Application of ginsenoside Rg5 in preparation of medicine for treating Alzheimer disease and evaluation method

Abstract

The invention discloses application of ginsenoside Rg5 in preparation of a medicine for treating Alzheimer's disease and an evaluation method, and relates to application of ginsenoside Rg5 in preparation of a medicine for treating Alzheimer's disease. The medicine improves A beta deposition, tau protein phosphorylation and neuron damage by adjusting the intestinal flora structure and metabolite level, and is used for relieving the pathological process of the Alzheimer's disease. The invention has the characteristics of multi-target effect, nerve protection, inflammation resistance and oxidation resistance.

Description

Technical Field

[0001] This invention belongs to the field of regulating gut microbiota-gut-brain axis to improve AD pathology, specifically involving the application and evaluation method of ginsenoside Rg5 in the preparation of drugs for treating Alzheimer's disease. Background Technology

[0002] Current medications for Alzheimer's disease (AD) mainly include cholinesterase inhibitors and N-methyl-D-aspartate (NMDA) receptor antagonists. Cholinesterase inhibitors, such as donepezil, improve cognitive function by inhibiting acetylcholinesterase, increasing the content of acetylcholine in the synaptic cleft. However, these drugs only temporarily relieve symptoms and cannot stop disease progression; long-term use may also cause adverse reactions such as nausea, vomiting, and diarrhea. NMDA receptor antagonists, such as memantine, mainly reduce excitotoxic damage by regulating glutamatergic neurotransmission. However, their efficacy in moderate to severe AD patients is limited, and they may cause side effects such as dizziness, headache, and drowsiness.

[0003] Furthermore, some emerging treatment strategies, such as β-amyloid (Aβ) vaccines and tau protein-targeted therapies, while theoretically promising, have encountered numerous setbacks in clinical trials. Early studies of Aβ vaccines revealed severe inflammatory responses in some patients after vaccination, forcing the trial to be discontinued. The challenges facing tau protein-targeted therapies include effectively penetrating the blood-brain barrier and precisely targeting abnormally phosphorylated tau proteins without affecting the function of normal tau proteins.

[0004] Meanwhile, a growing body of research indicates a close link between the gut microbiota and Alzheimer's disease (AD), known as the "brain-gut axis." Imbalances in the gut microbiota may affect AD pathological processes through multiple pathways, including neuroinflammation, neurotransmitter metabolism, Aβ deposition, and tau protein phosphorylation. However, drug development targeting AD by modulating the gut microbiota is still in its early stages, with most studies remaining at the animal testing level, and no mature drugs yet on the market.

[0005] Moreover, existing research has not fully elucidated the complex interaction mechanisms between gut microbiota and AD pathology, which has brought great difficulties to the development of related drugs. Summary of the Invention

[0006] In order to overcome the shortcomings of the existing technology, the purpose of this invention is to provide an application and evaluation method of ginsenoside Rg5 in the preparation of drugs for treating Alzheimer's disease, which has the characteristics of multi-target action, neuroprotection, anti-inflammation and anti-oxidation.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] Application of ginsenoside Rg5 in the preparation of drugs for treating Alzheimer's disease.

[0009] The drug improves Aβ deposition, tau protein phosphorylation, and neuronal damage by regulating gut microbiota structure and metabolite levels, thereby alleviating the pathological progression of Alzheimer's disease.

[0010] Ginsenosides are the main active ingredients of plants in the genus *Panax* of the Araliaceae family, belonging to the triterpenoid glycoside class. Based on their chemical structure and aglycone, ginsenosides are classified into dammarane-type, oleanane-type, and octylylene-type ginsenosides. Dammarane-type ginsenosides are the most prevalent type, including protopanaxadiol (PPD) and protopanaxatriol (PPT).

[0011] The ginsenoside Rg5 is classified as a rare PPD-type ginsenoside because its molecular skeleton has a PPD structure (glycosyl groups are attached at C-3 and C-20) and its content in natural ginseng is extremely low (it needs to be obtained by hydrolysis of the original saponin).

[0012] The ginsenoside Rg5 is derived from the Institute of Biomedical Research at Northwest University and can also be obtained commercially.

[0013] The Alzheimer's disease described is an animal model simulating the pathological features of sporadic AD, with core pathology consistent with clinical sporadic AD. The core pathological changes are characterized by two major landmark changes: β-amyloid (Aβ) deposition and abnormal phosphorylation of tau protein (p-tau), accompanied by hippocampal neuronal damage (reduction of Nissl bodies, neuronal disorganization) and spatial cognitive impairment (confirmed by the Morris water maze test). The model was established in C57BL / 6J mice by inducing AD through a combination of AlCl3·6H2O gavage and intraperitoneal injection of D-galactose, mimicking the progressive neurodegenerative changes of clinical sporadic AD.

[0014] The ginsenoside Rg5 is in the form of a solution and is administered by gavage (ig).

[0015] The preparation method of ginsenoside Rg5 includes the following steps;

[0016] Prepare a 120 mg / kg (±5%) ginsenoside Rg5 solution (final administration solution);

[0017] Take 0.40-0.45 ml of the saponin stock solution, add 2.35-2.45 ml of solvent, dissolve by sonication, and use immediately after preparation. The final dosage can be precisely controlled according to the animal's body weight by controlling the amount of stock solution used and the volume of solvent dilution.

[0018] The preparation of the drug includes the preparation of the solvent, specifically by weighing 12.0-14.0 g of poloxamer, 16.5-19.0 g of polyethylene glycol 400 (PEG 400), and 16.5-19.0 g of propylene glycol. The above components are dissolved by sonication in an 80°C water bath. After complete dissolution, an equal volume of physiological saline is added, and the mixture is sonicated again. The prepared solution is then stored at 4°C for later use. The solvent serves as the base carrier solution, constructing a composite solvent system with good dissolving power, biocompatibility, and physical stability, thus acting as the base carrier for the final drug solution.

[0019] The preparation of the drug includes the preparation of a 100 mg / ml saponin stock solution. Weigh 60.0-66.0 mg of ginsenoside Rg5 and add 0.60-0.66 ml of pharmaceutical-grade anhydrous ethanol to prepare a 100 mg / ml (±5%) saponin stock solution. Dissolve the solution using ultrasound and prepare immediately before use. The poorly soluble or low-soluble ginsenoside Rg5 is first dissolved in anhydrous ethanol (a co-solvent) to form a high-concentration stock solution, facilitating subsequent precise dilution to the target therapeutic concentration.

[0020] Alzheimer's disease mouse model was established using 6-8 week old C57BL / 6J mice by gavage administration of 50 mg / kg AlCl3·6H2O combined with intraperitoneal injection of 120 mg / kg D-Gal, in order to evaluate the therapeutic effect of the drug on Alzheimer's disease.

[0021] The method for evaluating the therapeutic effect of the drug on Alzheimer's disease includes the following steps:

[0022] Step 1: The Morris water maze test was used to assess the spatial cognitive ability of mice. The Morris water maze test consists of a navigation test and a spatial exploration test. By analyzing the escape latency of mice in the navigation test and the number of times they crossed the original platform location and the time spent in the target quadrant in the spatial exploration test, the effect of the drug on improving the spatial learning and memory ability of mice was evaluated. The Morris water maze test is a classic behavioral method for assessing the spatial learning and memory ability of animals. Its core principle is based on the animal's associative learning and memory retrieval of "environmental cues-target location".

[0023] The experimental environment was a circular pool (the water was stained to make the hidden platform invisible), with stable visual cues (such as laboratory wall markings, lights, etc.) set up around it. The animals needed to be trained repeatedly to establish a spatial association between these environmental cues and the location of the underwater hidden platform, forming a reference memory of "knowing where the platform is". This process relied on the spatial cognitive function of brain regions such as the hippocampus.

[0024] Orientation and navigation phase: Animals set off from different entry points multiple times a day to learn to find platforms; as the number of training sessions increases, normal animals gradually master spatial cues, and the "latency" (time) for finding the platform will gradually shorten, reflecting their spatial learning ability; if there is brain damage (such as in AD model mice), the latency will be significantly prolonged or show no decreasing trend.

[0025] Spatial exploration phase: After the platform is removed, the animals re-enter the water; normal animals will prioritize staying and moving around in the original platform area, which is reflected by "increased time spent in the target area" and "increased number of times crossing the original platform location", reflecting their ability to retain and retrieve the memory of the platform location; animals with cognitive impairment do not have this preference;

[0026] The experimental results are directly related to the function of brain regions closely related to spatial memory, such as the hippocampus. When there is neuronal damage or pathological protein deposition (such as Aβ and p-tau) in the brain region, the spatial learning and memory process is hindered, and characteristic abnormalities will appear in the experimental indicators (latency and exploratory behavior).

[0027] Step 2: Nissl staining was used to detect changes in the morphology and number of neurons in the brain, and the protective effect of the drug on neuronal damage was analyzed.

[0028] Step 3: Immunofluorescence staining (IF) and Western blotting (WB) were used to detect pathological markers of Alzheimer's disease such as Aβ deposition and tau phosphorylation, to clarify the ameliorative effect of drugs on the pathological characteristics of Alzheimer's disease; Ginsenoside Rg5 can reduce Aβ production and promote Aβ clearance in AD mice, inhibit tau phosphorylation, reduce tangle formation or promote tangle clearance.

[0029] Step 4: 16S rRNA sequencing was used to analyze the α-diversity index of the gut microbiota and the relative abundance changes of different phyla and genera. Combined with LEfSe analysis, the taxonomic units of the microbiota with significant differences among different groups were identified to evaluate the effect of the drug on the remodeling of the gut microbiota. Ginsenoside Rg5 can increase the abundance of beneficial bacteria such as Actinomycetota and Bifidobacterium, restore the α-diversity of the microbiota, and inhibit the proliferation of harmful bacteria such as Mycoplasmata.

[0030] Step 5: Metabolomics analysis of metabolites in mouse serum was performed using liquid chromatography-mass spectrometry (LC-MS) to identify differentially expressed metabolites and analyze their involved metabolic pathways. An exogenous Kyna supplementation experiment was conducted to verify the key role of Kyna in the neuroprotective effects of the drug. Ginsenoside Rg5 can increase Kyna levels through the tryptophan metabolic pathway, inhibit microglia activation, and reduce neuroinflammation.

[0031] Step 2 specifically involves:

[0032] Sample collection and fixation: Take fresh brain tissue (such as the hippocampus, cortex, etc.) and immediately fix it in 4% paraformaldehyde fixative for 24-48 hours (too long a fixation time may cause Nissl bodies to dissolve); after fixation, rinse the tissue with running water to remove residual fixative.

[0033] Tissue preparation: Immerse the tissue sequentially in 70%, 80%, 95%, and 100% ethanol for a gradient dehydration process, 5-10 minutes per stage (ethanol concentration must be strictly controlled to avoid excessive tissue shrinkage); clear the tissue twice with xylene, 5-10 minutes each time, until completely transparent. Immerse the tissue in molten paraffin wax, and after cooling, cut into 4-10 μm thick sections (sections that are too thick will affect the staining effect).

[0034] Section pretreatment: Dewax the paraffin sections in xylene for 5-10 minutes, repeat twice, until the sections are completely transparent; hydrate them successively with 100%, 95%, 80%, and 70% ethanol for 2-5 minutes each time, and finally rinse with distilled water for 2 minutes;

[0035] Staining: Place the sections in 1% toluidine blue solution (or tar purple solution) and stain in an incubator at 50-60℃ for 25-50 minutes (excessive temperature may cause the background to be stained too dark); after staining, rinse with distilled water and rapidly differentiate in 70% ethanol (about 5-10 seconds) until the background is nearly colorless and the Nissl bodies remain clearly visible; dehydrate successively with 95% and 100% ethanol for 2 minutes each, and then clear twice with xylene for 5 minutes each time;

[0036] Mount the slide for observation; add neutral resin to mount the slide, avoiding the formation of air bubbles; observe the morphology of Nissl body under a microscope (normally a dark blue or purplish-blue granular structure);

[0037] Normally, dense Nissl bodies are visible in the cytoplasm of neurons, distributed in patches or granules. The cell nucleus appears pale blue against a clear background with no excess staining. Abnormally, the number of Nissl bodies decreases, their size decreases, or they disappear.

[0038] The beneficial effects of this invention are:

[0039] 1. Improved cognitive function: The Morris water maze experiment confirmed that ginsenoside Rg5 significantly shortened the escape latency of AD mice and increased their dwell time and number of crossings in the target area, indicating that it has a significant effect on improving the spatial learning and memory ability of AD mice.

[0040] 2. Alleviating Pathological Damage: Nissl staining showed that Rg5 treatment increased the number of Nissl bodies in hippocampal neurons of AD mice, and the neuronal morphology and arrangement tended to be normal, suggesting its repair effect on neuronal damage. Immunofluorescence and Western blot confirmed that Rg5 significantly reduced the fluorescence intensity and protein expression levels of Aβ and p-tau, reducing their abnormal accumulation in brain tissue.

[0041] 3. Remodeling the Gut Microbiota: 16S rRNA sequencing showed that Rg5 intervention significantly restored the α-diversity of the gut microbiota in AD mice, with increased abundance of beneficial bacteria such as Actinomycetota and Bifidobacterium, while decreasing abundance of harmful bacteria such as Mycoplasmata. LEfSe analysis indicated that the Rg5 group was significantly enriched in key taxonomic units such as Bifidobacteriales, which regulate neuroinflammation and metabolic pathways through the brain-gut axis.

[0042] 4. Regulation of intestinal metabolites: Metabolomics analysis showed that Rg5 treatment significantly altered the intestinal metabolite profile in AD mice, particularly increasing the level of kyna in the tryptophan metabolism pathway. Kyna, as a neuroprotective factor, can alleviate neuroinflammation by inhibiting microglia activation. Validation experiments demonstrated that exogenous Kyna supplementation can mimic the neuroprotective effects of Rg5, confirming its importance as a key mediator. Attached Figure Description

[0043] Figure 1 This is a representative trajectory diagram for a positioning navigation experiment.

[0044] Figure 2 This diagram illustrates the escape latency period, the number of times the effective area is crossed, and the time spent in the effective area.

[0045] Figure 3 This is a schematic diagram of Nissl staining results for neurons in the hippocampus region.

[0046] Figure 4 This is a schematic diagram of the immunofluorescence of Aβ (red) and P-Tau (green) in the hippocampal region.

[0047] Figure 5 This diagram illustrates the α-diversity analysis of gut microbiota and the relative abundance changes at the phylum and genus levels.

[0048] Figure 6 This is linear discriminant analysis (LDA).

[0049] Figure 7 A schematic diagram of KEGG metabolic pathway enrichment analysis and ginsenoside Rg5 enriched metabolites.

[0050] Figure 8 The results of the experimental indicators were used to supplement exogenous Kyna. Detailed Implementation

[0051] The present invention will now be described in further detail with reference to the accompanying drawings.

[0052] Drug preparation:

[0053] 1. Solvent Preparation: Accurately weigh 13.0g of poloxamer, 17.9g of polyethylene glycol 400 (PEG 400), and 17.9g of propylene glycol. Dissolve them by sonication in an 80℃ water bath. After complete dissolution, add an equal volume of physiological saline and sonicate again. Store the prepared solution at 4℃ for later use. An 80℃ water bath accelerates dissolution, sonication promotes uniform dispersion of components, dilution with physiological saline followed by sonication ensures homogeneity, and storage at 4℃ maintains stability.

[0054] 2. Preparation of 100 mg / ml saponin stock solution: Based on a dosage of 21 mice weighing 25.0 g per day, weigh 63.0 mg of ginsenoside Rg5 and add 0.63 ml of pharmaceutical-grade anhydrous ethanol to prepare a 100 mg / ml saponin stock solution. Dissolve by sonication and use immediately. Precise dosage based on the number and weight of mice is crucial. Anhydrous ethanol has good dissolving properties, sonication promotes dissolution, and immediate use ensures activity.

[0055] Preparation of 3.120 mg / kg ginsenoside Rg5 solution: Prepare a solution according to the dosage for 14 mice weighing 25.0 g per day. Take 0.42 ml of the ginsenoside stock solution, add 2.38 ml of solvent, and dissolve by sonication. Prepare immediately before use. Administer 0.20 ml by gavage to each mouse. Accurately measure the stock solution and solvent, sonicate to ensure uniform dispersion, prepare immediately before use to ensure efficacy, and scientifically design the gavage dosage.

[0056] Model building:

[0057] A mouse model of Alzheimer's disease (AD) was successfully established by using 6-8 week old C57BL / 6J mice. The mice were induced by a combination of gavage administration of 50 mg / kg AlCl3·6H2O and intraperitoneal injection of 120 mg / kg D-Gal for 8 weeks. Mice in this age group exhibited stable physiological function, and the combined induction method effectively simulated the pathophysiological environment of AD.

[0058] The method for evaluating the therapeutic effect of the drug on Alzheimer's disease includes the following steps:

[0059] Step 1: The Morris water maze test was used to assess the spatial cognitive ability of mice. The Morris water maze test consists of a navigation test and a spatial exploration test. By analyzing the escape latency of mice in the navigation test and the number of times they crossed the original platform location and the time spent in the target quadrant in the spatial exploration test, the effect of the drug on improving the spatial learning and memory ability of mice was evaluated. The Morris water maze test is a classic behavioral method for assessing the spatial learning and memory ability of animals. Its core principle is based on the animal's associative learning and memory retrieval of "environmental cues-target location".

[0060] The experimental environment was a circular pool (the water was stained to make the hidden platform invisible), with stable visual cues (such as laboratory wall markings, lights, etc.) set up around it. The animals needed to be trained repeatedly to establish a spatial association between these environmental cues and the location of the underwater hidden platform, forming a reference memory of "knowing where the platform is". This process relied on the spatial cognitive function of brain regions such as the hippocampus.

[0061] Orientation and navigation phase: Animals set off from different entry points multiple times a day to learn to find platforms; as the number of training sessions increases, normal animals gradually master spatial cues, and the "latency" (time) for finding the platform will gradually shorten, reflecting their spatial learning ability; if there is brain damage (such as in AD model mice), the latency will be significantly prolonged or show no decreasing trend.

[0062] Spatial exploration phase: After the platform is removed, the animals re-enter the water; normal animals will prioritize staying and moving around in the original platform area, which is reflected by "increased time spent in the target area" and "increased number of times crossing the original platform location", reflecting their ability to retain and retrieve the memory of the platform location; animals with cognitive impairment do not have this preference;

[0063] The experimental results are directly related to the function of brain regions closely related to spatial memory, such as the hippocampus. When there is neuronal damage or pathological protein deposition (such as Aβ and p-tau) in the brain region, the spatial learning and memory process is hindered, and characteristic abnormalities will appear in the experimental indicators (latency and exploratory behavior).

[0064] Step 2: Nissl staining was used to detect changes in the morphology and number of neurons in the brain, and the protective effect of the drug on neuronal damage was analyzed.

[0065] Step 3: Immunofluorescence staining (IF) and Western blotting (WB) were used to detect pathological markers of Alzheimer's disease such as Aβ deposition and tau phosphorylation, to clarify the ameliorative effect of drugs on the pathological characteristics of Alzheimer's disease; Ginsenoside Rg5 can reduce Aβ production and promote Aβ clearance in AD mice, inhibit tau phosphorylation, reduce tangle formation or promote tangle clearance.

[0066] Step 4: 16S rRNA sequencing was used to analyze the α-diversity index of the gut microbiota and the relative abundance changes of different phyla and genera. Combined with LEfSe analysis, the taxonomic units of the microbiota with significant differences among different groups were identified to evaluate the effect of the drug on the remodeling of the gut microbiota. Ginsenoside Rg5 can increase the abundance of beneficial bacteria such as Actinomycetota and Bifidobacterium, restore the α-diversity of the microbiota, and inhibit the proliferation of harmful bacteria such as Mycoplasmata.

[0067] Step 5: Metabolomics analysis of metabolites in mouse serum was performed using liquid chromatography-mass spectrometry (LC-MS) to identify differentially expressed metabolites and analyze their involved metabolic pathways. An exogenous Kyna supplementation experiment was conducted to verify the key role of Kyna in the neuroprotective effects of the drug. Ginsenoside Rg5 can increase Kyna levels through the tryptophan metabolic pathway, inhibit microglia activation, and reduce neuroinflammation.

[0068] Step 2 specifically involves:

[0069] Sample collection and fixation: Take fresh brain tissue (such as the hippocampus, cortex, etc.) and immediately fix it in 4% paraformaldehyde fixative for 24-48 hours (too long a fixation time may cause Nissl bodies to dissolve); after fixation, rinse the tissue with running water to remove residual fixative.

[0070] Tissue preparation: Immerse the tissue sequentially in 70%, 80%, 95%, and 100% ethanol for a gradient dehydration process, 5-10 minutes per stage (ethanol concentration must be strictly controlled to avoid excessive tissue shrinkage); clear the tissue twice with xylene, 5-10 minutes each time, until completely transparent. Immerse the tissue in molten paraffin wax, and after cooling, cut into 4-10 μm thick sections (sections that are too thick will affect the staining effect).

[0071] Section pretreatment: Dewax the paraffin sections in xylene for 5-10 minutes, repeat twice, until the sections are completely transparent; hydrate them successively with 100%, 95%, 80%, and 70% ethanol for 2-5 minutes each time, and finally rinse with distilled water for 2 minutes;

[0072] Staining: Place the sections in 1% toluidine blue solution (or tar purple solution) and stain in an incubator at 50-60℃ for 25-50 minutes (excessive temperature may cause the background to be stained too dark); after staining, rinse with distilled water and rapidly differentiate in 70% ethanol (about 5-10 seconds) until the background is nearly colorless and the Nissl bodies remain clearly visible; dehydrate successively with 95% and 100% ethanol for 2 minutes each, and then clear twice with xylene for 5 minutes each time;

[0073] Mount the slide for observation; add neutral resin to mount the slide, avoiding the formation of air bubbles; observe the morphology of Nissl body under a microscope (normally a dark blue or purplish-blue granular structure);

[0074] Normally, dense Nissl bodies are visible in the cytoplasm of neurons, distributed in patches or granules. The cell nucleus appears pale blue against a clear background with no excess staining. Abnormally, the number of Nissl bodies decreases, their size decreases, or they disappear.

[0075] Experimental Groups:

[0076] The mice were divided into a normal control group (Con), a model group (AD), and a treatment group (Rg5). The normal control group consisted of healthy mice; the AD model mice in the model group exhibited typical AD pathological conditions of spatial cognitive dysfunction and neuronal damage; the treatment group was treated with ginsenoside Rg5 in addition to the AD model mice to observe the intervention effect.

[0077] Experimental methods:

[0078] Cognitive function testing: The Morris water maze test was used, which included a navigation experiment and a spatial exploration experiment. In the navigation experiment, mice were placed in the water from different quadrants for 5 consecutive days, and the escape latency was recorded to assess learning ability. In the spatial exploration experiment, the platform was removed, and mice were placed in the opposite quadrant. The number of times the original platform was crossed within 60 seconds and the time spent in the target quadrant were recorded to assess memory ability.

[0079] Pathological damage detection: Nissl staining was used to observe changes in the morphology and number of brain neurons; immunofluorescence staining (IF) was used to visually display the distribution and expression of Aβ protein and phosphorylated tau protein in brain tissue, and Western blotting (WB) was used to quantitatively analyze Aβ protein and phosphorylated tau protein.

[0080] Gut microbiota analysis: The α diversity index of gut microbiota was calculated by 16S rRNA sequencing to analyze the relative abundance changes of different phyla and genera; LEfSe analysis was used to identify microbiota taxa with significant differences among different groups.

[0081] Intestinal metabolite detection: Metabolites in mouse serum were analyzed using liquid chromatography-mass spectrometry (LC-MS) to identify differentially expressed metabolites and analyze the metabolic pathways they participate in. Exogenous Kyna supplementation experiments were conducted. A model was constructed using the same induction method. A model control group, an exogenous Kyna supplementation group, and a normal control group were set up. The exogenous Kyna supplementation group was given 100 mg / kg of Kyna by gavage for 4 weeks, and then the same indicators were detected.

[0082] This invention mainly focuses on the application of ginsenoside Rg5 in the preparation of drugs for treating Alzheimer's disease. The specific implementation process is described in detail below:

[0083] Laboratory animals and grouping

[0084] Six- to eight-week-old C57BL / 6J mice were selected as the research subjects. Mice were randomly divided into three groups: a normal control group (Con), a model group (AD), and a drug-treated group (Rg5). The number of mice in each group was reasonably set according to experimental statistical requirements, generally no less than 10 mice per group, to ensure the reliability of the experimental results. Mice in the normal control group received no special treatment. Mice in the model group and drug-treated group were induced to develop Alzheimer's disease by gavage administration of 50 mg / kg AlCl3·6H2O and intraperitoneal injection of 120 mg / kg D-Gal for 8 weeks to simulate the pathological characteristics of Alzheimer's disease, such as Aβ deposition and tau protein phosphorylation.

[0085] Administration method and dosage

[0086] Mice in the treatment group (Rg5) were treated with ginsenoside Rg5 by gavage for 4 weeks. Mice in the normal group (Con) and the model group (AD) were given the same amount of the solvent, with the same administration method and frequency.

[0087] Indicator Testing

[0088] Cognitive function testing

[0089] Morris water maze test: This is a classic experiment for assessing spatial learning and memory abilities in mice. The experiment consists of two phases: a navigation test and a spatial exploration test. In the navigation test, mice are placed in the water from different quadrants each day for five consecutive days, and the time it takes for the mouse to find the platform hidden underwater is recorded, i.e., the escape latency. By analyzing the daily changes in the escape latency, the learning ability of the mice can be assessed. In the spatial exploration test, the platform is removed, and the mouse is placed in the water from the quadrant opposite to the original platform. The number of times the mouse crosses the effective area within 60 seconds and the time spent in the effective area are recorded. These indicators reflect the mouse's memory ability.

[0090] Pathological damage detection

[0091] Nissl staining: After the experiment, mouse brain tissue was taken, fixed, dehydrated, embedded, and then prepared into paraffin sections. The number, morphology, and distribution of Nissl bodies in hippocampal neurons were observed under a microscope using Nissl staining.

[0092] Immunofluorescence and Western blot: Mouse brain tissue was used to observe the distribution and expression of Aβ and p-tau proteins in the brain tissue using immunofluorescence. Specific fluorescently labeled antibodies were used to bind to Aβ and p-tau proteins, and the fluorescence intensity was observed under a fluorescence microscope. Western blot was used to further quantify the expression levels of Aβ and p-tau proteins. Specifically, total protein was extracted from brain tissue, and after protein quantification, proteins were separated by SDS-PAGE electrophoresis. After transfer to a membrane, the protein was incubated with a specific antibody, and the grayscale value of the bands was detected using chemiluminescence to determine the relative expression levels of Aβ and p-tau proteins.

[0093] Gut microbiota analysis

[0094] 16S rRNA sequencing: Mouse fecal samples were collected, and total microbial DNA was extracted from the feces. PCR amplification was performed on specific regions of the 16S rRNA gene, and the amplified products were then sequenced using high-throughput sequencing. Bioinformatics analysis was used to calculate alpha diversity indices of the gut microbiota, such as the Chao1 index and Shannon index, to assess the richness and diversity of the microbiota. Simultaneously, the relative abundance changes of different phyla and genera were analyzed.

[0095] LEfSe analysis: Further LEfSe (linear discriminant analysis effect size) analysis was performed on the sequencing data to identify microbial taxa that showed significant differences between different groups.

[0096] Intestinal metabolite detection

[0097] Metabolomics analysis: Metabolites in mouse serum were analyzed using liquid chromatography-mass spectrometry (LC-MS). Differential metabolites were identified and their involved metabolic pathways were analyzed by comparing with a standard database and using relevant data analysis methods.

[0098] Verification Experiment: To confirm the key role of Kyna in the neuroprotective effect of ginsenoside Rg5, an exogenous Kyna supplementation experiment was conducted. An Alzheimer's disease mouse model was constructed using the same induction method as described above, with separate control groups, an exogenous Kyna supplementation group, and a normal control group. Mice in the exogenous Kyna supplementation group were administered an appropriate amount of 100 mg / kg of Kyna via gavage for 4 weeks. Then, the same indicators as those used in the cognitive function and pathological damage tests described above were performed.

[0099] Based on the above experiments, the significant effects of ginsenoside Rg5 in improving cognitive dysfunction, alleviating pathological damage, remodeling gut microbiota, and regulating intestinal metabolites in Alzheimer's disease mice were clarified, providing a solid experimental basis for the application of ginsenoside Rg5 in the preparation of drugs for the treatment of Alzheimer's disease.

[0100] Figure 1 , Figure 2 The figures show representative trajectories, escape latency, number of times the effective area was crossed, and time spent in the effective area for the navigation experiment. Mice treated with ginsenoside Rg5 showed a significantly shorter escape latency, an increased number of times the effective area was crossed, and a longer time spent in the effective area, indicating that ginsenoside Rg5 has an improving effect on cognitive function in mice.

[0101] Figure 3 The results show Nissl staining of neurons in the hippocampus. Normal neurons have abundant Nissl bodies, which appear as dark blue clumps or granules distributed in the cytoplasm. In Alzheimer's disease mouse models, hippocampal neurons showed a decrease in the number and abnormal morphology of Nissl bodies, while treatment with ginsenoside Rg5 increased the number of Nissl bodies and normalized neuronal morphology and arrangement, indicating that ginsenoside Rg5 has a repairing effect on neuronal damage.

[0102] Figure 4 Immunofluorescence of Aβ (red) and P-Tau (green) in the hippocampus. The fluorescence intensity and protein expression levels of Aβ and p-tau in mice treated with ginsenoside Rg5 were significantly reduced, indicating that ginsenoside Rg5 can reduce their abnormal accumulation in brain tissue.

[0103] Figure 5 This study analyzed the α-diversity of gut microbiota and the relative abundance changes at the phylum and genus levels. In the ginsenoside Rg5 treatment group, the α-diversity of gut microbiota in mice was significantly restored, with increased abundance of beneficial bacteria such as Actinomycetota and Bifidobacterium, while the abundance of harmful bacteria such as Mycoplasmata decreased, indicating that ginsenoside Rg5 has a remodeling effect on gut microbiota.

[0104] Figure 6 Linear discriminant analysis (LDA) was performed. The ginsenoside Rg5 group was significantly enriched with key taxonomic units such as Bifidobacteriales, indicating that these bacterial groups may play an important role in the regulation of neuroinflammation and metabolic pathways by ginsenoside Rg5.

[0105] Figure 7 This study focuses on the enrichment analysis of the KEGG metabolic pathway and the metabolites enriched by ginsenoside Rg5. Metabolomics analysis revealed that treatment with ginsenoside Rg5 significantly altered the intestinal metabolite profile in AD mice, particularly by a significant increase in kyna levels in the tryptophan metabolic pathway, suggesting that ginsenoside Rg5 may exert its neuroprotective effect by regulating tryptophan metabolism.

[0106] Figure 8The results of experimental indicators for exogenous Kyna supplementation are presented. Exogenous Kyna supplementation can mimic the neuroprotective effects of ginsenoside Rg5, such as improving cognitive function and reducing pathological damage in mice, further confirming the importance of Kyna as a key mediator.

Claims

1. Application of ginsenoside Rg5 in the preparation of drugs for treating Alzheimer's disease.

2. The application of ginsenoside Rg5 according to claim 1 in the preparation of drugs for treating Alzheimer's disease, characterized in that: The ginsenosides mentioned are rare PPD-type ginsenosides; The ginsenoside Rg5 is in the form of a solution and is administered by gavage.

3. The method for preparing ginsenoside Rg5 according to claim 1 or 2, characterized in that, Includes the following steps; Prepare a ginsenoside Rg5 solution; Take 0.40-0.45 ml of saponin mother liquor, add 2.35-2.45 ml of solvent, dissolve by sonication, and use immediately after preparation.

4. The method for preparing ginsenoside Rg5 according to claim 3, characterized in that, The solvent is prepared as follows: Weigh out 12.0-14.0g of poloxamer, 16.5-19.0g of polyethylene glycol 400 (PEG 400), and 16.5-19.0g of propylene glycol. Place the above components in an 80℃ water bath and sonicate to dissolve them. After they are fully dissolved, add an equal volume of physiological saline and sonicate again to mix. Store the prepared solution in a 4℃ refrigerator for later use.

5. The method for preparing ginsenoside Rg5 according to claim 3, characterized in that, The specific preparation method for the saponin mother liquor is as follows: Weigh 60.0-66.0 mg of ginsenoside Rg5, add 0.60-0.66 ml of pharmaceutical grade anhydrous ethanol to prepare a 100 mg / ml (±5%) saponin stock solution, dissolve by sonication, and use immediately after preparation.

6. A method for evaluating the therapeutic effect of the application based on claim 1 or 2 on Alzheimer's disease, characterized in that, Includes the following steps: Step 1: The experimental environment is a circular pool with stable visual cues around it; the animals need to be trained repeatedly to establish a spatial association between the environmental cues and the location of the hidden underwater platform, forming a reference memory of "knowing where the platform is"; Orientation and navigation phase: Animals depart from different entry points multiple times a day to learn to find platforms; as the number of training sessions increases, normal animals gradually master spatial cues, and the "latency" for finding the platform will gradually shorten, reflecting their spatial learning ability; if there is brain damage, the latency will be significantly prolonged or show no decreasing trend. Spatial exploration phase: After the platform is removed, the animals re-enter the water; normal animals will prioritize staying and moving around in the original platform area, which is reflected by "increased time spent in the target area" and "increased number of times crossing the original platform location", reflecting their ability to retain and retrieve memories of the platform location; animals with cognitive impairment do not have this preference; The experimental results are directly related to the function of brain regions such as the hippocampus that are closely related to spatial memory. When there is neuronal damage or pathological protein deposition in the brain regions, the spatial learning and memory process is hindered, and characteristic abnormalities will appear in the experimental indicators. Step 2: Nissl staining was used to detect changes in the morphology and number of neurons in the brain, and the protective effect of the drug on neuronal damage was analyzed. Step 3: Use immunofluorescence staining (IF) and Western blotting (WB) to detect pathological markers of Alzheimer's disease such as Aβ deposition and tau phosphorylation, and clarify the effect of drugs on improving the pathological characteristics of Alzheimer's disease; Step 4: Analyze the α diversity index of gut microbiota and the relative abundance changes of different phyla and genera by 16S rRNA sequencing, and combine with LEfSe analysis to identify microbial taxa with significant differences among different groups, and evaluate the effect of drugs on gut microbiota remodeling. Step 5: Metabolomics analysis of metabolites in mouse serum was performed using liquid chromatography-mass spectrometry (LC-MS) to identify differentially expressed metabolites and analyze the metabolic pathways they participate in. The key role of Kyna in the neuroprotective effect of drugs was verified by exogenous Kyna supplementation experiments.

7. The evaluation method according to claim 6, characterized in that, Step 2 specifically involves: Sample collection and fixation: Fresh brain tissue was immediately placed in 4% paraformaldehyde fixative for 24-48 hours; after fixation, the tissue was rinsed with running water to remove residual fixative. Organizational processing; The tissue was sequentially immersed in 70%, 80%, 95%, and 100% ethanol for graded dehydration, with each grade lasting 5-10 minutes. Clear the tissue twice with xylene, 5-10 minutes each time, until the tissue is completely transparent; immerse the tissue in molten paraffin, and after cooling, cut it into 4-10 μm thick sections; Slicing pretreatment; Dewax the paraffin sections in xylene for 5-10 minutes, repeat twice, until the sections are completely transparent; hydrate them successively with 100%, 95%, 80%, and 70% ethanol for 2-5 minutes each time, and finally rinse with distilled water for 2 minutes. Staining: Place the sections in 1% toluidine blue solution and stain in an incubator at 50-60℃ for 25-50 minutes; after staining, rinse with distilled water, and rapidly differentiate in 70% ethanol until the background is nearly colorless and the Nissl bodies remain clearly visible; dehydrate with 95% and 100% ethanol successively, 2 minutes each time, and then clear with xylene twice, 5 minutes each time; Mount the slide for observation; add neutral resin to mount the slide, avoiding the formation of air bubbles; observe the morphology of Nissl bodies under a microscope; Normally, dense Nissl bodies are visible in the cytoplasm of neurons, distributed in patches or granules, with pale blue nuclei, a clear background, and no excess staining; abnormally, the number of Nissl bodies is reduced, their size is smaller, or they disappear.

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