water. Disperse the powders in the pure water by vortexing and sonication (20 kHz frequency, 5 min). Filter the solutions using a 0.45 μm aqueous syringe filter. Perform dynamic light scattering analysis on the aqueous solutions prepared from the ethanol extracts at different concentrations. The particle size, PDI, and Zeta potential are shown in Table 1. PDI reflects the particle size distribution width; a smaller value indicates more uniform particle size and a more concentrated distribution. Zeta potential is a measure of the strength of the repulsive or attractive forces between particles; a higher absolute value of Zeta potential indicates a more stable system. The 95% ethanol extract has a larger particle size and exhibits aggregation. The particle size of other total extracts is around 200 nm. However, the 70% ethanol extract has a larger absolute value of Zeta potential compared to the other extracts, indicating better stability.

[0063] Table 1. Particle size, PDI and potential of total extract

[0064]

[0065] Neuroprotective activity assay of total extract

[0066] A human neuroblastoma neuronal injury model induced by H2O2 was established using the MTT assay. 30% H2O2 was stored protected from light and heat. Before use, a 3mM stock solution of H2O2 was prepared in serum-free DMEM medium, and then serially diluted with serum-free DMEM medium to H2O2 concentrations of 800μM, 600μM, 300μM, 200μM, and 100μM. Human neuroblastoma SH-SY5Y cells grown to the logarithmic growth phase were harvested, digested with trypsin, and then subjected to a 5×10⁻⁶ ppm solution. 4Cells were seeded at a density of approximately 5000 cells / mL in 96-well plates. 100 μL of DMEM medium containing 10% serum was added to each well. Six wells were in vitro per group. Cells were incubated overnight. After cell attachment, the supernatant was removed. For the experimental group, 100 μL of serum-free DMEM medium with different concentrations of H2O2 was added, and incubation continued for 8 hours. For the control group, 100 μL of serum-free DMEM medium without H2O2 was added, and incubation continued for 8 hours. The 96-well plates were then removed, the supernatant discarded, and 100 μL of 0.5 mg / mL MTT solution was added in the dark. Incubation continued for 2 hours, the supernatant discarded, and 100 μL of DMSO was added. The absorbance of each well was measured at 492 nm using a Thermo microplate reader, and the values ​​were recorded. Cell viability was calculated.

[0067] Cell viability (%) = (Experimental group absorbance value / control group average absorbance value) × 100%

[0068] Cell viability after H2O2-induced oxidative damage is shown in the figure. Figure 1 The cell survival rate of the experimental group with H2O2 concentration of 600 μM was about 50%, so H2O2 concentration of 600 μM incubation for 8 h was selected as the in vitro oxidative damage model.

[0069] The neuroprotective activity of different concentrations of ethanol extracts was detected using the MTT assay. Lyophilized powders of different concentrations of ethanol extracts were accurately weighed and dissolved in dimethyl sulfoxide (DMSO) using sonication to prepare solutions with a concentration of 120 mg / mL. These solutions were then serially diluted with serum-free DMEM medium on a clean bench to concentrations of 250 μg / mL, 125 μg / mL, and 62.5 μg / mL. Human neuroblastoma SH-SY5Y cells grown to the logarithmic growth phase were digested with trypsin and then subjected to a 5 × 10⁻⁶ ppm solution. 4Cells were seeded at a density of approximately 5000 cells / mL in 96-well plates, with 100 μL of DMEM medium containing 10% serum added to each well. Six wells were prepared per group. After overnight culture in a CO2 incubator, once cells have adhered, the supernatant is removed, and 100 μL of drug-containing solutions of different concentrations are added for further incubation for 3 hours. Both the control and model groups are incubated for 3 hours with 100 μL of DMEM medium (without drug solution and serum). The 96-well plates are then removed, and the supernatant is discarded. Different concentrations of drug-containing solutions and H2O2 solutions are added to the drug-containing groups simultaneously, bringing the final H2O2 concentration to 600 μM and the final drug concentrations to 250 μg / mL, 125 μg / mL, and 62.5 μg / mL, respectively. The control group is incubated with 100 μL of serum-free DMEM medium, and the model group is incubated with 100 μL of H2O2 solution (final concentration 600 μM). Incubation continues for 8 hours. The 96-well plates are then removed, the supernatant is discarded, and 100 μL of 0.5 mg / mL MTT solution is added in the dark. Incubation continues for 2 hours, the supernatant is discarded, and 100 μL of [unclear text - possibly a specific ingredient or solution] is added. The absorbance of each well in DMSO solution was measured at 492 nm using a Thermo microplate reader. The values ​​were recorded, and the cell viability was calculated.

[0070] Cell viability (%) = (Experimental group absorbance value / control group average absorbance value) × 100%

[0071] The experimental group consisted of a treatment group and a model group.

[0072] Table 2. Protective effects of different total extracts on oxidatively damaged nerve cells

[0073]

[0074] Table 2 shows the cell viability of different concentrations of ethanol extracts from Uncaria rhynchophylla. It can be seen that the 70% ethanol total extract has a higher cell viability and better neuroprotective activity compared with other total extracts.

[0075] Based on the characterization results, the most likely candidate for active nanoparticles is the 70% ethanol total extract.

[0076] Example 2

[0077] I. Preparation of Uncaria rhynchophylla NPs

[0078] Take 50g of dried Uncaria rhynchophylla, add 1L of 70% ethanol, soak at room temperature for 30min, reflux for 1h, filter with gauze, add 1L of 70% ethanol to the residue again and reflux for 1h, combine the two extracts, concentrate under reduced pressure at 60℃ until most of the solvent is evaporated, and then evaporate naturally at room temperature to obtain a total extract of 70% ethanol; disperse 2g of the total extract of 70% ethanol in 100mL of pure water, place in an ultrasonic cleaner, ultrasonic frequency at 20kHz for 20min, then centrifuge at 14000rpm for 20min at 4℃, discard the supernatant, and resuspend the precipitate in 100mL of pure water by pipette, and centrifuge at 4℃. Centrifuge at 5000 rpm for 5 min to remove the precipitate and retain the supernatant. Then, put the supernatant into a dialysis bag with a molecular weight cutoff of 3500 Da and dialyze it in 1 L of pure water for 24 h to remove free substances. Change the pure water every 8 h. Take the solution in the dialysis bag to obtain a solution containing traditional Chinese medicine supramolecular nanoparticles (i.e., Uncaria rhynchophylla NPs). Freeze dry in a freeze dryer at a temperature of about -60℃ and a vacuum value of about 0.3 mbar to obtain freeze-dried powder, i.e., Uncaria rhynchophylla supramolecular nanoparticles (Uncaria rhynchophylla NPs), and store at 4℃ for later use.

[0079] II. Characterization of Uncaria rhynchophylla NPs

[0080] The NPs of Uncaria rhynchophylla were characterized by methods including Tyndall effect, transmission electron microscopy, ultraviolet spectroscopy, infrared spectroscopy and X-ray diffraction.

[0081] Redissolving 1 mg of Uncaria rhynchophylla NPs lyophilized powder in 1 mL of pure water to prepare a 1 mg / mL Uncaria rhynchophylla NPs solution, a significant Tyndall effect (e.g., ...) was observed. Figure 2 (As shown).

[0082] The particle size, PDI, and Zeta potential in the solution were determined using a Malvern particle size analyzer. The particle size distribution of Uncaria rhynchophylla NPs is shown below. Figure 3 As shown, the Uncaria rhynchophylla NPs have a uniform particle size distribution, with a particle size of 152.1±1.8 nm, a polydispersity index (PDI) of 0.171±0.054, and a zeta potential of -24.9±3.1 mV.

[0083] Transmission electron microscopy image of Uncaria rhynchophylla NPs as shown below Figure 4 As shown, the morphology of Uncaria rhynchophylla NPs is uniform spherical nanoparticles.

[0084] The UV spectrum of Uncaria rhynchophylla NPs is shown in [reference needed]. Figure 5 The results showed that, compared to the 70% ethanol extract of Uncaria rhynchophylla, the UV absorption of Uncaria rhynchophylla NPs in the 300-350 nm range disappeared; the infrared spectrum is shown below. Figure 6 This indicates that, compared to the 70% ethanol total extract of Uncaria rhynchophylla, the NPs of Uncaria rhynchophylla showed 1440 and 1520 cm⁻¹.-1 The infrared absorption peaks correspond to the C=N and C=C stretching vibrations on the imidazole ring, indicating the formation of new bonds. The X-ray diffraction pattern of Uncaria rhynchophylla NPs is shown below. Figure 7 The results indicate that the 70% ethanol extract of Uncaria rhynchophylla contains crystalline peaks, while the crystalline peaks in Uncaria rhynchophylla NPs disappear and a broad peak of about 15-25 degrees appears, which is an amorphous scattering peak. This indicates that there are some ordered structures in the Uncaria rhynchophylla NPs sample, indicating the formation of nanoparticles.

[0085] III. Detection of Neuroprotective Activity of Uncaria rhynchophylla NPs

[0086] The neuroprotective activity of Uncaria rhynchophylla NPs was detected by the MTT assay.

[0087] Accurately weigh the lyophilized Uncaria rhynchophylla NPs powder and prepare a solution with a concentration of 100 mg / mL using dimethyl sulfoxide (DMSO) via ultrasonication. Then, on a clean bench, use serum-free DMEM medium to serially dilute the solution to concentrations of 50 μg / mL, 25 μg / mL, and 12.5 μg / mL.

[0088] Human neuroblastoma SH-SY5Y, grown to the logarithmic growth phase, was digested with trypsin and then treated with 5×10⁻⁶ mol / L saturates. 4 Cells were seeded at a density of approximately 5000 cells / mL in 96-well plates, with 100 μL of DMEM medium containing 10% serum added to each well. Six cells were cultured in duplicate per group. Cells were incubated overnight. After cell attachment, the supernatant was removed. The drug-treated groups were incubated for 3 hours with 100 μL of the previously prepared drug-containing solution (concentrations of 50 μg / mL, 25 μg / mL, and 12.5 μg / mL, respectively). The control and model groups were each incubated with 100 μL of serum-free DMEM medium. Under the same conditions, the cells were cultured, and the 96-well plates were removed. The supernatant was discarded. For each drug treatment group, 100 mL of a mixture of drug-containing solutions and H2O2 solutions of different concentrations were added simultaneously, so that the final H2O2 concentration in all drug treatment groups was 600 μM, and the final drug concentrations were 50 μg / mL, 25 μg / mL, and 12.5 μg / mL, respectively. The control group was given 100 μL of serum-free DMEM medium, and the model group was given 100 μL of H2O2 solution with a final concentration of 600 μM. The cells were incubated for another 8 h. The 96-well plates were then removed, the supernatant was discarded, and 100 μL of 0.5 mg / mL MTT solution was added in the dark. The cells were incubated for another 2 h, the supernatant was discarded, and 100 μL of LDMSO was added. The absorbance of each well at 492 nm was measured using a Thermo microplate reader, and the values ​​were recorded. Cell viability was calculated.

[0089] Cell viability (%) = (Experimental group absorbance value / control group average absorbance value) × 100%

[0090] The in vitro antioxidant activity of Uncaria rhynchophylla NPs is shown in [reference needed]. Figure 8 H2O2 induces oxidative damage in nerve cells. With increasing drug concentration, the cell viability of oxidatively damaged cells increases, demonstrating that Uncaria rhynchophylla NPs have a neuroprotective effect and can achieve better antioxidant effect when the drug concentration is significantly lower than that of 70% total ethanol extract.

[0091] IV. Chemical Composition Analysis of Uncaria rhynchophylla NPs

[0092] Preparation of the test sample solution: Accurately weigh Uncaria rhynchophylla NPs, sonicate with methanol to prepare a solution with a concentration of 5 mg / mL, centrifuge at 13000 rpm for 15 min at 4℃, take the supernatant as the test sample solution, and use it for high performance liquid chromatography and mass spectrometry analysis.

[0093] HPLC analysis conditions for Uncaria rhynchophylla NPs: Mobile phase: 0.1% formic acid water (A) — acetonitrile (B); Elution conditions: 0-5 min, 5% B-15% B; 5-40 min, 15% B-7% B; 40-42 min, 27% B-100% B; 42-52 min, 100% B; Flow rate: 1 mL / min; Detection wavelength: 254 nm and 210 nm; Injection volume: 15 μL.

[0094] Mass spectrometry analysis conditions for Uncaria rhynchophylla NPs: Mobile phase: 0.1% formic acid water (A) — acetonitrile (B); Elution conditions: 0-5 min, 5% B-15% B; 5-40 min, 15% B-27% B; 40-42 min, 27% B-100% B; 42-52 min, 100% B; Flow rate: 1 mL / min; Detection wavelength: 254 nm and 210 nm; Injection volume: 15 μL; Positive ion mode and negative ion mode.

[0095] The HPLC chromatogram of Uncaria rhynchophylla NPs is shown below. Figure 9 The HPLC-ESI-QTOF-MS / MS results are shown in the figure. Figure 10 Qualitative analysis results are shown in Table 3. A total of 14 chemical components were identified from Uncaria rhynchophylla NPs, among which the main chemical components were acetylcholine, chlorogenic acid, and rhynchophylline.

[0096] Table 3. Identification and analysis results of chemical composition of Uncaria rhynchophylla nanoparticles

[0097]

[0098] V. Digestion Stability Testing of Uncaria Rhizoma Supramolecular Nanoparticles

[0099] After calibrating the pH meter using standard calibration solutions of pH 4.00, pH 6.86, and pH 9.18 from Leici, a phosphate buffer solution was prepared to adjust the pH to 2.1 using NaH₂PO₄ solution, a phosphate buffer solution was prepared to adjust the pH to 6.8 using a mixture of NaH₂PO₄ and Na₂HPO₄ solutions, and a phosphate buffer solution was prepared to adjust the pH to 7.4 using Na₂HPO₄ solution. The pH 2.1 phosphate buffer solution was used to simulate the gastric acid environment in vivo, the pH 6.8 phosphate buffer solution was used to simulate the colonic environment in vivo, and the pH 7.4 phosphate buffer solution was used to simulate the small intestinal environment in vivo.

[0100] 10 mg of Uncaria rhynchophylla NPs lyophilized powder was dissolved in 1 mL of pure water to prepare a 10 mg / mL Uncaria rhynchophylla NPs solution. 200 μL of this solution was added to 1800 μL of phosphate buffer solution with pH values ​​of 2.1, 6.8, and 7.4. Particle size and PDI were measured at time points of 0 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, and 48 h. The particle size and PDI at each time point are shown below. Figure 11 As shown in the figure, in environments with pH 6.8 and 7.4, the particle size of Uncaria rhynchophylla NPs is less than 200 nm, and the particle size variation is relatively stable. However, in a solution simulating gastric acid at pH 2.1, the particle size of Uncaria rhynchophylla NPs is less than 200 nm for the first 4 hours, and then the particle size increases with a larger fluctuation. Considering that the oral formulation stays in the stomach for 1-2 hours, then in the small intestine for 2-4 hours, and finally reaches the colon for 4-6 hours, the Uncaria rhynchophylla NPs basically meet the absorption requirements of the oral formulation and can be stably present in the gastrointestinal tract during digestion.

[0101] VI. Storage stability testing of Uncaria rhynchophylla supramolecular nanoparticles

[0102] Accurately weigh 1 mg of Uncaria rhynchophylla NPs lyophilized powder, dissolve it in 1 mL of pure water to prepare an Uncaria rhynchophylla NPs solution with a concentration of 1 mg / mL, and place it in a 4℃ refrigerator. The particle size and dispersion index (PDI) were measured at different time points: 1 hour, 1 day, 3 days, 5 days, 7 days, and 14 days. The results are shown in the figure. Figure 12 This indicates that the particle size and particle size distribution of Uncaria rhynchophylla NPs did not change significantly after two weeks of storage, demonstrating high stability.

[0103] VII. Establishment of a Monosodium Glutamate-Induced Neurological Injury Model

[0104] KM mice were randomly divided into four groups: a control group (CTL), an intraperitoneal group (administered via intraperitoneal injection), a subcutaneous group (administered via subcutaneous injection), and a gavage group (administered via gavage), with six mice in each group. Monosodium glutamate (MSG) powder was dissolved in physiological saline and heated in a water bath until completely dissolved. Mice in the intraperitoneal, subcutaneous, and gavage groups were administered MSG for 10 consecutive days at a dose of 3 g / kg / day. Mice in the control group received no other treatment. The degree of neurological damage was determined using a water maze test to select the optimal modeling method.

[0105] Behavioral Experiment - Morris Water Maze Experiment: The Morris water maze is a circular pool with black inner walls. The water depth is 30 cm, and the water temperature is maintained at (22±2)℃. The room lighting is stable, with no strong direct sunlight shining into the pool. The pool is divided into four quadrants. A circular black platform is placed 35 cm from the pool wall in the target quadrant (quadrant 3), submerged 2 cm below the water surface. A small amount of ink is added to the pool water to make the platform invisible. The latency period is set to 90 seconds. KM mice are placed in the water from quadrant 1. A curtain is drawn to prevent the animals from being affected by other visible reference points. Simultaneously, another person clicks "start recording trajectory" on a computer. If the mouse finds the platform within 90 seconds and stays on it for more than 5 seconds, recording automatically stops. If the mouse does not find the platform within 90 seconds and stays on it for more than 5 seconds, the recording time is recorded as 90 seconds. The water maze experiment was conducted for 5 consecutive days. The first four days were the training phase, during which each mouse was placed on the platform in the third quadrant for 5 seconds to familiarize itself with the platform. Each mouse repeated the operation three times. The fifth day was the testing phase, during which each mouse performed the operation once.

[0106] The results of the water maze using three different modeling methods are as follows: Figure 13 As shown, the mice in the subcutaneous administration group exhibited disordered trajectories in the water maze experiment, with a significantly reduced time spent in the third quadrant and an increased latency period. Therefore, subcutaneous injection was chosen as the method for modeling.

[0107] VIII. In vivo neuroprotective pharmacological effects of Uncaria rhynchophylla supramolecular nanoparticles

[0108] 1) Animal grouping

[0109] KM mice were randomly divided into 8 groups: control group (C or CTL), model group (M or model), low-dose Uncaria rhynchophylla NPs group (M+D), medium-dose Uncaria rhynchophylla NPs group (M+Z), high-dose Uncaria rhynchophylla NPs group (M+G), 70% ethanol total extract of Uncaria rhynchophylla group (M+T), positive control group (M+Y) and negative control group (NA).

[0110] 2) Drug preparation and administration process

[0111] The lyophilized Uncaria rhynchophylla NPs powder was dissolved in physiological saline using ultrasound (20 kHz, 10 min) to prepare a 10 mg / mL Uncaria rhynchophylla NPs solution. Mice were administered the solution via gavage for 45 consecutive days. The low-dose group received 62.5 mg / kg / day of Uncaria rhynchophylla NPs, the medium-dose group received 125 mg / kg / day, and the high-dose group received 250 mg / kg / day. Starting on day 16, mice were subcutaneously injected with sodium monoglutamate solution (dissolved in physiological saline and aided by dissolution in an 80°C water bath) at a dose of 3 g / kg / day, every other day, for a total of 15 injections until day 45. The 70% ethanol total extract group of Uncaria rhynchophylla: The lyophilized powder of the 70% ethanol total extract was dispersed in physiological saline by ultrasound (ultrasound frequency 20kHz, ultrasound time 10min) to prepare a solution with a concentration of 10mg / mL. This was administered by gavage for 45 consecutive days at a dose of 250mg / kg / day. Starting on day 16, sodium monoglutamate solution was injected subcutaneously at a dose of 3g / kg / day, every other day, ending on day 45, for a total of 15 injections. The positive control group: Memantine hydrochloride was dissolved in physiological saline to prepare a solution with a concentration of 5mg / mL. This was administered by intraperitoneal injection for 45 consecutive days at a dose of 20mg / kg / day. Starting on day 16, sodium monoglutamate solution was injected subcutaneously at a dose of 3g / kg / day, every other day, ending on day 45, for a total of 15 injections. Negative control group: Uncaria rhynchophylla NPs solution (10 mg / mL) was administered by gavage for 45 consecutive days at a dose of 250 mg / kg / day. Subcutaneous injection of normal saline was started on day 16, with injections every other day until day 45, for a total of 15 injections. Control group: Normal saline was administered by gavage for 45 consecutive days. Subcutaneous injection of normal saline was started on day 16, with injections every other day until day 45, for a total of 15 injections.

[0112] 3) In vivo safety evaluation

[0113] Blood biochemistry and routine blood tests

[0114] After drug administration, blood was collected from the eyes of mice in each group in 1.5 mL EP tubes. The tubes were incubated at room temperature for 2 hours, centrifuged at 4000 rpm for 10 minutes, and the supernatant (serum) was collected and stored at -80℃ for the measurement of blood biochemical indicators. Liver function indicators such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and renal function indicators such as blood urea nitrogen (UREA) and creatinine (CREA) were measured. The results of the blood biochemical indicators are shown in Table 4. The results indicate that there were no significant changes in the levels of these indicators among the groups, suggesting that Uncaria rhynchophylla NPs do not affect liver and kidney function.

[0115] Blood was collected from the eyes of mice in each group in tubes containing EDTA anticoagulant. The blood was immediately mixed and stored at 4°C for the determination of complete blood count (CBC) parameters, including hemoglobin (HGB, normal range 110-143), white blood cells (WBC, normal range 0.8-6.8), platelets (PLT, normal range 450-1590), monocytes (Mon, normal range 0.0-0.3), lymphocytes (Lynph, normal range 0.7-5.7), and neutrophils (Gran, normal range 0.1-1.8). The CBC results are shown in Table 5. The results indicate that the levels of these parameters fluctuated within the normal range across groups, suggesting that Uncaria rhynchophylla NPs do not affect normal cellular function.

[0116] Table 4. Blood biochemical index detection

[0117]

[0118] Table 5. Blood routine index detection

[0119]

[0120] Histopathological H&E staining

[0121] After blood collection, the animals were dissected, and the heart, liver, spleen, lungs, and kidneys were separated. The tissues were rinsed with physiological saline, excess water was blotted with filter paper, and the weight was recorded. The tissues were then preserved in 4% paraformaldehyde at room temperature for fixation. The paraformaldehyde solution was washed away three times with running water for 5 minutes each time. The tissues were then dehydrated successively with 30% ethanol, 50% ethanol, 70% ethanol, 95% ethanol, and anhydrous ethanol. Clearing was then performed three times with 50% ethanol + xylene solution and xylene. Paraffin was melted and kept at 55°C. The tissues were immersed in paraffin three times, the first time for 15 minutes, and the next two times for 40 minutes each. The tissues were then embedded in high-purity paraffin sheets and incubated overnight at 4°C. Frozen sections were then prepared. The sections were successively soaked in xylene, anhydrous ethanol, 95% ethanol, 80% ethanol, 70% ethanol, and distilled water for 5 minutes each. They were then stained with 5% hematoxylin and eosin solution prepared with distilled water for 10 minutes, rinsed with distilled water, and placed in 1%... Rinse with NaHCO3 solution until blue-purple, stain with eosin for 1 min, wash with distilled water, then pass through acetone twice, and acetone-xylene (2:1) three times, 3-5 min each time. Finally, dehydrate with xylene for 5-10 min, mount with DPX resin mounting medium, and observe under a slide scanner. Histopathological H&E staining results are shown below. Figure 14 This indicates that there were no significant pathological differences among the tissues, suggesting that Uncaria rhynchophylla NPs are safe in vivo.

[0122] 4) Behavioral experimental testing

[0123] Water maze experiment: After drug administration, a water maze experiment was conducted for 5 consecutive days, including a training phase of 4 days and a testing phase on day 5. Water was added and the water temperature was adjusted to 22-24℃. A platform was placed in the third quadrant, with the water level approximately 2cm above the platform. Ink was added to the water to prevent non-specific responses. From day 1 to day 4, mice were released from the first quadrant, and their trajectories were recorded on a computer screen. The latency period was set to 90 seconds, and each test lasted a maximum of 90 seconds. The test was stopped when the mouse found the platform in the third quadrant and remained there for 5 seconds. During the training phase, after each mouse completed the test, it was manually placed on the platform for 5 seconds to familiarize it with the platform. After all mice completed the test, the test was repeated twice, for a total of three rounds. Day 5 was the testing phase. Each mouse only needed to be tested once, and after the test, it was not necessary to place it on the platform to familiarize it with the platform.

[0124] Figure 15 (A) is the trajectory diagram of each group during the water maze test assessment phase; (B) is the percentage of time each group spent in the target quadrant during the assessment phase; (C) is the number of times each group crossed platforms during the assessment phase; (D) is a schematic diagram showing the changes in the escape latency of each group over 5 consecutive days of water maze testing. The escape latency represents the time from when the mouse enters the water to when it successfully finds the platform. The shorter the time, the better the long-term memory of the experimental animal. A longer time spent in the target quadrant and a higher number of times the mouse crosses platforms also indicate that the experimental mouse has good memory.

[0125] Experimental results showed that the number of times mice in the model group crossed platforms was significantly lower than that in the blank control group, the time spent in the target quadrant was reduced, and the escape latency was significantly increased, indicating that MSG caused neurological damage in mice. High doses of Uncaria rhynchophylla NPs significantly improved MSG-induced memory impairment, manifested in shortening the escape latency and increasing the time spent in the target quadrant.

[0126] Y-maze experiment: After drug administration, the Y-maze experiment was conducted. The Y-maze consists of three arms (I, II, III) of equal length, arranged at 120-degree angles to each other, forming a radial maze box. Each arm is 23 cm long. There is a central area at the intersection of the three arms, with stable lighting above, without strong direct light. The three arms and the central area of ​​the Y-maze device were designated as areas 1, 2, 3, and the center, respectively. The experiment time was set to 5 minutes. Mice were placed in the central area, and the start button was clicked on the computer. The experimental trajectory of the mice was recorded within 5 minutes. After each test, the inner walls and bottom of the arms were cleaned to remove any residual animal scent. The spontaneous alternation rate was calculated for each group of mice after the experiment. The spontaneous alternation rate is used to assess short-term spatial memory; a higher spontaneous alternation rate indicates stronger short-term spatial working memory in the experimental animal.

[0127] Spontaneous Alternation Rate % = (Number of times entering arms 1, 2, and 3 in sequence + Number of times entering arms 1, 3, and 2 in sequence + Number of times entering arms 2, 3, and 1 in sequence + Number of times entering arms 2, 1, and 3 in sequence + Number of times entering arms 3, 1, and 2 in sequence + Number of times entering arms 3, 2, and 1 in sequence) / (Number of times entering arm 1 + Number of times entering arm 2 + Number of times entering arm 3 - 2) * 100%

[0128] Figure 16 (A) shows the trajectory diagram of the Y maze for each group, and (B) shows the spontaneous alternation rate for each group. The experimental results show that the percentage of spontaneous alternation in the model group was lower than that in the control group (P = 0.0977). MSG caused short-term memory impairment in mice, while high doses of Uncaria rhynchophylla NPs and total extracts could improve short-term memory impairment induced by monosodium glutamate.

[0129] Novel object recognition experiment: After drug administration, a novel object recognition experiment was conducted. The bottom and inside of a square box (100cm*100cm*40cm) were black. Two cube-shaped objects were placed in a fixed position near one side wall of the box. Both objects were familiar to the mice. The mice were allowed to become familiar with the objects for 5 minutes. Then, one of the square objects was replaced with a cylinder, which was recorded as the new object. The trajectory was recorded. After each test, the inner wall and bottom of the box were cleaned to remove any traces of the animals' previous scent. The novelty preference index and the relative discrimination index were calculated. Both quantify the cognitive ability of the experimental animals. The higher the value, the stronger the cognitive ability.

[0130] Novelty Preference Index = Novel Object Exploration Time / (Novel Object Exploration Time + Familiar Object Exploration Time)

[0131] Relative Discrimination Index = (Novelty Exploration Time - Familiarity Exploration Time) / Total Time

[0132] Figure 17 (A) is a trajectory diagram for new object recognition; (B) is a comparison diagram with the novelty preference index on the ordinate; (C) is a schematic diagram with the relative discrimination index on the ordinate. Experimental results show that the novelty preference index and relative discrimination index in the model group were significantly lower than those in the control group, proving that monosodium glutamate (MSG)-induced cognitive impairment in mice. Compared with the model group, the preference index and relative discrimination index of mice in the Uncaria rhynchophylla NPs group and the positive drug group showed an increasing trend, which can improve MSG-induced cognitive impairment to some extent.

[0133] 5) Nissl staining for histopathological examination

[0134] Prepare a 4% chloral hydrate solution using physiological saline and store it protected from light. Inject mice intraperitoneally with 9 μL / g of the 4% chloral hydrate solution. When the mice stop struggling, open the thoracic cavity to expose the heart. Cut open the right atrial appendage with surgical scissors, then insert a 5 mL syringe into the heart through the left ventricle to begin injecting physiological saline. Observe the blood flow from the atrial appendage until the blood flowing from the atrial appendage becomes physiological saline, indicating the end of cardiac perfusion. At this point, replace the physiological saline syringe with paraformaldehyde solution and inject about 10 mL. Fix the brain tissue, separate the brain tissue, and store it at room temperature in 4% paraformaldehyde for fixation. Wash off the paraformaldehyde solution three times with running water, 5 minutes each time. Dehydrate the tissue successively with 30% ethanol, 50% ethanol, 70% ethanol, 95% ethanol, and anhydrous ethanol. Then, dehydrate the tissue with 50% ethanol + xylene solution and xylene... The tissue was cleared by benzene treatment three times, then the paraffin was melted and kept at 55°C. The tissue was then immersed in the paraffin three times, the first time for 15 minutes and the next two times for 40 minutes. The tissue was then embedded in high-purity sheet paraffin and incubated overnight at 4°C. The tissue was then frozen and sectioned. The sections were then soaked in xylene, anhydrous ethanol, 95% ethanol, 80% ethanol, 70% ethanol and distilled water for 5 minutes each. Nissl staining was performed with 1% thionine for 30 minutes. The sections were washed with distilled water and then dehydrated by 70% ethanol, 80% ethanol and 95% ethanol for 2 minutes each. The sections were then soaked in anhydrous ethanol twice for 5 minutes each time and xylene twice for 10 minutes each time. The sections were then mounted with DPX resin mounting medium. Finally, the number of neurons in the CA1, CA3 and DG regions of the hippocampus was observed under a slide scanner to analyze the degree of hippocampal neuronal damage in each group.

[0135] Figure 18 (A) Nissl staining scans of the CA1, CA3, and DG regions of the hippocampus in each group; (B) quantitative maps of hippocampal neurons in each region. The results showed that the number of hippocampal neurons in the model group was significantly reduced compared to the control group. However, Uncaria rhynchophylla NPs (low, medium, and high doses), 70% ethanol extract, and positive control drugs increased the number of oxidatively damaged neurons to some extent. Furthermore, compared to 70% ethanol extract and positive control drugs, the high-dose Uncaria rhynchophylla NPs group showed a greater trend of increasing hippocampal neurons, indicating that Uncaria rhynchophylla NPs can improve the degree of nerve damage and has a superior neuroprotective effect compared to 70% ethanol extract and positive control drugs.

Claims

1. A supramolecular nanoparticle of traditional Chinese medicine with anti-nerve damage effect, characterized in that: It uses Uncaria rhynchophylla as the raw material and 68-72% ethanol as the extraction solvent. First, it is extracted by heating and reflux with 68-72% ethanol, filtered, and the solvent is removed from the extract to obtain the total extract. The total extract is then ultrasonically dispersed in pure water, centrifuged at high speed, the precipitate is resuspended in pure water, centrifuged at low speed, and the supernatant is placed in a dialysis bag for dialyzing and drying. The high-speed centrifugation is performed at 13000-15000 rpm for 15-20 minutes at 2-8℃; the low-speed centrifugation is performed at 4000-6000 rpm for 5-20 minutes at 2-8℃; the molecular weight cutoff of the dialysis bag is 2000-5000 Da; the dialysis time is 20-28 hours, with the pure water being changed twice during dialysis.

2. A method for preparing supramolecular nanoparticles according to claim 1, characterized in that: include: Using Uncaria rhynchophylla as the raw material, and 68-72% ethanol (v / v) as the extraction solvent, the extraction was carried out by heating and reflux. After filtration, the solvent was removed from the extract to obtain the total extract. The total extract was ultrasonically dispersed in pure water and centrifuged at high speed. The precipitate was resuspended in pure water and centrifuged at low speed. The supernatant was then placed in a dialysis bag, and the solution in the dialysis bag was freeze-dried to obtain supramolecular nanoparticles of traditional Chinese medicine. The high-speed centrifugation was carried out at 13,000-15,000 rpm for 15-20 min at 2-8℃. The low-speed centrifugation was carried out at 4,000-6,000 rpm for 5-20 min at 2-8℃. The molecular weight cutoff of the dialysis bag was 2,000-5,000 Da. The dialysis time was 20-28 h, and the pure water was changed twice during the dialysis.

3. The method for preparing supramolecular nanoparticles according to claim 2, characterized in that: The ratio of Uncaria rhynchophylla to the extraction solvent is 1:10-1:25 g / mL; the number of heating and reflux extractions is 1-3 times; and the time for each heating and reflux extraction is 0.5-2 h.

4. The method for preparing supramolecular nanoparticles according to claim 3, characterized in that: The ratio of Uncaria rhynchophylla to the extraction solvent is 1:20 g / mL; the extraction is performed twice by heating and reflux, and each heating and reflux extraction lasts for 1 hour.

5. The method for preparing supramolecular nanoparticles according to claim 2, characterized in that: The extraction solvent is 70% ethanol by volume.

6. The method for preparing supramolecular nanoparticles according to claim 2, characterized in that: The ratio of total extract to pure water is 1:50 g / mL; based on the total extract, the ratio of precipitate to pure water is 1:50 g / mL.

7. The method for preparing supramolecular nanoparticles according to claim 2, characterized in that: The molecular weight cutoff of the dialysis bag is 3500 Da.

8. The use of the traditional Chinese medicine supramolecular nanoparticles according to claim 1 in the preparation of anti-nerve damage drugs.

9. A pharmaceutical composition, characterized in that: The pharmaceutical composition contains the traditional Chinese medicine supramolecular nanoparticles as described in claim 1 and a pharmaceutically acceptable carrier.

Images