Application of American ginseng total protein in preparation of neuroinflammation treatment medicine

Abstract

The invention relates to application of American ginseng total protein in preparation of neuroinflammation treatment drugs. Six proteins with the highest content in the American ginseng total protein are ribonuclease-like main storage protein, main latex-like protein, phosphoglycerate kinase 1, glyceraldehyde-3-phosphate dehydrogenase, disease course related protein 10 and ribonuclease 2. The American ginseng total protein is used as an active ingredient of the neuroinflammation treatment medicine, compared with existing western medicines, the neuroinflammation treatment effect can be guaranteed, meanwhile, side effects are avoided, and the American ginseng total protein is easier to digest and absorb; compared with an existing traditional Chinese medicine composition, the composition is definite in component and controllable in quality, and interference of ineffective components is reduced.

Description

Technical Field

[0001] The present invention belongs to the field of neuroinflammation therapeutic drug development, and specifically relates to an application of American ginseng total protein in the preparation of neuroinflammation therapeutic drugs. Background Art

[0002] Neuroinflammation refers to an inflammatory response in the central nervous system (CNS) or peripheral nervous system (PNS). It is a complex pathological process involving the interaction of multiple cell types, cytokines, and signaling pathways. Neuroinflammation can induce, influence, or exacerbate many psychiatric disorders, such as Alzheimer's disease, stroke, epilepsy, anxiety, and depression. Research and treatment of neuroinflammation are crucial for the effective prevention, treatment, and control of various neuropsychiatric disorders.

[0003] The core of neuroinflammation is the activation of immune cells. In the central nervous system, microglia are the main immune cells and the key factors of neuroinflammatory response. Microglia maintain the homeostasis of the central nervous system by inducing inflammatory response and promoting neurogenesis. Under normal circumstances, microglia are in a resting state. When the central nervous system is damaged, microglia are activated into two phenotypes, M1 and M2. The M1 type can secrete pro-inflammatory factors such as IL-6, IL-1β, and TNF-α, and the M2 type can secrete anti-inflammatory factors such as IL-10 and IL-13. Studies have shown that activated microglia around DA neurons are usually pro-inflammatory, and the activation of microglia increases with the degeneration and necrosis of dopaminergic neurons.

[0004] Currently, most drugs used to treat neuroinflammation are Western medicines, such as gabapentin, pregabalin, ibuprofen, diclofenac, and camrelizumab, but most Western medicines have side effects. Traditional Chinese medicine compositions, on the other hand, are often complex, their mechanisms of action are difficult to clearly elucidate, and their efficacy is difficult to predict and control. Traditional Chinese medicine extracts are active ingredients extracted from traditional Chinese medicines through physical or chemical methods. They have clear ingredients, controllable quality, and can reduce interference from ineffective ingredients. Traditional Chinese medicine extracts are one of the important achievements of the modernization of traditional Chinese medicine. They combine the advantages of traditional Chinese medicine with modern scientific and technological means, making the use of traditional Chinese medicine more scientific, efficient, and safe. Therefore, the development of a traditional Chinese medicine extract with a definite therapeutic effect on neuroinflammation will be of great clinical significance.

[0005] American ginseng, a traditional Chinese medicine, has a cooling effect, with a sweet and slightly bitter taste. It is commonly used clinically to treat symptoms such as internal heat, asthenic fever, fatigue, cough, asthma, and dry mouth and throat caused by qi and yin deficiency. It can both replenish qi and nourish yin, demonstrating unique therapeutic effects in improving deficient constitutions. Research has shown that American ginseng has a wide range of pharmacological activities, including regulating blood sugar, improving immune function, controlling blood pressure, alleviating fatigue, inhibiting oxidative stress, and fighting tumors.

[0006] The main chemical components of American ginseng include ginsenosides, polysaccharides, flavonoids, fatty acids, polyalkynes, volatile oils, proteins, and other compounds, as well as nutrients such as vitamins and amino acids. Polysaccharides are the most abundant, and saponins are currently recognized as the main active ingredients in American ginseng. Currently, research on the therapeutic effects of American ginseng extracts on neuroinflammation is relatively limited, with research primarily focusing on ginsenosides. Summary of the Invention

[0007] The purpose of the present invention is to provide an application of American ginseng total protein in the preparation of a drug for treating neuroinflammation.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] The first aspect of the present invention provides an application of American ginseng total protein in the preparation of a drug for treating neuroinflammation, wherein the six proteins with the highest content in the American ginseng total protein are ribonuclease-like major storage protein, major latex-like protein, phosphoglycerate kinase 1, glyceraldehyde-3-phosphate dehydrogenase, pathogenesis-related protein 10, and ribonuclease 2.

[0010] In an embodiment of the present invention, the neuroinflammation therapeutic drug is a lyophilized agent of an aqueous solution of total protein from American ginseng, and the protein purity of the aqueous solution of total protein from American ginseng is ≥80%, for example, the protein purity of the aqueous solution of total protein from American ginseng is 80% to 85%.

[0011] In an embodiment of the present invention, the pH value of the American ginseng total protein aqueous solution is 6.8-7.2.

[0012] In an embodiment of the present invention, the American ginseng total protein aqueous solution is prepared by extraction with cold water at 3-5° C., and the obtained extract is purified by isoelectric precipitation and ultrafiltration.

[0013] During the 3-5° C. cold water extraction, the material-liquid ratio (w / v) of the American ginseng dry powder to water is 1:15-20, and the pH value of the material liquid is 6.8-7.2.

[0014] In an embodiment of the present invention, the American ginseng dry powder is a powder of dried roots of American ginseng with a particle size of ≤24 mesh.

[0015] In an embodiment of the present invention, the pH value used in the isoelectric precipitation is 3.4 to 3.6, and the ultrafiltration uses an ultrafiltration cut-off tube with a molecular weight of 2.5 to 3.5 kD.

[0016] According to some specific embodiments of the present invention, the preparation method of the American ginseng total protein aqueous solution is as follows: American ginseng dry powder and distilled water are evenly mixed according to a material-liquid ratio (w / v) of 1:15-20, the mixed solution is adjusted to a pH of 6.8-7.2 with HCl and NaOH, cold-soaked and extracted at 3-5°C for 10-15h, centrifuged at 7000-9000rpm for 10-20min, the supernatant is collected, and the residue is repeated with the above steps, cold-soaked and extracted twice, the supernatants of the two times are combined, the pH of the supernatant is adjusted to 3.4-3.6 with HCl, precipitated at 3-5°C overnight, centrifuged to separate the precipitate and supernatant, the precipitate is redissolved, and the pH is adjusted to 6.8-7.2 with NaOH again, the ultrafiltration protein is intercepted with an ultrafiltration cutoff tube with a molecular weight of 2.5-3.5kD, centrifuged at 4000-6000r / min for 25-35min, and the supernatant is collected, which is the American ginseng total protein aqueous solution.

[0017] The second aspect of the present invention provides a drug for treating neuroinflammation, the active ingredient of which is American ginseng total protein, and the six proteins with the highest content in the American ginseng total protein are ribonuclease-like major storage protein, major latex-like protein, phosphoglycerate kinase 1, glyceraldehyde-3-phosphate dehydrogenase, pathogenesis-related protein 10, and ribonuclease 2.

[0018] In an embodiment of the present invention, the neuroinflammation treatment drug is a lyophilized agent, and the purity of the American ginseng total protein is ≥80%, for example, the protein purity of the American ginseng total protein aqueous solution is 80% to 85%.

[0019] Due to the adoption of the above technical solution, the present invention has the following advantages compared with the prior art:

[0020] The present invention provides the use of American ginseng total protein in the preparation of a drug for treating neuroinflammation. The six proteins with the highest content in American ginseng total protein are ribonuclease-like major storage protein, major latex-like protein, phosphoglycerate kinase 1, glyceraldehyde-3-phosphate dehydrogenase, pathogenesis-related protein 10, and ribonuclease 2. Experiments have shown that the American ginseng total protein of the present invention can not only regulate the state of microglia and then regulate the release of inflammatory factors to activate the immune system, but also can regulate neuroinflammation by improving abnormalities in the arachidonic acid pathway, improving abnormalities in the sphingolipid metabolic pathway, improving abnormalities in the primary bile acid biosynthesis metabolic pathway, and regulating the phenylalanine metabolic pathway, thereby exerting a neuroprotective effect. The present invention uses American ginseng total protein as an active ingredient in a drug for treating neuroinflammation. Compared with existing Western medicines, it can avoid side effects while ensuring the therapeutic effect of neuroinflammation, and it is easier to digest and absorb; compared with existing Chinese medicine compositions, its ingredients are clear, the quality is controllable, and the interference of ineffective ingredients is reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the infrared spectrum of the American ginseng total protein in Example 1;

[0022] Figure 2 This is the ultraviolet absorption spectrum of the American ginseng total protein in Example 1;

[0023] Figure 3 This is the intrinsic fluorescence spectrum of the American ginseng total protein in Example 1;

[0024] Figure 4 The CD spectrum of the American ginseng total protein in Example 1 at 190-260 nm;

[0025] Figure 5 This is a liquid chromatogram of the amino acid composition of the American ginseng total protein of Example 1;

[0026] Figure 6 This is a two-dimensional electrophoresis diagram of the total protein of American ginseng in Example 1;

[0027] Figure 7 The effect of American ginseng total protein on the pathological changes of the hippocampus and substantia nigra in mice;

[0028] Figure 8 Effects of American ginseng total protein on inflammatory factors in mouse serum (A) Changes in IL-1β content in each group of mice (B) Changes in TNF-α content in each group of mice (C) Changes in IL-6 content in each group of mice (D) Changes in IL-10 content in each group of mice;

[0029] Figure 9 PCA and OPLS-DA diagrams of serum metabolism of mice in each group (A) PCA diagram under positive and negative ion modes (B) OPLS-DA diagram under positive and negative ion modes;

[0030] Figure 10 Serum displacement test and S-plot of each group of mice (A) displacement test of serum of each group of mice in positive and negative ion modes (B) S-plot of serum of each group of mice in positive and negative ion modes;

[0031] Figure 11 Volcano plot of potential differential metabolites in the serum of mice in each group;

[0032] Figure 12 This is the Venn diagram of the common differential metabolites in the MPTP vs. Control group and the AGP vs. MPTP group;

[0033] Figure 13 Cluster heat map analysis of serum differential metabolites;

[0034] Figure 14 This is a bubble diagram of mouse serum metabolic pathways;

[0035] Figure 15 PCA and OPLS-DA diagrams of fecal metabolism of mice in each group (A) PCA diagram under positive and negative ion modes (B) OPLS-DA diagram under positive and negative ion modes;

[0036] Figure 16 Fecal metabolic displacement test and S-plot diagram of each group of mice (A) displacement test diagram under positive and negative ion modes (B) S-plot diagram under positive and negative ion modes;

[0037] Figure 17 Volcano analysis of potential differential metabolites in feces of mice in each group;

[0038] Figure 18 This is the Venn diagram of the common differential metabolites in the MPTP vs. Control group and the AGP vs. MPTP group;

[0039] Figure 19 Cluster heat map analysis of differential metabolites in feces;

[0040] Figure 20 This is a bubble diagram of the metabolic pathways in mouse feces. DETAILED DESCRIPTION

[0041] The technical solution of the present invention is further described below in conjunction with specific implementation cases, but the present invention is not limited to the following examples. The implementation conditions used in the examples can be further adjusted according to specific requirements, and the implementation conditions not specified are usually the conditions in routine experiments.

[0042] In the present invention, the total protein of American ginseng is abbreviated as "AGP".

[0043] In the present invention, the water used is distilled water.

[0044] In the following examples and comparative examples, unless otherwise specified, the instruments, raw materials, and reagents used are all commercially available.

[0045] American ginseng (3-year-old sun-dried ginseng) was purchased from Teyuan Ginseng and Rong in Changchun City. It was identified as the dried root of Panax quinquefolius L., a plant of the Araliaceae family. The particle size of the American ginseng powder was ≤24 mesh.

[0046] The experimental animals were male C57BL / 6 mice (weighing approximately 20 g each) provided by Changchun Yisi Laboratory Animal Technology Co., Ltd. All mice were housed in a temperature- and humidity-controlled room with a 12-h light-dark cycle. Throughout the study, all mice had free access to feed and drinking water and were acclimated to the laboratory environment for one week prior to the experiment.

[0047] Example 1:

[0048] Weigh 10 g of American ginseng powder, add distilled water according to a material-liquid ratio (w / v) of 1:15, stir evenly, adjust the solution to pH 7.0 with HCl and NaOH, cold-soak and extract at 4 ° C for 12 h, centrifuge at 8000 rpm for 15 min, collect the supernatant, repeat the above steps with the residue, extract twice, combine the two supernatants, adjust the pH of the supernatant to 3.5 with HCl, precipitate at 4 ° C overnight, centrifuge and separate the precipitate and supernatant, redissolve the precipitate, adjust the pH to 7.0 with NaOH again, retain the ultrafiltration protein with an ultrafiltration cutoff tube with a molecular weight of 3 kD, centrifuge at 5000 r / min for 30 min, collect the supernatant, and test the protein purity to 82.36%, the yield is 2.59%, freeze-dried and set aside.

[0049] Structural characterization and identification of American ginseng total protein (AGP):

[0050] (1) Fourier transform infrared spectroscopy (FTIR)

[0051] The secondary structure of American ginseng total protein was determined by Fourier transform infrared spectrophotometry. The freeze-dried American ginseng total protein powder was mixed with an appropriate amount of potassium bromide powder and pressed into tablets. The full-band scanning was performed using Vetex70 Fourier transform infrared spectrometer. The parameter setting was: scanning range 500-4000 cm -1 , with a resolution of 4cm -1 , scan times 64 times.

[0052] The infrared absorption spectrum of American ginseng total protein was tested, and the results were as follows: Figure 1 As shown, 3274cm -1 The broad peak at 2921 cm is a typical absorption peak caused by the stretching vibration of the HO bond. -1 The peak at 1634 cm is caused by the antisymmetric stretching vibration of -CH2-. -1 The absorption peak at 1527 cm is caused by the protein amide I band. -1 and 1453cm -1 The absorption peak at 1392 cm is produced by protein amide II band. -1 The absorption peak at 1239 cm is generated by the symmetrical stretching vibration of free COO- in the protein. -1 The absorption peak at is caused by protein amide III band. The infrared spectrum results show that the total protein of American ginseng has a typical protein structure.

[0053] (2) UV spectrum scanning

[0054] Dissolve 1 mg of freeze-dried American ginseng total protein powder in distilled water (pH 7.0) to a final concentration of 2 mg / mL. Scan using a UV-2550 UV-visible spectrophotometer with a scan range of 250–500 nm and a scan speed of 60 nm / min.

[0055] The UV absorption spectrum of total protein of American ginseng is as follows Figure 2 As shown, the maximum UV absorption peak of American ginseng total protein is at 280nm. This is because specific aromatic amino acid residues in proteins contain conjugated double bonds. The maximum absorption peak of protein in the UV spectrum usually appears around 280nm. The results show that American ginseng total protein has protein UV absorption properties.

[0056] (3) Endogenous fluorescence spectroscopy detection

[0057] Prepare a 0.5 mg / mL solution of freeze-dried American ginseng total protein powder and measure the protein fluorescence spectrum using a fluorescence spectrophotometer. Parameter settings: excitation wavelength 280 nm, emission wavelength range 300-550 nm, and slit 5 nm.

[0058] Fluorescence spectra can reflect the conformational changes of the tertiary structure of proteins, which are mainly determined by the ultraviolet absorption and fluorescence emission characteristics of aromatic amino acids. Figure 3 As shown in the figure, the maximum absorption peak of the fluorescence spectrum of American ginseng total protein under 280nm excitation at pH 5.0, 7.0, and 9.0 is located near 340nm. The maximum absorption peak of American ginseng total protein at different pH values ​​does not show a significant red shift, indicating that its tertiary structure remains stable in acidic and alkaline environments.

[0059] (4) Circular dichroism (CD) detection

[0060] Weigh 1 mg of freeze-dried American ginseng total protein powder and add 100 mL of distilled water. Adjust the pH of the solution to 5.0, 7.0, and 9.0, respectively. Scan using a circular dichroism spectrometer. Parameter settings: scanning wavelength 190–260 nm (far-UV region), scan speed 100 nm / min, bandwidth 1.0 nm, response time 0.5 s, and step resolution 1.0 nm.

[0061] CD is one of the most widely used methods to study the secondary structure of proteins. It can show the existence of α-helix, β-sheet, β-turn and random coil. In view of this, CD spectroscopy was used to analyze the spatial conformation of American ginseng total protein. Figure 4 As shown in the figure, under the conditions of pH 5.0, 7.0 and 9.0, the CD spectra of American ginseng total protein showed a strong positive band at 195 nm and an obvious negative band at 205 nm, indicating that it mainly contained β-sheet structure.

[0062] (5) Amino acid composition analysis

[0063] 30 mg of freeze-dried American ginseng total protein powder was added to 10 mL of 6 mol / L HCl and hydrolyzed at 110°C for 24 h. After hydrolysis, the hydrolyzate was concentrated to dryness using vacuum. Sodium citrate buffer was added, and the mixture was shaken and mixed thoroughly in an ultrasonic cleaner. The filtrate was filtered through a 0.22 μm filter membrane and collected for later use. Amino acid composition was analyzed using an automatic amino acid analyzer according to the test method in accordance with GB5009.124-2016.

[0064] Figure 5 The results showed that 17 amino acids were detected in the total protein of American ginseng. These amino acids are divided into essential amino acids (EAAs) and non-essential amino acids (NEAAs) based on whether they can be synthesized by the human body. EAAs include Thr, Val, Met, Ile, Leu, Phe, Lys, and His. NEAAs include Asp, Ser, Glu, Gly, Ala, Cys, Tyr, Arg, and Pro. Among them, Asp and Leu are the most abundant in the total protein of American ginseng. In addition, the total protein of American ginseng contains a certain proportion of aromatic amino acids, branched-chain amino acids, and sulfur-containing amino acids.

[0065] (6) Two-dimensional electrophoresis

[0066] Take 200 μL of a 10 mg / mL aqueous solution of American ginseng total protein, add 1800 μL of protein lysis buffer and dilute 10-fold. Load the sample into the IPG strip tank and hydrate overnight. After hydration, place the strip into the isoelectric focusing instrument and perform first-dimension isoelectric focusing electrophoresis. Set the isoelectric focusing program with voltages of 30 V, 60 V, 100 V, 500 V, 1000 V, 5000 V, 8000 V, and 500 V for 6 h, 6 h, 1 h, 1 h, 1 h, 2 h, 2 h, and 10 h, respectively. For the second-dimension SDS-PAGE, first set the electrophoresis parameters to 70 V, 22 mA, and a time of 40 min, then set the electrophoresis parameters to 225 V, 67 mA, and a time of 5 h.

[0067] After electrophoresis, the gel strips are stained and destained, and the selected protein bands are removed and pre-treated according to LC-MS / MS requirements, including reductive alkylation, in-gel digestion with trypsin, peptide extraction with peptide extraction buffer, and desalting. The samples are then ready for analysis.

[0068] Peptides were detected using an Orbitrap Exploris 480 high-resolution mass spectrometer. Mobile phase A consisted of 0.1% formic acid in water, and mobile phase B consisted of 0.1% formic acid in water and 80% acetonitrile in water. The flow rate was 300 nL / min, and the mobile phase gradient elution was as follows: 4% to 8% phase B (0-2 min); 8% to 28% phase B (2-35 min); 28% to 40% phase B (35-55 min); 40% to 95% phase B (55-56 min); and 95% phase B (56-66 min). Mass spectrometry was then performed over a full scan range of 300-1800 m / z, with a primary MS resolution of 120,000 and a secondary MS resolution of 15,000. Protein names and amino acid sequences were identified by comparison with a protein database.

[0069] Figure 6 The results showed that the main protein with the highest content in the total protein of American ginseng falls in the molecular weight range of 10 to 75 kD. Six high-content protein bands were subjected to gel excision sequencing and analyzed using in-gel digestion combined with high-resolution mass spectrometry. The target protein in each band was determined based on the number of peptides, number of identified proteins, and molecular weight, according to the scoring values ​​and actual conditions. The results are shown in Table 1 (arranged from high to low content): major ribonuclease-like storage protein, major latex-like protein, phosphoglycerate kinase 1 (PGK1), glyceraldehyde-3-phosphate dehydrogenase, pathogenesis-associated protein 10, and ribonuclease 2.

[0070] Table 1

[0071]

[0072]

[0073] Example 2: Animal Experiment

[0074] MPTP can cross the blood-brain barrier and is converted into MPP by astrocytes in the brain. + , MPP + It is selectively taken up by dopaminergic cells, disrupts the mitochondrial transport chain, and triggers an inflammatory process characterized by microglial activation in the substantia nigra (SN) and striatum, thereby inducing neurodegeneration.

[0075] Animal grouping and drug administration

[0076] After one week of adaptive feeding, mice were randomly divided into the following groups: control group, MPTP group, low-dose AGP group (40 mg / kg, AGPL), medium-dose AGP group (80 mg / kg, AGPM), and high-dose AGP group (120 mg / kg, AGPH), with 10 mice in each group. The control group was intraperitoneally injected with 0.2 mL of normal saline for 28 consecutive days. Mice in the other groups were intraperitoneally injected with MPTP at a concentration of 35 mg / kg in a volume of 0.2 mL for 28 consecutive days. On the first day after model establishment, AGP was dissolved in normal saline and administered. The AGPL, AGPM, and AGPH groups were intragastrically administered with 40 mg / kg, 80 mg / kg, and 120 mg / kg of American ginseng total protein, respectively, in a volume of 0.2 mL for 28 consecutive days. The control and MPTP groups were intragastrically administered with 0.2 mL of normal saline. After the administration was completed, the mice were euthanized, and serum, brain tissue, and fecal samples were collected.

[0077] (1) H&E staining of brain tissue

[0078] After euthanasia, the mice were skull-opened and the intact brains removed. The brains were perfused with 4% paraformaldehyde and fixed for 48 hours. The sections were then dehydrated with graded ethanol, cleared with xylene, embedded in paraffin, and sectioned. Paraffin sections were dewaxed with xylene and ethanol, stained with hematoxylin for 10 minutes, differentiated with 0.7% hydrochloric acid-ethanol, and stained with eosin for 3 minutes. Subsequently, the sections were dehydrated with graded ethanol, cleared with xylene, and mounted with neutral gum. Finally, the hippocampus and substantia nigra regions were observed and photographed under a light microscope.

[0079] like Figure 7 As shown, the hippocampal nerve cells in the Control group were tightly organized and spherical, with normal cell membrane and nucleus development, and no obvious swelling and necrosis were observed. The hippocampal nerve cell tissue in the MPTP group was obviously disordered and distorted, with uneven size and structure, and reduced number. After treatment with American ginseng total protein, the necrosis of the mouse nerve cells was improved, the cells were arranged relatively tightly and neatly, and the number was relatively increased. This shows that the treatment with American ginseng total protein can improve the morphology and number of relevant brain tissue cells in mice. The nuclei of the cells in the substantia nigra region of the Control group mice were clear, while the nuclei of the cells in the MPTP group mice were blurred, the number of neurons was significantly reduced, and the morphology was abnormal. After treatment with American ginseng total protein, the cell pathology in the substantia nigra region was significantly improved, the number of neuronal cells increased, the cell nuclei were clearer, and the morphology was more complete. This shows that American ginseng total protein has a certain improvement effect on neuronal apoptosis in the substantia nigra region of the mouse brain.

[0080] (2) Levels of inflammatory factors

[0081] Eye blood was collected from each group of mice. After standing for 1.5 hours, the blood was centrifuged at 12,000 rpm for 10 minutes at 4°C. The supernatant was collected and frozen at -80°C until further use. The levels of IL-6, IL-1β, TNF-α, and IL-10 in mouse serum were determined according to the ELISA kit instructions.

[0082] like Figure 8 As shown in the results, compared with the Control group, the MPTP group mice significantly increased the levels of pro-inflammatory factors IL-1β, TNF-α and IL-6 (IL-1βp<0.001, TNF-αp<0.001, IL-6p<0.01), and reduced the level of anti-inflammatory factor IL-10 (p<0.001); after treatment with American ginseng total protein, compared with the MPTP group, the levels of IL-1β, TNF-α and IL-6 were significantly decreased, and the level of IL-10 was significantly increased, and all the treatment groups were statistically significant.

[0083] Microglia, the primary immune cells in the central nervous system, play a central role in regulating neuroinflammation and maintaining homeostasis. Under normal physiological conditions, they exist in a steady-state state, but under pathological stimulation, they switch to a reactive state. After MPTP administration to mice, microglia are able to perform synaptic pruning, while astrocytes produce factors that cause neurons to release complement components. Increased production and release of complement proteins lead to microglial phagocytosis of neurons. At this point, neurodegeneration begins to manifest its pathophysiological characteristics. In the reactive state, microglia are activated into two phenotypes: M1 and M2. The M1 phenotype secretes proinflammatory cytokines such as IL-6, IL-1β, and TNF-α, while the M2 phenotype secretes anti-inflammatory cytokines such as IL-10 and IL-13. Serum levels of IL-1β, IL-6, and TNF-α were upregulated in the MPTP group, indicating that MPTP induces microglia in the reactive state, in which M1 microglia dominate and exert proinflammatory effects. After treatment with American ginseng total protein, the expression levels of IL-1β, IL-6 and TNF-α were downregulated, while the expression level of IL-10 was upregulated. M2 microglia took a dominant position and exerted an anti-inflammatory effect, indicating that American ginseng total protein can regulate the state of microglia, thereby regulating the release of inflammatory factors and activating the immune system.

[0084] (3) Serum metabolomics and fecal metabolomics studies

[0085] After the administration, the mice were killed by dislocation of the neck, and the blood was collected. The blood was centrifuged at 12000 rpm for 30 minutes at 4°C, and the supernatant was taken and stored at -80°C for later use. For sample analysis, the serum sample was thawed at 4°C, 100 μL of serum was taken, an equal volume of methanol was added, and the sample was vortexed for 60 seconds to mix it thoroughly. Centrifuged at 12000 rpm for 10 minutes at 4°C, the supernatant was collected, and dried with nitrogen. Then, it was re-dissolved with 300 μL of mass spectrometry-grade acetonitrile, and the solution was centrifuged again at 12000 rpm for 10 minutes at 4°C. Finally, 200 μL was accurately transferred from the obtained supernatant and transferred to a liquid phase vial for injection and analysis.

[0086] During the last week of oral administration, feces were collected and stored at -80°C until use. For sample analysis, feces were removed. Fecal sample preparation was performed at 4°C. The collected fecal sample was ground into a powder. 300 mg of the powder was added to 1200 μL of ice-cold methanol, vortexed for 2 minutes, sonicated for 20 minutes, and centrifuged at 14,000 rpm for 10 minutes. The supernatant was collected and filtered through a 0.22 μm filter membrane. 200 μL of the supernatant was placed in a liquid phase vial and prepared for analysis.

[0087] Serum metabolic liquid chromatography conditions

[0088] Chromatographic column: Thermo Syncronis C18 (100 mm × 2.1 mm, 1.7 μm), mobile phase: phase B is acetonitrile, phase C is 0.1% formic acid in water, column temperature: 30°C, sample plate temperature: 4°C, flow rate: 0.4 mL / min, injection volume: 5 μL, gradient elution was selected, and the mobile phase ratio is shown in Table 2.

[0089] Table 2

[0090] Time (min) B(％) C(％) 0-4 5-30 95-70 4-7 30-50 70-50 7-11 50-70 50-30 11-14 70-80 30-20 14-15 80 20 15-18 80-40 20-60 18-19 40-5 60-95 19-20 5 95

[0091] Fecal metabolite liquid chromatography conditions

[0092] Column: Waters ACQUITY HSS T3 (2.1 mm × 150 mm, 1.8 μm), mobile phase: phase B is acetonitrile, phase C is 0.1% formic acid aqueous solution, column temperature: 30 ° C, sample plate temperature: 4 ° C, flow rate: 0.3 mL / min, injection volume: 5 μL, gradient elution was selected, and the mobile phase ratio is shown in Table 3.

[0093] Table 3

[0094] Time (min) B(％) C(％) 0-2 5-30 95-70 2-4 30-60 70-40 4-6 60 40 6-7 60-90 40-10 7-13 90-5 10-95 13-15 5 95

[0095] Mass spectrometry conditions

[0096] Serum mass spectrometry conditions: ESI electrospray ionization source, data acquisition in positive and negative ion modes, mass-to-charge ratio range: m / z 150-1500, resolution: 70,000, scan frequency: 1, automatic gain control (AGC): 1e6, sheath gas flow rate: 40, auxiliary gas flow rate: 10, spray voltage: 4.00 V, capillary temperature: 350°C, S-lens RF level: 55. Fecal mass spectrometry conditions: The mass-to-charge ratio range: m / z 100-1500, other conditions are the same as those for serum mass spectrometry.

[0097] Data processing and analysis

[0098] The raw data were imported into XploreMET software (v3.0 Metabo-Profifile, Shanghai, China) for peak detection and alignment. A data set containing sample name, retention time (RT), accurate molecular weight, and peak intensity was generated. The data were normalized and imported into SIMCA-P software (v13.0, Umetrics, Umea, Sweden). Principal component analysis (PCA) was used to represent the distribution of the raw data, and orthogonal partial least squares discriminant analysis (OPLS-DA) was used to show the separation between the groups. Multivariate statistical analysis was performed, and the reliability of the above model was verified by permutation test. Potential differential metabolites were identified based on the variable importance in projection (VIP) calculated by the OPLS-DA model. Visualization analysis was performed using S-polt. Potential differential metabolites that met the conditions of VIP>1.0, P-value<0.05, and fold changes (FC)>1.2 were preliminarily screened. The Human Metabolomics Database (HMDB) (http: / / www.hmdb.ca) was used to match the accurate MS and MS / MS information of potential differential metabolites. MetaboAnalyst 6.0 (https: / / www.metaboanalyst.ca / ) was used to create heat maps and annotate the metabolic pathways of differential metabolites.

[0099] The PCA analysis results of serum samples of mice in each group are shown in Figure 2. Figure 9As shown in Figure 3-3B, the results showed that the MPTP group and the Control group could be clearly distinguished, indicating that the MPTP group had a significant disorder in serum metabolism. In addition, the distance between the MPTP group and the other groups was significantly widened, and the groups were highly clustered, while the AGPL group, AGPM group, and AGPH group tended to be close to the Control group, indicating that the condition of the mice was significantly improved after treatment with American ginseng total protein. To improve the discriminant analysis ability of the model and further determine the compositional differences between the MPTP group and the other groups, an OPLS-DA (Orthogonal Partial Least Squares Discriminant Analysis) model was constructed. The results are shown in Figure 3-3B. The Control group, AGPL group, AGPM group, and AGPH group can all be clearly separated from the MPTP group, indicating that the serum metabolic components of the MPTP group are significantly different from those of the other groups.

[0100] Permutation test is often used to evaluate the fitness and predictive ability of OPLS-DA model. 200 permutation tests were performed on the OPLS-DA model. 2 and Q 2 Used to indicate the data's ability to explain the model. Figure 10 As shown in Figure A, in the permutation test analysis, in the positive ion mode, the Control group vs. MPTP group R2 = 0.999, Q2 = 0.917; the MPTP group vs. AGPL group R2 = 0.999, Q2 = 0.939; the MPTP group vs. AGPM group R2 = 0.978, Q2 = 0.922; the MPTP group vs. AGPH group R2 = 0.994, Q2 = 0.909. In the negative ion mode, the Control group vs. MPTP group R 2 =0.929, Q 2 =0.538; MPTP group vs. AGPL group R 2 =0.999, Q 2 =0.831; MPTP group vs. AGPM group R 2 =0.965, Q 2 =0.841; MPTP group vs. AGPH group R 2 =0.77, Q 2 =0.32. Q 2 The value is below the origin, the ordinate intercept is less than 0, and R 2 In Q 2 The above shows that the OPLS-DA model is not overfitting and has good predictive ability. The model is suitable for subsequent optimization analysis. Based on the OPLS-DA analysis, the S-polt diagram is established, as shown in Figure 10 As shown in Figure B, VIP>1.0 and P-value<0.05 were set as the criteria for preliminary screening of differential metabolites.

[0101] The volcano plot can show the overall distribution of metabolite differences between different groups, so the volcano plot is used to display the screening results of differential metabolites. Figure 11 As shown, the MPTP group was compared pairwise with the control, AGPL, AGPM, and AGPH groups. Significantly elevated metabolites are highlighted in red, significantly downregulated metabolites are highlighted in blue, and metabolites with no significant differences are highlighted in gray. As shown in the figure, in serum metabolomics, the number of differentially upregulated metabolites in each group was 194, 53, 89, and 78, respectively, while the number of differentially downregulated metabolites was 125, 227, 228, and 165, respectively.

[0102] Differential metabolites between the comparison groups were screened using criteria such as VIP > 1.0, P-value < 0.05, and FC > 1.2. Metabolite types were identified by matching differential metabolites with m / z, RT, and fragment ion information using the HMDB database. Results showed that 19 significantly differential biomarkers were identified in the MPTP group compared to the control group, while 25 significantly differential biomarkers were identified between the AGP group and the MPTP group. Analysis revealed that these significantly differential metabolites primarily encompassed fatty alcohols and fatty acids, fatty amides, amino acid-like compounds, and other organic compounds.

[0103] The Venn diagram uses sets to represent the relationship between groups, and intuitively displays the intersection and difference of data between groups. Figure 12 As shown in the figure, the Venn diagram results showed that in serum metabolomics, 19 differential metabolites were identified in the M vs. C group, 25 differential metabolites were identified in the AGP vs. M group, and there were 10 common differential metabolites between the two groups.

[0104] In order to analyze the changing trends of the common differential metabolites in each group and determine the effects of American ginseng total protein on mouse serum and metabolites, Metabo Analyst 6.0 online website was used to draw heat maps for visual analysis. Figure 13 As shown in the figure, serum metabolomics heatmap results revealed that compared with the control group, eight endogenous metabolites were upregulated and two were downregulated in the MPTP group. Upregulated metabolites included dihexyl adipate, N-hexadecanoylserine, and sphingosine, while downregulated metabolites included tridecanol and lysoPE. After treatment with American ginseng total protein, the levels of nine common differentially expressed metabolites in serum decreased.

[0105] The serum common differential metabolite data were uploaded to Metabo Analyst 6.0 for metabolic pathway analysis. Figure 14The results showed that serum differential metabolites mainly involved four metabolic pathways: sphingolipid metabolism, glycerophospholipid metabolism, fatty acid degradation, and arachidonic acid metabolism. This suggests that American ginseng total protein may play a therapeutic role in neuroinflammation by regulating these key metabolic pathways.

[0106] The fecal metabolism data of each group of mice were subjected to PCA analysis, as shown in Figure 15 As shown in Figure A, the results showed that the fecal metabolic components of the control group and the MPTP group were significantly separated, indicating that the fecal metabolism of the MPTP group was disordered, and the American ginseng total protein groups had different degrees of closeness to the control group. In order to screen potential differential metabolites in fecal metabolism, OPLS-DA analysis was performed on the fecal metabolic data. The results are shown in Figure 1. Figure 15 As shown in Figure B, the MPTP group was clearly separated from the Control group, AGPL group, AGPM group, and AGPH group.

[0107] like Figure 16 As shown in Figure A, in the permutation test analysis, the R of the Control group vs. MPTP group in the positive ion mode 2 =0.976, Q 2 =0.838; MPTP group vs. AGPL group R 2 =0.935, Q 2 =0.743; MPTP group vs. AGPM group R 2 =0.981, Q 2 =0.814; MPTP group vs. AGPH group R 2 =0.995, Q 2 =0.846, Control group vs. MPTP group in negative ion mode R 2 =0.893, Q 2 =0.636; MPTP group vs. AGPL group R 2 =0.998, Q 2 =0.714; MPTP group vs. AGPM group R 2 =1,Q 2 =0.917; MPTP group vs. AGPH group R 2 =0.999, Q 2 =0.809. Q 2 The value is below the origin, the ordinate intercept is less than 0, and R 2 In Q 2 The above shows that the OPLS-DA model is not overfitting, has good predictive ability, and is reliable, so further analysis can be performed. Based on the OPLS-DA analysis, an S-polt diagram is established, as shown in Figure 16As shown in Figure B, VIP>1.0 and P-value<0.05 were set as the conditions for preliminary screening of differential metabolites.

[0108] The volcano plot shows our screening results of differential metabolites. Figure 17 As shown, in each comparison group of fecal metabolism, the numbers of up-regulated differential metabolites were 181, 392, 735, and 622, respectively, and the numbers of down-regulated differential metabolites were 443, 202, 268, and 208, respectively.

[0109] Using the serum metabolite identification method to identify differential metabolites, we analyzed metabolites with VIP > 1.0, P-value < 0.05, and FC > 1.2. The results showed that, in terms of the fecal metabolome, 53 significantly differential biomarkers were identified in the MPTP group compared to the control group, while 117 significantly differential biomarkers were identified in the AGP group compared to the MPTP group. Analysis revealed that these significantly differential metabolites primarily included amino acids, fatty acids, isoflavones, steroid acids, fatty alcohols, esters, purines, and other organic compounds.

[0110] like Figure 18 As shown in the figure, the Venn diagram shows that in the fecal metabolomics, 53 differential metabolites were identified in the M vs. C group, 117 differential metabolites were identified in the AGP vs. M group, and 42 common differential metabolites were identified between the two groups. American ginseng total protein may play a protective role in PD mice by regulating the content of common differential metabolites. MetaboAnalyst 6.0 was used to draw a heat map of the common differential metabolite data of fecal metabolomics and perform a visual analysis. The results are shown in the figure. Figure 19 As shown in the results, compared with the control group, there were 12 up-regulated and 30 down-regulated endogenous metabolites in the MPTP group. The up-regulated metabolites included 2,6-pyridinedicarboxylic acid and 1,2,8-trihydroxy-3-methylanthraquinone, while the down-regulated metabolites included 11-hydroxyeicosatetraenoic acid glyceride, isodeoxycholic acid and β-tyrosine. In order to analyze the regulatory pathway of American ginseng total protein on fecal metabolism in PD mice, metabolic pathway analysis of common differential metabolites was performed. The results are shown in the figure. Figure 20 As shown, the differential metabolites in feces mainly involved nine metabolic pathways: phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine metabolism, niacin and nicotinamide metabolism disorders, ether lipid metabolism, unsaturated fatty acid biosynthesis, tyrosine metabolism, arachidonic acid metabolism, primary bile acid biosynthesis, and purine metabolism.

[0111] The above results show that serum metabolomics involves four major metabolic pathways, while fecal metabolomics mainly involves nine major metabolic pathways. This study found that arachidonic acid metabolism is a metabolic pathway that is jointly participated by serum and fecal metabolites, sphingolipid metabolism contributes significantly to the serum metabolic pathway, and phenylalanine and primary bile acid biosynthesis contribute more significantly to fecal metabolism.

[0112] Arachidonic acid metabolic pathway

[0113] Arachidonic acid (AA) is a 20-carbon chain, omega-6 (n-6) polyunsaturated fatty acid. Free arachidonic acid influences neuronal growth and differentiation by stimulating and inhibiting the function of ion channels, receptors, and enzymes, regulating G protein-mediated second messenger signal transduction, and modulating gene expression. When cell membranes are stimulated by inflammation, arachidonic acid is released from the phospholipid pool of the cell membrane and metabolized through three pathways: cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 (CYP). 12-Hydroxyeicosatetraenoic acid (12-HETE) is an important metabolite of arachidonic acid catalyzed by lipoxygenase. Studies have shown that 12-HETE can promote the activation and nuclear translocation of NF-κB through the integrin-linked kinase (ILK) pathway, thereby activating the NF-κB pathway and triggering an inflammatory response. In this example, the metabolite content of the arachidonic acid metabolic pathway changed significantly. The high level of 12-HETE in the serum of the MPTP group mice indicated that the arachidonic acid pathway was activated and an inflammatory response occurred. The content of 11-hydroxyeicosatetraenoate glyceryl ester (11-Hydroxyeicosatetraenoate glyceryl ester), which can inhibit the release of inflammatory mediators and reduce the inflammatory response, in the feces of the MPTP mice was reduced, indicating that the anti-inflammatory ability of the MPTP group mice was weakened. After treatment with American ginseng total protein, the expression levels of 12-HETE and 11-Hydroxyeicosatetraenoate glycerylester were restored, indicating that American ginseng total protein can improve the abnormalities of the arachidonic acid pathway, regulate neuroinflammation, and exert a neuroprotective effect.

[0114] Sphingolipid metabolism pathway

[0115] Sphingolipids are a class of lipids that are highly enriched in the central nervous system. They are diverse and complex and are related to the development and function of the central nervous system. Sphingolipid metabolism begins in the endoplasmic reticulum, where serine reacts with palmitoyl-CoA to generate 3-ketosphingosine under the action of serine palmitoyltransferase (SPT), which is then converted into sphingosine (Sph) by the action of reductase. Sph reacts with fatty acyl-CoA under the catalysis of dihydroceramide synthase to generate dihydroceramide, and finally forms ceramide (Cer) through the action of desaturase. Related studies have shown that Sph can activate the sphingolipid metabolic pathway and promote Cer generation. In this embodiment, the metabolite content of the sphingolipid metabolic pathway changed significantly, and the Sph content in the serum of mice in the MPTP group increased significantly, indicating that the sphingolipid metabolic pathway was activated and Cer synthesis increased. Cer is highly expressed in neurons and can regulate neuronal signaling, cell metabolism, neuron-glia interaction and cell survival. Studies have shown that through the mitochondrial pathway, elevated Cer levels activate protein phosphatase 2A (PP2A). Activated PP2A activates the pro-apoptotic protein Bax, dephosphorylating it and degrading proteases, ultimately leading to cell apoptosis. Cer is also involved in neuroinflammatory responses. Cer induces the expression of NF-κB, activates the NF-κB signaling pathway

[125] , and promotes the expression of pro-inflammatory factors such as IL-6, IL-1β, and TNF-α. In this example, the elevated levels of IL-6, IL-1β, and TNF-α in the serum of mice in the MPTP group also demonstrate the occurrence of inflammatory responses. After treatment with American ginseng total protein, the sphingosine content in the serum of mice in the MPTP group decreased, indicating that American ginseng total protein can improve the abnormalities of the sphingolipid metabolic pathway, regulate neuroinflammation and cell apoptosis, and play a role in neurodegenerative diseases.

[0116] Phenylalanine metabolic pathway

[0117] Phenylalanine is an essential amino acid that the human body cannot produce. One of its main functions is to serve as a precursor of tyrosine, indirectly participating in the synthesis of multiple important neurotransmitters such as dopamine, norepinephrine and epinephrine, and playing a key role in maintaining the normal function of the nervous system. The core pathway of phenylalanine metabolism in the human body is that phenylalanine is irreversibly degraded by being hydroxylated by phenylalanine hydroxylase (PAH). This process is considered to be the rate-limiting step in dopamine synthesis and can generate L-tyrosine. Studies have shown that when phenylalanine cannot be normally converted into tyrosine, it is metabolized by bypass to generate toxic products such as phenylpyruvic acid and phenyllactic acid, leading to nervous system damage. In the present embodiment, phenylpyruvic acid, as a product of abnormal phenylalanine metabolism, is elevated in the fecal metabolism of mice in the MPTP group. This change can hinder the metabolic pathway of phenylalanine produced by PAH-mediated tyrosine production, thereby affecting the normal metabolism of dopamine. In addition, it can also cause the toxic accumulation of dopamine metabolism intermediate 3,4-dihydroxyphenylacetaldehyde (DOPAL). Furthermore, this shift in the phenylalanine metabolic pathway also produces more ammonia, a neurotoxic substance that can increase oxidative stress in neurons and promote neurodegeneration. Treatment with American ginseng total protein downregulated phenylpyruvic acid levels, suggesting that American ginseng total protein can play a therapeutic role in neuroinflammation by regulating the phenylalanine metabolic pathway.

[0118] Primary bile acid biosynthesis metabolic pathway

[0119] Bile acids (BAs) are amphiphilic molecules synthesized from cholesterol in the liver and are the main component of bile. Bile acids can regulate physiological functions such as glucose and lipid metabolism and homeostasis, the intestinal microbiome, the intestinal barrier, inflammation, the immune system, and the nervous system through interactions with receptors and the intestinal microbiota, thereby affecting metabolic and nutritional health. Studies have shown that bile acid signaling plays a neuroprotective role in neurological diseases. Ursodeoxycholic acid significantly alleviates motor deficits and microglial activation in mice, inhibiting oxidative stress and neuroinflammation. Deoxycholic acid can inhibit the expression of TNF-α and IL-β, reduce MDA and iron ion concentrations, and upregulate TGR5 receptors, inhibit the NF-κB signaling pathway, and reduce the phosphorylation of IκB and P65 proteins, thereby alleviating the inflammatory response. Taurodeoxycholic acid (TUDCA) is an endogenous bile acid that regulates cell death by interrupting the classical apoptotic pathway. In this example, the contents of ursodeoxycholic acid, isodeoxycholic acid, 3β, 12β-dihydroxy-5β-cholic acid, and α-muricholic acid in the feces of mice in the MPTP group were significantly reduced, and the inhibitory effects on neuroinflammatory response and oxidative stress response were reduced. After treatment with American ginseng total protein, the contents of ursodeoxycholic acid, isodeoxycholic acid, 3β, 12β-dihydroxy-5β-cholic acid, and α-muricholic acid were significantly increased, thereby enhancing the inhibitory effects on neuroinflammatory response and oxidative stress response. This indicates that American ginseng total protein can improve the abnormalities of the primary bile acid biosynthesis metabolic pathway, improve the abnormalities of the arachidonic acid pathway in mice, regulate neuroinflammation, and exert a neuroprotective effect.

[0120] The above detailed description of the present invention is intended to enable those skilled in the art to understand the contents of the present invention and implement them, but it does not limit the scope of protection of the present invention. The present invention is not limited to the above embodiments. Any equivalent changes or modifications made based on the spirit of the present invention should be included in the scope of protection of the present invention.

Claims

1. An application of American ginseng total protein in the preparation of a drug for treating neuroinflammation, characterized in that: The six proteins with the highest content in the American ginseng total protein are ribonuclease-like major storage protein, major latex-like protein, phosphoglycerate kinase 1, glyceraldehyde-3-phosphate dehydrogenase, pathogenesis-related protein 10, and ribonuclease 2.

2. The use of the American ginseng total protein according to claim 1 in preparing a drug for treating neuroinflammation, characterized in that: The neuroinflammation treatment drug is a freeze-dried agent of an aqueous solution of total protein from American ginseng, and the protein purity of the aqueous solution of total protein from American ginseng is ≥80%.

3. The use of the American ginseng total protein in the preparation of a drug for treating neuroinflammation according to claim 2, characterized in that: The pH value of the American ginseng total protein aqueous solution is 6.8-7.

2.

4. The use of the American ginseng total protein in the preparation of a drug for treating neuroinflammation according to claim 2, characterized in that: The American ginseng total protein aqueous solution is prepared by extraction with 3-5°C cold water, and the obtained extract is purified by isoelectric precipitation and ultrafiltration.

5. The use of the American ginseng total protein in the preparation of a drug for treating neuroinflammation according to claim 4, characterized in that: During the 3-5° C. cold water extraction, the material-liquid ratio (w / v) of the American ginseng dry powder to water is 1:15-20, and the pH value of the material liquid is 6.8-7.

2.

6. The use of the American ginseng total protein in the preparation of a drug for treating neuroinflammation according to claim 5, characterized in that: The American ginseng dry powder is powder of dried roots of American ginseng with a particle size of ≤24 meshes.

7. The use of the American ginseng total protein in the preparation of a drug for treating neuroinflammation according to claim 4, characterized in that: The pH value adopted in the isoelectric precipitation is 3.4-3.6, and the ultrafiltration adopts an ultrafiltration cut-off tube with a molecular weight of 2.5-3.5 kD.

8. The use of the American ginseng total protein in the preparation of a drug for treating neuroinflammation according to claim 4, characterized in that: The preparation method of the American ginseng total protein aqueous solution is as follows: American ginseng dry powder and distilled water are uniformly mixed at a material-liquid ratio (w / v) of 1:15-20, HCl and NaOH are used to adjust the mixed solution to a pH of 6.8-7.2, cold-immersion extraction is performed at 3-5°C for 10-15 hours, centrifugation is performed at 7000-9000 rpm for 10-20 minutes, the supernatant is collected, and the residue is repeated with the above steps, cold-immersion extraction is performed twice, the supernatants of the two times are combined, the pH of the supernatant is adjusted to 3.4-3.6 with HCl, precipitation is carried out at 3-5°C overnight, the precipitate and supernatant are separated by centrifugation, the precipitate is redissolved, and NaOH is used again to adjust the pH to 6.8-7.2, ultrafiltration protein is intercepted with an ultrafiltration cutoff tube with a molecular weight of 2.5-3.5 kD, centrifugation is performed at 4000-6000 r / min for 25-35 minutes, and the supernatant is collected to obtain the American ginseng total protein aqueous solution.

9. A drug for treating neuroinflammation, characterized in that: The active ingredient of the neuroinflammation treatment drug is American ginseng total protein, and the six proteins with the highest content in the American ginseng total protein are ribonuclease-like major storage protein, major latex-like protein, phosphoglycerate kinase 1, glyceraldehyde-3-phosphate dehydrogenase, pathogenesis-related protein 10, and ribonuclease 2.

10. The drug for treating neuroinflammation according to claim 9, characterized in that The neuroinflammation treatment drug is a freeze-dried agent, and the purity of the American ginseng total protein is ≥80%.

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