A composition and its use in the preparation of a medicament for treating Parkinson's disease

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a composition and application thereof in preparation of a medicine for treating Parkinson's disease. The composition comprises the following raw materials: palladium-loaded graphdiyne nanoscale enzyme, short small snake grass glycoside and tetrastigma hemsleyanum polysaccharide; the mass ratio of the palladium-loaded graphdiyne nanoscale enzyme, the short small snake grass glycoside and the tetrastigma hemsleyanum polysaccharide is 1: (0.3-0.5): (0.05-0.1). The composition can improve the levels of DA and 5-HT in the striatum of a Parkinson's disease model mouse, inhibit the activity of MAO-B, and effectively reduce the neuroinflammatory response.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a composition and its application in the preparation of drugs for treating Parkinson's disease. Background Technology

[0002] Parkinson's disease (PD) is a common neurodegenerative disorder with an incidence that increases significantly with age, severely impacting patients' quality of life. The main pathological feature of the disease is the progressive loss of dopaminergic neurons in the substantia nigra pars compacta of the midbrain, and the aggregation of α-synuclein in the remaining neurons to form Lewy bodies. This leads to a significant decrease in striatal dopamine levels, causing typical motor symptoms such as resting tremor, rigidity, and bradykinesia. Furthermore, multiple pathological mechanisms, including oxidative stress, mitochondrial dysfunction, neuroinflammation, and excitotoxicity, are intertwined and collectively drive the progression of the disease.

[0003] Currently, the main clinical treatment for Parkinson's disease is levodopa replacement therapy. While this method can temporarily relieve motor symptoms, it cannot stop the continuous degeneration of dopaminergic neurons, and long-term use can easily induce serious complications such as end-of-dose phenomenon and dyskinesia. Therefore, developing novel drugs with neuroprotective effects that can delay or reverse disease progression has become a research hotspot.

[0004] In recent years, nanozymes, as a class of nanomaterials with enzyme-like catalytic activity, have shown great promise in the treatment of Parkinson's disease due to their advantages such as high stability, low cost, and multifunctional integration. Graphdiyne nanozymes loaded with palladium nanoparticles are a novel composite nanomaterial. Graphdiyne is a novel two-dimensional carbon material with a unique sp / sp² hybrid carbon network structure, high specific surface area, and good biocompatibility, making it an ideal carrier. Palladium nanoparticles possess multiple enzyme-like activities, mimicking superoxide dismutase and catalase, and can efficiently scavenge reactive oxygen species and inhibit neuroinflammation, thereby protecting dopaminergic neurons.

[0005] Nevertheless, single-nanozyme therapy still falls short of comprehensively addressing the complex pathological network of Parkinson's disease. Combining nanozymes with synergistic natural active ingredients holds promise for synergistic effects across multiple dimensions, including antioxidant activity, monoamine oxidase inhibition, and neurotransmitter restoration. Therefore, this invention provides a composition comprising palladium-loaded graphyne nanozyme, eugenol, and trifoliate orange polysaccharide, aiming to synergistically exert therapeutic effects on Parkinson's disease from multiple dimensions. Summary of the Invention

[0006] In order to overcome the shortcomings of the prior art, the first objective of the present invention is to provide a composition that can increase the levels of DA and 5-HT in the striatum of Parkinson's disease model mice, inhibit MAO-B activity, and effectively reduce neuroinflammatory response.

[0007] The second objective of this invention is to provide a method for preparing a composition that is simple and easy to operate.

[0008] The third objective of this invention is to provide an application of the composition, which has broad application prospects.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] A composition comprising the following raw materials: palladium-loaded graphynyne nanozyme, eugenol, and trifoliate polysaccharide; wherein the mass ratio of the palladium-loaded graphynyne nanozyme, eugenol, and trifoliate polysaccharide is 1:(0.3-0.5):(0.05-0.1).

[0011] Furthermore, the preparation method of the palladium-loaded graphynyne nanozyme includes the following steps:

[0012] A potassium tetrachloropalladium solution was added to a dispersion of graphyne nanoparticles, and the mixture was stirred under ice bath conditions and purified to obtain the palladium-loaded graphyne nanozyme.

[0013] Furthermore, the volume ratio of the potassium tetrachloropalladium aqueous solution to the graphylene nanoparticle dispersion is 1:(8-10); the stirring time is 4-6 hours.

[0014] Furthermore, the concentration of the graphyne nanoparticle dispersion is 0.5-2 mg / mL; and the concentration of the potassium tetrachloropalladate aqueous solution is 25-30 mg / mL.

[0015] Furthermore, the preparation method of the *Trifolium repens* polysaccharide includes the following steps:

[0016] After pulverizing *Trifolium repens*, it was added to an ethanol-water solution for extraction, and the residue was collected. The residue was then extracted with water, filtered, and the filtrate was collected, concentrated, precipitated with alcohol, washed, and dried to obtain the *Trifolium repens* polysaccharide.

[0017] Furthermore, the volume fraction of the ethanol-water solution is 90%; the extraction temperature is 70-80℃, and the extraction is performed 3-4 times; the water extraction temperature is 90-100℃, and the extraction is performed 2-3 times.

[0018] Furthermore, the ratio of the amount of *Trifolium repens* to the ethanol aqueous solution is 1g:(10-15)mL; during water extraction, the ratio of the amount of residue to water is 1g:(10-15)mL.

[0019] The method for preparing the above composition includes the following steps:

[0020] According to the stated mass ratio, the palladium-loaded graphynyne nanozyme, eugenol, trifoliate polysaccharide, and pharmaceutically acceptable excipients are mixed evenly.

[0021] The above composition is used in the preparation of drugs for treating Parkinson's disease.

[0022] Compared with the prior art, the main advantages of the present invention are:

[0023] 1. This invention provides a composition comprising palladium-loaded graphynyne nanozyme, eugenol, and trifoliate polysaccharide. This composition can increase the levels of dopamine (DA) and 5-HT in the striatum of Parkinson's disease model mice, inhibit MAO-B activity, and effectively reduce neuroinflammatory responses. Specifically, the palladium-loaded graphynyne nanozyme exerts antioxidant and anti-inflammatory effects, eugenol reduces dopamine degradation by inhibiting MAO-B, and trifoliate polysaccharide directly or indirectly promotes neurotransmitter recovery. Through synergistic effects across multiple targets and pathways, these three components significantly enhance the comprehensive therapeutic effect of Parkinson's disease.

[0024] 2. The composition of the present invention has no significant effect on liver function at effective doses, and has good biosafety and therapeutic potential, providing a new, highly efficient, low-toxicity, multi-component synergistic intervention strategy for the treatment of Parkinson's disease. Attached Figure Description

[0025] Figure 1 This is a morphological diagram of PdNPs / GDY;

[0026] Figure 2 This is an elemental distribution diagram of PdNPs / GDY;

[0027] Figure 3 This is the XPS full spectrum of PdNPs / GDY;

[0028] Figure 4 These are the Raman spectra of GDY and PdNPs / GDY;

[0029] Figure 5 These are FTIR plots of GDY and PdNPs / GDY;

[0030] Figure 6 The graph shows the particle size and zeta potential of GDY and PdNPs / GDY. Detailed Implementation

[0031] The technical solution of the present invention will be further described below with reference to specific embodiments. However, those skilled in the art should understand that the following embodiments are only for illustrating the present invention and should not be regarded as limiting the present invention. Specific conditions not specified in the embodiments are performed according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the reagents or instruments used are all conventional products obtained through commercial channels.

[0032] (a) Preparation example

[0033] Preparation Example 1

[0034] A palladium-loaded graphynyne nanozyme is prepared as follows:

[0035] Graphdiyne was added to deionized water at a mass ratio of 1:6, and the mixture was ultrasonically treated with a 500W probe for 11 hours. After centrifugation, washing, and drying, graphdiyne nanoparticles were obtained. The graphdiyne nanoparticles were then added to deionized water to prepare a 1 mg / mL graphdiyne nanoparticle dispersion. A 28 mg / mL potassium tetrachloropalladate aqueous solution was added dropwise to the graphdiyne nanoparticle dispersion at a volume ratio of 1:9. The mixture was stirred in an ice bath for 5 hours, centrifuged, washed sequentially with deionized water and anhydrous ethanol, and freeze-dried to obtain palladium-loaded graphdiyne nanozyme (PdNPs / GDY).

[0036] Preparation Example 2

[0037] A palladium-loaded graphynyne nanozyme is prepared as follows:

[0038] Graphdiyne was added to deionized water at a mass ratio of 1:5, and the mixture was ultrasonically treated with a 400W probe for 12 hours. After centrifugation, washing, and drying, graphdiyne nanoparticles were obtained. The graphdiyne nanoparticles were then added to deionized water to prepare a 0.5 mg / mL graphdiyne nanoparticle dispersion. A 25 mg / mL potassium tetrachloropalladate aqueous solution was added dropwise to the graphdiyne nanoparticle dispersion at a volume ratio of 1:8. The mixture was stirred in an ice bath for 4 hours, centrifuged, washed sequentially with deionized water and anhydrous ethanol, and freeze-dried to obtain palladium-loaded graphdiyne nanozyme.

[0039] Preparation Example 3

[0040] A palladium-loaded graphynyne nanozyme is prepared as follows:

[0041] Graphdiyne was added to deionized water at a mass ratio of 1:7, and the mixture was ultrasonically treated with a 600W probe for 12 hours. After centrifugation, washing, and drying, graphdiyne nanoparticles were obtained. The graphdiyne nanoparticles were then added to deionized water to prepare a 2 mg / mL graphdiyne nanoparticle dispersion. A 30 mg / mL potassium tetrachloropalladate aqueous solution was added dropwise to the graphdiyne nanoparticle dispersion at a volume ratio of 1:10. The mixture was stirred in an ice bath for 6 hours, centrifuged, washed sequentially with deionized water and anhydrous ethanol, and freeze-dried to obtain palladium-loaded graphdiyne nanozyme.

[0042] Preparation Example 4

[0043] A polysaccharide from *Ilex trifoliata* is prepared as follows:

[0044] After pulverizing *Tripterygium wilfordii*, add it to an ethanol-water solution (90%, v / v) at a material-to-liquid ratio of 1g:12mL and extract four times at 75℃ for 2.5h each time. Collect the residue and air-dry it. Add the obtained residue to hot water at 95℃ at a material-to-liquid ratio of 1g:12mL and extract twice for 1.6h each time. Filter and combine the filtrates, centrifuge, and take the supernatant for vacuum filtration. Concentrate under reduced pressure to obtain a concentrated solution. Add three times the volume of ethanol-water solution (90%, v / v) to the concentrated solution, let it stand overnight, centrifuge to collect the precipitate, wash the precipitate with anhydrous ethanol, and freeze-dry it under vacuum to obtain *Tripterygium wilfordii* polysaccharide.

[0045] Preparation Example 5

[0046] A polysaccharide from *Ilex trifoliata* is prepared as follows:

[0047] After pulverizing *Trifolium repens*, add it to an ethanol-water solution (90%, v / v) at a material-to-liquid ratio of 1g:10mL and extract three times at 70℃ for 3 hours each time. Collect the residue and air-dry it. Add the obtained residue to hot water at 90℃ at a material-to-liquid ratio of 1g:10mL and extract twice for 2 hours each time. Filter and combine the filtrates, centrifuge, and take the supernatant for vacuum filtration. Concentrate under reduced pressure to obtain a concentrated solution. Add 2 times the volume of ethanol-water solution (90%, v / v) to the concentrated solution, let it stand overnight, centrifuge to collect the precipitate, wash the precipitate with anhydrous ethanol, and freeze-dry it under vacuum to obtain *Trifolium repens* polysaccharide.

[0048] Preparation Example 6

[0049] A polysaccharide from *Ilex trifoliata* is prepared as follows:

[0050] After pulverizing *Tripterygium wilfordii*, add it to an ethanol-water solution (90%, v / v) at a material-to-liquid ratio of 1g:15mL and extract four times at 80℃ for 2 hours each time. Collect the residue and air-dry it. Add the obtained residue to hot water at 100℃ at a material-to-liquid ratio of 1g:15mL and extract three times for 1.5 hours each time. Filter and combine the filtrates, centrifuge, and take the supernatant for vacuum filtration. Concentrate under reduced pressure to obtain a concentrated solution. Add three times the volume of ethanol-water solution (90%, v / v) to the concentrated solution, let it stand overnight, centrifuge to collect the precipitate, wash the precipitate with anhydrous ethanol, and freeze-dry it under vacuum to obtain *Tripterygium wilfordii* polysaccharide.

[0051] (II) Implementation Examples

[0052] Example 1

[0053] A composition comprising the following raw materials: palladium-loaded graphynyne nanozyme of Preparation Example 1, eugenol, and *Trifolium repens* polysaccharide of Preparation Example 4. The mass ratio of the palladium-loaded graphynyne nanozyme, eugenol, and *Trifolium repens* polysaccharide is 1:0.4:0.07.

[0054] The method for preparing the above composition includes the following steps:

[0055] According to the mass ratio described, palladium-loaded graphynyne nanozyme, eugenol, and trifoliate polysaccharide are mixed evenly. Based on the total mass of the active ingredient, 10 times the amount of lactose is added and mixed evenly. Then, 0.5 times the amount of ethanol aqueous solution (85%, v / v) is added to prepare a soft material. The material is then wet-granulated, dried, and sized to obtain the final product.

[0056] Example 2

[0057] A composition comprising the following raw materials: palladium-loaded graphynyne nanozyme of Preparation Example 2, eugenol, and *Trifolium repens* polysaccharide of Preparation Example 5. The mass ratio of the palladium-loaded graphynyne nanozyme, eugenol, and *Trifolium repens* polysaccharide is 1:0.3:0.05.

[0058] The method for preparing the above composition includes the following steps:

[0059] According to the stated mass ratio, palladium-loaded graphynyne nanozyme, eugenol, and trifoliate polysaccharide are mixed evenly. Based on the total mass of the active ingredient, 30 times the amount of starch and 0.3 times the amount of povidone are added, mixed evenly, and directly compressed into tablets to obtain the final product.

[0060] Example 3

[0061] A composition comprising the following raw materials: palladium-loaded graphynyne nanozyme of Preparation Example 3, eugenol, and *Trifolium repens* polysaccharide of Preparation Example 6. The mass ratio of the palladium-loaded graphynyne nanozyme, eugenol, and *Trifolium repens* polysaccharide is 1:0.5:0.1.

[0062] The method for preparing the above composition includes the following steps:

[0063] According to the stated mass ratio, palladium-loaded graphynyne nanozyme, eugenol, and trifoliate polysaccharide are mixed evenly. Based on the total mass of the main drug, 30 times the amount of microcrystalline cellulose and 0.2 times the amount of magnesium stearate are added, mixed evenly, and then filled into capsules to obtain the final product.

[0064] (III) Comparative Example

[0065] Comparative Example 1

[0066] The difference between Comparative Example 1 and Example 1 is that short-root ginsenoside is omitted.

[0067] Comparative Example 2

[0068] The difference between Comparative Example 2 and Example 1 is that the polysaccharide of *Trifolium repens* is omitted.

[0069] (iv) Experimental Examples

[0070] Experimental Example 1

[0071] The morphology of the palladium-loaded graphynyne nanozyme (PdNPs / GDY) obtained in Example 1 was observed using transmission electron microscopy, and the results are as follows: Figure 1 As shown.

[0072] Figure 1 These are morphology images of PdNPs / GDY. In image a, morphology of graphdiene is shown; images b and c are morphology images of PdNPs / GDY at different magnifications. (Observation) Figure 1 It is known that graphyne exhibits a typical nanosheet structure, with palladium nanoparticles of approximately 5-10 nm in size uniformly loaded on the surface of the sheets. The particles are well dispersed and show no obvious aggregation. This result indicates that palladium nanoparticles have been successfully loaded onto the surface of graphyne nanosheets.

[0073] Experiment Example 2

[0074] Elemental analysis of PdNPs / GDY yielded the following results: Figure 2 As shown.

[0075] Figure 2 This is an elemental distribution diagram of PdNPs / GDY. Observe... Figure 2 The presence of uniformly distributed carbon and palladium elements on the nanosheet surface further indicates that palladium nanoparticles have been successfully loaded and are well dispersed.

[0076] Experimental Example 3

[0077] XPS analysis of PdNPs / GDY was performed using X-ray photoelectron spectroscopy, and the results are as follows: Figure 3 As shown.

[0078] Figure 3 This is the XPS full spectrum of PdNPs / GDY. In the image, a is the full spectrum, b is the high-resolution spectrum of Pd 3d, and c is the peak fitting plot. Observation... Figure 3 It is known that palladium has been successfully loaded. The characteristic peaks of C 1s and Pd 3d are clearly visible in the full spectrum, confirming the presence of carbon and palladium on the sample surface. In the high-resolution Pd 3d spectrum, Pd 3d... 5 / 2 and Pd 3d 3 / 2 The binding energies are located at 335.5 eV and 340.8 eV, respectively, compared to metallic Pd. 0 The shift of the peak value (335.0 eV) towards higher binding energy indicates an electronic interaction between the palladium nanoparticles and the graphyne support. Peak fitting results show that palladium mainly exists in the metallic state (Pd). 0 The presence of palladium in this form is beneficial for its enzyme-like activity. This result further confirms that palladium has been successfully loaded onto the surface of graphyne.

[0079] Experiment Example 4

[0080] Graphdiyne (GDY) and PdNPs / GDY were measured using Raman spectroscopy and Fourier transform infrared spectroscopy, respectively. The results are as follows: Figure 4-5 As shown.

[0081] Figure 4 These are the Raman spectra of GDY and PdNPs / GDY. Figure 5 These are FTIR plots of GDY and PdNPs / GDY. Observe... Figure 4 It is known that GDY is approximately 1340 cm tall. -1 and 1580 cm -1 Characteristic peaks of the D and G bands appeared at the positions, corresponding to the defect structure of carbon materials and the ordered vibration of sp² carbon, respectively. After loading palladium, the peak positions of the D and G bands remained essentially unchanged, indicating that loading palladium nanoparticles did not alter the bulk structure of graphyne. (Observation) Figure 5 It is known that GDY is approximately 2100 cm. -1 The characteristic stretching vibration peak of C≡C appears at approximately 1600 cm⁻¹. -1 A C=C stretching vibration peak appears at approximately 1200 cm. -1 A stretching vibration peak of COC was observed. After palladium loading, the positions of these characteristic peaks did not shift significantly, indicating that the loading process did not disrupt the chemical structure of graphyne. In summary, PdNPs / GDY maintained the original chemical framework of graphyne, and no destructive changes occurred during the loading process.

[0082] Experimental Example 5

[0083] The particle size and zeta potential of GDY and PdNPs / GDY were determined using a dynamic light scattering particle size analyzer, and the results are as follows: Figure 6 As shown.

[0084] Figure 6 Figures a and b show the particle size and zeta potential results for GDY and PdNPs / GDY. Figure a represents the particle size distribution of GDY, figure b represents the particle size distribution of PdNPs / GDY, and figure c compares the zeta potentials of GDY and PdNPs / GDY. Figures a and b show that the average hydrated particle size of GDY is approximately 220 nm, while that of PdNPs / GDY is approximately 258 nm, showing a slight increase in particle size, consistent with the nanosheet structure and palladium nanoparticle loading observed by TEM. The particle size distribution of PdNPs / GDY is relatively concentrated, with a polydispersity index (PDI) of less than 0.2, indicating good dispersion of the sample in solution. As shown in Figure c, the Zeta potential of GDY is approximately -25.6 mV, and the Zeta potential of PdNPs / GDY is approximately -28.3 mV. Both exhibit negative charge and have an absolute value greater than 25 mV, indicating that the samples have good colloidal stability in aqueous solution, which is beneficial for subsequent biological applications.

[0085] Experimental Example 6

[0086] 6.1 Laboratory Animals

[0087] Seventy-five healthy C57BL / 6 mice, weighing 22-25g, were selected and kept in an SPF-grade laboratory for 7 days with alternating 12-hour light and 12-hour dark cycles and a controlled temperature of 22-25℃.

[0088] 6.2 Modeling and Drug Intervention

[0089] The animals were randomly divided into a normal control group, a model group, Example 1 group, Example 2 group, Example 3 group, Comparative Example 1 group, and Comparative Example 2 group, with 10 animals in each group, and drug intervention was carried out in the following manner.

[0090] Normal control group and model group: The same volume of normal saline was administered by gavage once a day for 21 consecutive days.

[0091] Example 1 group: The composition of Example 1 was administered by gavage once a day for 21 consecutive days, based on a dosage of 25 mg / kg of palladium-loaded graphynyne nanozyme.

[0092] Example 2 group: The composition of Example 2 was administered by gavage once a day for 21 consecutive days, based on a dosage of 25 mg / kg of palladium-loaded graphynyne nanozyme.

[0093] Example 3 group: The composition of Example 3 was administered by gavage once a day for 21 consecutive days, based on a dosage of 25 mg / kg of palladium-loaded graphynyne nanozyme.

[0094] Comparative Example 1: The composition of Comparative Example 1 was administered by gavage once a day for 21 consecutive days, based on a dosage of 25 mg / kg of palladium-loaded graphynyne nanozyme.

[0095] Comparative Example 2: The composition of Comparative Example 2 was administered by gavage once a day for 21 consecutive days, based on a dosage of 25 mg / kg of palladium-loaded graphynyne nanozyme.

[0096] In the control group, an equal volume of physiological saline was injected into the peritoneum 1 hour after administration of the drug on days 8-14. Except for the control group, the mice in the other groups were injected into the peritoneum with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, 20 mg / kg / d) for 7 days. The mice were observed to have arched backs, piloerection, obvious body tremors, and slowed movement, indicating that the Parkinson's disease model was successfully established.

[0097] 6.3 Indicator Testing

[0098] 6.3.1 Effects of the composition on mouse toxicity

[0099] After the last administration, blood was drawn from the carotid artery of each group of mice, left to stand at room temperature for 30 min, and then centrifuged at 10,000 rpm at 4°C. The serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were detected using the microplate method. The results are shown in Table 1.

[0100] 6.3.2 Detection of inflammatory factor levels, dopamine, serotonin content, and monoamine oxidase activity in the striatum

[0101] After blood collection, mice in each group were anesthetized and euthanized. The bilateral striatums of the mice were completely separated on ice and placed in liquid nitrogen for later use. Tissue from the right striatum was taken, and dopamine (DA) and serotonin (5-HT) in the striatum were determined by HPLC. The results are shown in Table 2.

[0102] Left striatum tissue was collected, and the striatum was lysed with lysis buffer containing protease inhibitor and phosphatase inhibitor to extract proteins. After standing at 4°C for 30 min, the tissue was centrifuged at 12,000 rpm for 30 min at 4°C to remove insoluble impurities. The activity of monoamine oxidase (MAO-B) was detected using the Amplex™ Red monoamine oxidase assay kit according to the kit instructions. The results are shown in Table 2.

[0103] The supernatant of the remaining lysis buffer from the left striatum was used for MAO-B detection. The levels of IL-6, IL-8, and TNF-α in the striatum were detected according to the ELISA kit instructions. The results are shown in Table 3.

[0104] Table 1

[0105]

[0106] Table 2

[0107]

[0108] Table 3

[0109]

[0110] ALT and AST are the most direct indicators of liver function, directly reflecting whether liver function is normal. Therefore, the serum levels of AST and ALT in mice were measured to assess the toxic effects of the composition on MPTP-induced Parkinson's disease C57BL / 6 mice. The results are shown in Table 1. Compared with the normal control group, the compositions in Examples 1-3 and Comparative Examples 1-2 had no significant effect on the serum AST and ALT levels in mice.

[0111] Table 2 shows that compared with the normal control group, the levels of DA and 5-HT in the striatum of the model group mice were decreased, while the MAO-B activity was increased. This indicates that the MPTP-induced Parkinson's disease mouse model was successfully established, consistent with the pathological characteristics of dopaminergic neuron damage and abnormally increased MAO-B activity in Parkinson's disease. Compared with the model group, the levels of DA and 5-HT in groups 1-3 of Examples were increased, while the MAO-B activity was decreased, indicating that the composition of the present invention can stably and effectively restore the neurotransmitter levels in the striatum and inhibit MAO-B activity.

[0112] The DA and 5-HT contents of Comparative Example 1 (omitting basidiosin) and Comparative Example 2 (omitting Trifolium repens polysaccharide) were lower than those of Example 1, while the MAO-B activity was higher, indicating that the absence of any single component significantly reduced the efficacy. Specifically, Comparative Example 1 showed higher MAO-B activity and lower dopamine neurotransmitter content, verifying that basidiosin mainly exerts its effect by inhibiting MAO-B. The degree of serotonin neurotransmitter recovery in Comparative Example 2 was slightly lower than that in Comparative Example 1, indicating that Trifolium repens polysaccharide has a regulatory effect on neurotransmitter levels.

[0113] Table 3 shows that compared with the normal control group, the levels of IL-6, IL-8, and TNF-α in the striatum of the model group mice were all increased, indicating that the Parkinson's disease model mice had a significant neuroinflammatory response. Compared with the model group, the levels of IL-6, IL-8, and TNF-α in Examples 1-3 were all decreased, indicating that the composition of the present invention has a strong inhibitory effect on neuroinflammation.

[0114] The levels of inflammatory factors in Comparative Examples 1 and 2 were higher than those in Example 1, indicating that the absence of any component weakened the anti-inflammatory effect. Specifically, the inflammatory factor levels in Comparative Example 1 were higher than those in Comparative Example 2, consistent with the trend of increased MAO-B activity shown in Table 2, suggesting that eugenol may indirectly exert its anti-inflammatory effect by inhibiting MAO-B and reducing oxidative stress products. The absence of *Trifolium repens* polysaccharide also exacerbated the inflammatory response, indicating that it also possesses anti-inflammatory properties.

[0115] In summary, the composition of the present invention can significantly restore the levels of DA and 5-HT in the striatum of Parkinson's disease model mice, inhibit MAO-B activity, and effectively reduce neuroinflammatory responses.

[0116] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. The basic principles and main features of the present invention have been described above with specific implementation schemes. Based on the present invention, some modifications or substitutions can be made, but these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of protection claimed by the present invention.

Claims

1. A composition, characterized in that, It contains the following raw materials: palladium-loaded graphynyne nanozyme, eugenol, and trifoliate polysaccharide; the mass ratio of the palladium-loaded graphynyne nanozyme, eugenol, and trifoliate polysaccharide is 1:(0.3-0.5):(0.05-0.1).

2. The composition according to claim 1, characterized in that, The preparation method of the palladium-loaded graphynyne nanozyme includes the following steps: A potassium tetrachloropalladium solution was added to a dispersion of graphyne nanoparticles, and the mixture was stirred under ice bath conditions and purified to obtain the palladium-loaded graphyne nanozyme.

3. The composition according to claim 2, characterized in that, The volume ratio of the potassium tetrachloropalladium aqueous solution to the graphylene nanoparticle dispersion is 1:(8-10); the stirring time is 4-6 hours.

4. The composition according to claim 3, characterized in that, The concentration of the graphyne nanoparticle dispersion is 0.5-2 mg / mL; the concentration of the potassium tetrachloropalladate aqueous solution is 25-30 mg / mL.

5. The composition according to claim 1, characterized in that, The preparation method of the *Trifolium repens* polysaccharide includes the following steps: After pulverizing *Trifolium repens*, it was added to an ethanol-water solution for extraction, and the residue was collected. The residue was then extracted with water, filtered, and the filtrate was collected, concentrated, precipitated with alcohol, washed, and dried to obtain the *Trifolium repens* polysaccharide.

6. The composition according to claim 5, characterized in that, The volume fraction of the ethanol-water solution is 90%; the extraction temperature is 70-80℃, and the extraction is performed 3-4 times; the water extraction temperature is 90-100℃, and the extraction is performed 2-3 times.

7. The composition according to claim 5, characterized in that, The ratio of *Trifolium repens* to ethanol aqueous solution is 1g:(10-15)mL; during water extraction, the ratio of residue to water is 1g:(10-15)mL.

8. A method for preparing the composition according to any one of claims 1-7, characterized in that, Includes the following steps: According to the stated mass ratio, the palladium-loaded graphynyne nanozyme, eugenol, trifoliate polysaccharide, and pharmaceutically acceptable excipients are mixed evenly.

9. The use of the composition according to any one of claims 1-7, characterized in that, It is used in the preparation of drugs for treating Parkinson's disease.

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