Natural medicine mixture for treating alzheimer's disease and use thereof

Abstract

The application discloses a natural medicine compound for treating Alzheimer's disease and application thereof, and the compound comprises the following components: 3-6 parts of Gastrodia elata, 3-6 parts of Polygala, 3-6 parts of Radix Notoginseng, 3-6 parts of Uncaria, 9-15 parts of Radix Rehmanniae Preparata, 9-15 parts of Radix Anemarrhenae, 6-10 parts of Fructus Schisandrae, 6-9 parts of Fried Semen Ziziphi Preparatum, 2-5 parts of Chuanxiong Rhizoma, 3-6 parts of Radix Paeoniae Alba, 9-15 parts of Radix et Rhizoma Bambusae, 1-5 parts of Carthamus, 6-10 parts of Atractylodes, 6-9 parts of Poria, 9-15 parts of Ophiophagus, 3-9 parts of Angelica, 9-15 parts of Radix et Rhizoma Ginseng, 2-5 parts of Salvia, and 2-5 parts of Radix Glycyrrhizae. The application further discloses a pharmacological action mechanism of the Tianqi granules for treating AD and application of the Tianqi granules in preventing and / or treating Alzheimer's disease. The application is a natural medicine, contains multiple active components, has scientific dosage, no side effect, and provides a new effective natural medicine compound for treating AD, and plays a significant role in benefiting a large number of patients and benefiting human health.

Description

Technical Field

[0001] This invention relates to a natural drug compound for treating Alzheimer's disease and its application, belonging to the field of pharmaceutical technology. Background Technology

[0002] Alzheimer's disease (AD) is a multifactorial neurodegenerative disease characterized by progressive cognitive decline and neuropathological changes such as β-amyloid plaque deposition.

[0003] This disease is a neurodegenerative disorder characterized by its early, insidious, and persistent nature, posing a significant challenge to neuroscience and medicine. In the preclinical stage, neuroinflammatory responses, abnormal protein metabolism, and the deposition of β-amyloid plaques are early events in its pathological process. During the clinical symptomatic stage, the pathological spread of Tau protein, synaptic dysfunction, and progressive decline in cognitive function occur.

[0004] Currently, there are still no effective drugs for the prevention and treatment of Alzheimer's disease in clinical practice, and the development of new drugs remains a challenging task. Internationally recognized drugs for treating Alzheimer's disease include the acid inhibitor donepezil, the receptor inhibitor memantine, the acetylcholine receptor inhibitor rivastigmine, and the acid inhibitor galantamine. These drugs can only improve some symptoms in Alzheimer's patients; they cannot prevent neuronal damage or repair damaged neurons in the brain. Moreover, most drugs currently used clinically for the treatment of Alzheimer's disease are single-target drugs. While these drugs can temporarily and partially improve cognitive impairment, they cannot effectively halt disease progression and are accompanied by serious side effects such as hallucinations, dizziness, and liver toxicity, failing to meet clinical needs. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention, based on the clinical manifestations and pathological characteristics of Alzheimer's disease and targeting the multi-target nature of Alzheimer's disease pathogenesis, screened multiple effective medicinal materials from natural drugs, traditional Chinese medicine, and herbal medicines, and verified their active effects through experimental research, creatively forming a natural drug compound for treating Alzheimer's disease and its application. This drug compound is named "Tianqi Granules".

[0006] To achieve the above objectives, the technical solution adopted in this application is as follows: A natural herbal medicine compound for treating Alzheimer's disease, composed of the following ingredients in parts by weight: Gastrodia elata 3-6 parts, Polygala tenuifolia 3-6 parts, Curcuma longa 3-6 parts, Uncaria rhynchophylla 3-6 parts, Rehmannia glutinosa 9-15 parts, Achyranthes bidentata 9-15 parts, Lycium barbarum 6-10 parts, Ziziphus jujuba var. spinosa 6-9 parts, Ligusticum chuanxiong 2-5 parts, Paeonia lactiflora 3-6 parts, Zaocys dhumnades 9-15 parts, Carthamus tinctorius 1-5 parts, Atractylodes macrocephala 6-10 parts, Poria cocos 6-9 parts, Ophiopogon japonicus 9-15 parts, Angelica sinensis 3-9 parts, Panax ginseng 9-15 parts, Salvia miltiorrhiza 2-5 parts, Glycyrrhiza uralensis 2-5 parts.

[0007] Preferably, it includes an effective dose of the pharmaceutically acceptable excipient.

[0008] Preferably, the raw material is a prepared slice of Chinese medicinal material, an extract of Chinese medicinal material, or an effective part of Chinese medicinal material.

[0009] Preferably, the drug combination can be formulated as any one of oral liquid, granules, powder, tablets, capsules or pills.

[0010] Preferably, the dosage form of the compound is granules.

[0011] The use of any of the above-mentioned natural drug mixtures in the preparation of medicines for the prevention and / or treatment of Alzheimer's disease.

[0012] In Traditional Chinese Medicine (TCM), Alzheimer's disease falls under the category of "forgetfulness, forgetfulness, and dementia." TCM believes that "the brain is the sea of ​​marrow," and only when the brain marrow is clear can a person think normally and have good memory. The spleen governs the transformation and transportation of food and water, and governs Yang Qi; therefore, the energy for clearing the marrow comes from the spleen. If the spleen is weak and unable to generate sufficient nutrients, Yin essence and Yin blood will be depleted, ultimately leading to weakened marrow, brain despondency, and loss of mental function, eventually developing into dementia. This natural herbal compound invigorates the brain, tonifies the spleen and kidneys, replenishes Qi and blood, and promotes blood circulation. It features multiple components, multiple targets, and multiple pathways, demonstrating the advantage of "treating both the symptoms and the root cause." Gastrodin in Gastrodia elata helps alleviate microglia activation and improve energy metabolism. Glycyrrhizin in Polygala tenuifolia can reduce Aβ levels and reverse the decline in learning and memory during the development of Alzheimer's disease; Polygala tenuifolia saponins can activate microglia, improve antioxidant status, and play a neuroprotective role; glycyrrhizic acid has anti-inflammatory and immunomodulatory effects, can improve cognitive ability, regulate the cholinergic system, and reduce oxidative stress damage, playing a neuroprotective role against cognitive impairment. Curcumin in turmeric can reduce oxidative stress and inflammatory responses to protect neurons, thereby improving cognitive function. Uncaria rhynchophylla's main active ingredient, rhynchophylline, can effectively inhibit the excessive production of β-amyloid precursors and amyloid protein, protecting nerves and improving learning and memory. It can also improve cognitive ability by regulating glycolysis metabolism to counteract immune responses and neuroinflammation; isorhynchophylline can reduce and inhibit hippocampal neuronal apoptosis, providing some protection for learning and memory. Uncaria rhynchophylla, when combined with Gastrodia elata, can reduce liver damage and enhance the liver-calming and yang-suppressing effects of Gastrodia elata and Uncaria rhynchophylla. Rehmannia glutinosa, a representative Chinese herb for "replenishing essence and marrow," contains multiple active ingredients such as catalpol, verbascoside, and echinacoside, possessing multiple pharmacological effects including neuroprotection, antioxidation, anti-inflammation, and immunomodulation. Catalpol can reduce oxidative stress damage to cells, verbascoside can inhibit the activation of microglia and astrocytes, and echinacoside can reduce the production and accumulation of Aβ in the brain, improving memory impairment. Achyranthes bidentata contains achyranthes polypeptides, which have neuroprotective effects. The polysaccharides in wolfberry have a neuroprotective effect. Fried jujube seeds can regulate metabolism and improve learning and memory impairment. Ligustrazine in chuanxiong can inhibit microglia activation, reduce neuroinflammation, decrease neuronal apoptosis, and improve behavioral deficits. White peony, with its bidirectional regulatory properties of "nourishing blood and regulating menstruation, calming the liver and harmonizing the collaterals," bridges the gap between classic blood-tonifying effects and modern multi-target integrated pharmacology. Its core components play a role in neuroprotection through an anti-inflammatory, antioxidant, immunomodulatory, and metabolic intervention network. Paeoniflorin, a potential pharmacodynamic component of white peony, can improve cognitive function by inhibiting inflammatory responses, reducing oxidative stress damage, decreasing neuronal apoptosis, and repairing learning and memory functions. Black-striped snake has anti-inflammatory and immunomodulatory effects. Safflower contains safflower yellow pigment, hydroxysafflower yellow pigment A, kaempferol, quercetin, and polysaccharides, which can regulate lipid metabolism, inhibit inflammatory responses, resist oxidative stress, and inhibit apoptosis.The lactone chemical components in Atractylodes macrocephala exert neuroprotective effects through multiple pathways, including inhibiting Aβ production and deposition, improving mitochondrial function, and reducing neuroinflammatory responses. Poria cocos contains poria acid, which effectively clears β-amyloid protein accumulation and reduces neuroinflammatory effects; poria polysaccharides have antioxidant and immunomodulatory effects. Ophiopogon japonicus contains polysaccharides with antioxidant and anti-inflammatory activity. Angelica sinensis contains angelicin, which accelerates Aβ efflux, effectively preventing Aβ aggregation-induced brain tissue toxicity and improving Tau protein hyperphosphorylation; angelica sinensis polysaccharides can inhibit the release of pro-inflammatory factors to protect neurons from damage and improve learning and memory deficits. Ginsenosides in ginseng have anti-inflammatory effects. Salvia miltiorrhiza has anti-inflammatory, anti-apoptotic, and immune microenvironment-regulating effects. Glycyrrhizin in licorice has antioxidant effects. Beneficial effects

[0013] This invention demonstrates a significant therapeutic effect on 3xTg-AD model mice. In behavioral experiments, mice treated with Tianqi granules showed significantly reduced immobility time and enhanced exploratory behavior in the open field test; improved recognition index in the new object recognition test; increased female mouse alternation rate and new arm dwell time in the Y-maze test; and shortened escape latency and significantly increased platform crossing frequency in the Morris water maze test. These findings indicate that the drug can alleviate anxiety and improve learning and memory abilities, spatial cognition, and spatial learning and memory deficits. Morphological examination revealed a significant decrease in β-amyloid protein deposition in the prefrontal cortex of the treated mice (p<0.05), and a significant decrease in the fluorescence intensity of GFAP, IBA1, IL-1β, and LC3 in the hippocampus. This suggests that Tianqi granules can reduce abnormal glial cell activation, alleviate neuroinflammation, and regulate autophagy. Transcriptomic analysis further revealed that Tianqi granules exert their effects through multi-target synergy, sequentially initiating three stages in male mice: defense and protection, repair and remodeling, and homeostasis consolidation; and sequentially initiating three stages in female mice: protection and defense, immune remodeling, and homeostasis maintenance. It systematically regulates immune inflammation, protein metabolism, nuclear homeostasis, lipid metabolism, and energy metabolism, thereby delaying the pathological progression of Alzheimer's disease (AD). Attached Figure Description

[0014] Figure 1The images show the open field results of 3xTg-AD mice after administration of the natural drug compound of this invention. A represents the statistical results (n=30) of male mice in the open field (OFT) 21 days after administration, including 10-minute resting time, number of times entering the central region, total movement distance, movement time in the central region, and movement distance in the central region. B represents a representative map of the 10-minute activity trajectory of male mice in the OFT 21 days after administration. C represents the statistical results (n=30) of female mice in the open field (OFT) 21 days after administration. D represents a representative map of the 10-minute activity trajectory of female mice in the OFT 21 days after administration. Figure 2 The image shows the results of the new object recognition experiment in 3xTg-AD mice after intervention with the natural drug compound of the present invention; where A is the recognition index and representative activity trajectory image of male mice in the new object recognition test, and B is the recognition index and representative activity trajectory image of female mice in the new object recognition test. Figure 3 The image shows the Y-maze test results of 3xTg-AD mice after intervention with the natural drug compound of the present invention; where A represents the percentage of spontaneous alternation, the time spent in the new arm, and the representative movement trajectory of male mice in the Y-maze test, and B represents the percentage of spontaneous alternation, the time spent in the new arm, and the representative movement trajectory of female mice in the Y-maze test. Figure 4 The image shows the results of the Morris water maze test in male 3xTg-AD mice after intervention with the natural drug compound of this invention; where A is the escape latency of male mice in the 4-day MWM training test, B is the statistical results of escape latency, number of platform crossings, distance traveled in the target quadrant, time spent in the target quadrant, and distance traveled in the exploration test, and C is a representative activity trajectory image of male mice in the exploration test; Figure 5 The image shows the results of the Morris water maze test in female 3xTg-AD mice after intervention with the natural drug compound of this invention; where A is the escape latency of female mice in the 4-day MWM training test, B is the statistical results of escape latency, number of platform crossings, distance traveled in the target quadrant, time spent in the target quadrant, and distance traveled in the exploration test, and C is a representative activity trajectory image of female mice in the exploration test; Figure 6The figures show the results of the β-amyloid deposition experiment in 3xTg-AD mice after intervention with the natural drug compound of the present invention. In the figures, A is a representative image of thio-S staining showing Aβ plaques (green) in brain slices of male 3xTg-Veh and 3xTg-TQ mice, and a magnified view of the cortical region; B is a representative image of thio-S staining showing Aβ plaques (green) in brain slices of female 3xTg-Veh and 3xTg-TQ mice, and a magnified view of the cortical region; C is a quantitative analysis of the area of ​​Thio-S positive Aβ in the cortex of male 3xTg-Veh and 3xTg-TQ mice; and D is a quantitative analysis of the area of ​​Thio-S positive Aβ in the cortex of female 3xTg-Veh and 3xTg-TQ mice. Figure 7 The image shows the results of the glial cell activation experiment in male 3xTg-AD mice after intervention with the natural drug compound of this invention; where A is a representative immunofluorescence image of a hippocampal slice, with GFAP (green) labeling astrocytes and Iba1 (red) labeling microglia; B is the quantitative analysis of the immunofluorescence intensity of GFAP and Iba1 cells in the dentate gyrus (DG), CA1 and CA3 subregions of the hippocampus. Figure 8 The image shows the results of the glial cell activation experiment in female 3xTg-AD mice after intervention with the natural drug compound of this invention; where A is a representative immunofluorescence image of a hippocampal slice, with GFAP (green) labeling astrocytes and Iba1 (red) labeling microglia; B is the quantitative analysis of the immunofluorescence intensity of GFAP and Iba1 cells in the dentate gyrus (DG), CA1 and CA3 subregions of the hippocampus. Figure 9 The figures show the results of the neurological tissue inflammation experiment in 3xTg-AD mice after intervention with the natural drug compound of this invention; where A is a representative immunofluorescence image of the CA3 region of the hippocampus in male mice, B is a representative immunofluorescence image of the CA3 region of the hippocampus in female mice, IL-1β (red) labels the neurological tissue inflammation, NeuN (green) labels the neurons, C is the quantitative analysis of the immunofluorescence intensity of IL-1β in male mice, and D is the quantitative analysis of the immunofluorescence intensity of IL-1β in female mice; Figure 10 The figures show the autophagy results of 3xTg-AD mice after intervention with the natural drug compound of this invention; where A is a representative immunofluorescence image of the CA3 region of the hippocampus of male mice, B is a representative immunofluorescence image of the CA3 region of the hippocampus of female mice, LC3 (red) is used to label autophagy, NeuN (green) is used to label neurons, C is a quantitative analysis of the immunofluorescence intensity of LC3 in male mice, and D is a quantitative analysis of the immunofluorescence intensity of LC3 in female mice. Figure 11This image shows the PCA and GO enrichment results of sequencing in male 3xTg-AD mice after intervention with the natural drug compound of this invention. A represents principal component analysis (PCA) at different time points after intervention in the male 3xTg-TQ group; B represents a volcano plot of differentially expressed genes between male 3xTg-TQ mice and their respective 3xTg-Veh control groups (red indicates upregulated genes, blue indicates downregulated genes); C represents the biological processes (BP) of the top 10 enriched GO analyses in male 3xTg-TQ mice compared to the 3xTg-Veh control group; D represents the cellular components (CC) of the top 10 enriched GO analyses in male 3xTg-TQ mice compared to the 3xTg-Veh control group; and E represents the molecular functions (MF) of the top 10 enriched GO analyses in male 3xTg-TQ mice compared to the 3xTg-Veh control group. Figure 12 This image shows the PCA and GO enrichment results of sequencing in female 3xTg-AD mice after intervention with the natural drug compound of this invention. A represents principal component analysis (PCA) at different time points after intervention in the female 3xTg-TQ group; B represents a volcano plot of differentially expressed genes between female 3xTg-TQ mice and their respective 3xTg-Veh control groups, with red indicating upregulated genes and blue indicating downregulated genes; C represents the biological processes (BP) of the top 10 enriched GO analyses in female 3xTg-TQ mice compared to the 3xTg-Veh control group; D represents the cellular components (CC) of the top 10 enriched GO analyses in female 3xTg-TQ mice compared to the 3xTg-Veh control group; and E represents the molecular functions (MF) of the top 10 enriched GO analyses in female 3xTg-TQ mice compared to the 3xTg-Veh control group. Figure 13 This is a diagram showing the results of sequencing the differentially expressed gene network related to protein metabolism in male 3xTg-AD mice after intervention with the natural drug compound of this invention; where A is the expression and regulation network of ribosome-related genes, B is the expression and regulation network of endoplasmic reticulum-related genes, C is the expression and regulation network of Golgi-related genes, D is the expression and regulation network of lysosome-related genes, and E is the expression and regulation network of endosome-related genes. Red indicates upregulated genes and blue indicates downregulated genes. Figure 14 This is a graph showing the changes in protein metabolism-related transcriptional modules in male 3xTg-AD mice after intervention with the natural drug compound of this invention; where A is the time trend of the significance (-log10 p value) of protein metabolism-related GO items enriched in male 3xTg-TQ mice, and B is the time dynamic analysis of GSEA related to protein metabolism pathways in male 3xTg-TQ mice. The numbers next to the bars represent the normalized enrichment scores (NES). Figure 15This is a diagram showing the results of sequencing the differentially expressed gene network related to protein metabolism in female 3xTg-AD mice after intervention with the natural drug compound of this invention; where A is the expression and regulation network of ribosome-related genes, B is the expression and regulation network of endoplasmic reticulum-related genes, C is the expression and regulation network of Golgi-related genes, D is the expression and regulation network of lysosome-related genes, and E is the expression and regulation network of endosome-related genes. Red indicates upregulated genes and blue indicates downregulated genes. Figure 16 This is a graph showing the changes in protein metabolism-related transcriptional modules in female 3xTg-AD mice after intervention with the natural drug compound of this invention; where A is the time trend of the significance (-log10 p value) of enriched protein metabolism-related GO items in female 3xTg-TQ mice, and B is the time dynamic analysis of GSEA related to protein metabolism pathways in female 3xTg-TQ mice. The numbers next to the bars represent the normalized enrichment scores (NES). Detailed Implementation

[0015] The present invention will be further described below with reference to embodiments. These embodiments are only used to more clearly illustrate the technical solutions of the present invention and should not be construed as limiting the scope of protection of the present invention.

[0016] "Stir-fried jujube seed" and "wine-processed black snake" are processed products, and all components used in this invention were purchased from Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0017] Example 1

[0018] A natural herbal medicine compound for treating Alzheimer's disease is composed of the following ingredients in parts by weight: Gastrodia elata 3 parts, Polygala tenuifolia 3 parts, Curcuma longa 3 parts, Uncaria rhynchophylla 3 parts, Rehmannia glutinosa 9 parts, Achyranthes bidentata 9 parts, Lycium barbarum 6 parts, Ziziphus jujuba var. spinosa 6 parts, Ligusticum chuanxiong 2 parts, Paeonia lactiflora 3 parts, Zaocys dhumnades 9 parts, Carthamus tinctorius 2 parts, Atractylodes macrocephala 9 parts, Poria cocos 6 parts, Ophiopogon japonicus 9 parts, Angelica sinensis 3 parts, Panax ginseng 9 parts, Salvia miltiorrhiza 2 parts, Glycyrrhiza uralensis 2 parts.

[0019] The preparation method of the above-mentioned natural drug compound involves adding the weighed component granules to double-distilled water, heating to dissolve and mix, cooling to room temperature to obtain a solution with a final concentration of 0.728 g / ml, then dispensing, sealing, and storing at -20°C. Before each administration, it should be thawed and warmed in a 37°C water bath.

[0020] Example 2: The therapeutic effect of the natural drug mixture prepared in Example 1 on 3xTg-AD mice.

[0021] Materials: Natural herbal compound prepared in Example 1 (14.56 g / kg / d); wild-type blank mice (WT-Veh); 3xTg-AD model mouse blank group (3xTg-Veh); 3xTg-AD model mouse herbal medicine group (3xTg-TQ). 3xTg-AD (B6;129-Tg(APPSwe,tauP301L)1Lfa Psen1) tm1Mpm / Mmjax mice (RRID: MMRRC_034830-JAX) were purchased from Jackson Lab in the United States. The wild-type blank mice were bred from Jackson Lab's C57BL / 6J female mice (catalog number: 000664) and 129X1 / SvJ male mice (catalog number: 000691). The breeding process can be followed according to the procedures published on the Jax Lab website.

[0022] Methods: Thirty female and thirty male mice were randomly divided into three groups: wild-type control mice (WT-Veh), a 3xTg-AD model mouse control group (3xTg-Veh), and a 3xTg-AD model mouse herbal medicine group (3xTg-TQ). Animals were randomly assigned to each group using a randomized block design. The treatment groups received herbal medicine by gavage at the same time each day for 31 days, while the wild-type control group and the 3xTg-AD group received the same volume of water by gavage daily. On day 21 of treatment, a 10-day behavioral test was initiated. Mice were deeply anesthetized with a mouse anesthetic and then euthanized by decapitation in an unconscious physiological state. Brain samples were then harvested for Thio-S staining (TS staining) and immunofluorescence staining. Statistical analysis was performed using Graph-Pad Prism 8.0 software; quantitative data are expressed as mean ± standard deviation (Mean ± SD). One-way ANOVA was used to compare the means among multiple independent groups, with 3xTg-Veh as the control group. A p-value < 0.05 was considered statistically significant.

[0023] Results: The open field experiment results show that ( Figure 1 Regardless of male mice ( Figure 1 A, Figure 1 B) or female mouse ( Figure 1 C Figure 1 (D) Compared to the WT-Veh group, the 3xTg-Veh group showed a significant increase in immobility time, a significant decrease in the number of times mice entered and exited the central region, and a significant decrease in the distance traversed within the central region. After treatment with the natural herbal compound, the 3xTg-TQ group showed a significant decrease in immobility time, a decrease in the number of times mice entered and exited the central region, and a decrease in the distance traversed within the central region compared to the 3xTg-Veh group. These data suggest that the natural herbal compound can alleviate anxiety and enhance exploratory behavior in mice.

[0024] The results of the new object recognition experiment show that ( Figure 2 Regardless of male mice ( Figure 2 A) or female mouse ( Figure 2 B) The recognition index of the 3xTg-Veh group mice was significantly lower than that of the WT-Veh group mice, while the recognition index of the 3xTg-TQ group was significantly higher than that of the 3xTg-Veh group mice, indicating that the natural drug compound has an improving effect on the learning and memory ability of AD mice.

[0025] The results of the Y-maze experiment show that ( Figure 3 ), male mice ( Figure 3 A) The alternation rate and time to new arm were not statistically significant among the WT-Veh, 3xTg-Veh, and 3xTg-TQ groups, but the 3xTg-TQ group showed an increasing trend. Female mice ( Figure 3 Analysis of the Y-maze data in B) showed that the 3xTg-NC group mice had a lower alternation rate and shorter neoarm time than the WT-Veh and 3xTg-TQ groups. This indicates that the natural drug combination has an improving effect on the spatial cognitive ability of AD mice.

[0026] Morris water maze experiment showed that regardless of male mice ( Figure 4 ) or female mouse ( Figure 5 On day 4 of the training period, significant differences were observed in the learning curves of mice in the WT-Veh and 3xTg-Veh groups. During the test period, compared to WT-Veh, mice in the 3xTg-Veh group had a longer escape latency, significantly fewer platform crossings, and significantly less movement distance within the platform quadrant, indicating that 3xTg-Veh resulted in spatial learning and memory impairment. Significant differences were also observed in the learning curves of the 3xTg-Veh and 3xTg-TQ groups on day 4 of the training period. During the test period, compared to 3xTg-Veh, mice in the 3xTg-TQ group had a shorter escape latency, significantly more platform crossings, and significantly increased movement distance within the platform quadrant. The time spent in the platform quadrant and the total distance moved were also increased, indicating that the natural drug combination significantly improved spatial learning and memory deficits in AD mice.

[0027] Morphological examination and TS staining results showed that ( Figure 6 Compared with the model group, the treatment with the natural herbal medicine compound significantly reduced the deposition of β-amyloid protein in the prefrontal cortex of 3xTg-AD mice (p<0.05).

[0028] Immunofluorescence staining results showed that GFAP was used to label astrocyte markers and IBA1 was used to label microglia markers. Regardless of male mice ( Figure 7 ) or female mouse ( Figure 8Compared to WT-Veh, the fluorescence signal intensity of GFAP and IBA1 in the hippocampal DG, CA1, and CA3 regions of mice in the 3xTg-Veh group was significantly enhanced. Intervention with Tianqi granules reduced the fluorescence signal intensity of GFAP in the DG, CA1, and CA3 regions and significantly reduced the fluorescence signal intensity of IBA1 in the CA1 and CA3 regions. This indicates that treatment with the natural herbal compound can reduce the abnormal activation of astrocytes and microglia.

[0029] Immunofluorescence staining results showed that regardless of male mice ( Figure 9 A, Figure 9 C) or female mouse ( Figure 9 B Figure 9 (D) The expression of IL-1β in CA3 of 3xTg-Veh mice was significantly increased compared to WT-Veh mice, while the fluorescence intensity of IL-1β in CA3 region of 3xTg-TQ mice was decreased compared to 3xTg-Veh mice. This indicates that the natural drug compound can alleviate neuroinflammation.

[0030] The role of autophagy in AD is complex. LC3-associated endocytosis and phagocytosis play crucial roles in clearing neurotoxic protein aggregates, but excessive autophagy can stress cells, leading to excessive degradation of organelles and impairing normal cellular function. In 3xTg-Veh male and female mice ( Figure 10 The LC3 fluorescence intensity in both neuronal and non-neuronal regions of the CA3 area was significantly higher than that in the WT-Veh group, while the LC3 fluorescence intensity in the 3xTg-TQ group was significantly lower than that in the 3xTg-Veh group. These data suggest that traditional Chinese medicine intervention may help restore normal autophagy function and thus alleviate the pathological phenotype of AD.

[0031] Example 3: Mechanism of therapeutic effect of the natural drug mixture prepared in Example 1 on 3xTg-AD mice. Materials: Natural herbal compound prepared in Example 1 (14.56 g / kg / d); wild-type blank mice (WT-Veh); 3xTg-AD model mouse blank group (3xTg-Veh); 3xTg-AD model mouse herbal medicine group (3xTg-TQ). 3xTg-AD (B6;129-Tg(APPSwe,tauP301L)1Lfa Psen1) tm1Mpm The Mmjax mice (RRID: MMRRC_034830-JAX) were purchased from Jacksonlab in the United States. The wild-type blank mice were bred from Jacksonlab's C57BL / 6J female mice (catalog number: 000664) and 129X1 / SvJ male mice (catalog number: 000691). The breeding process can be followed according to the procedures published on the Jax Lab website.

[0032] Methods: Thirty female and thirty male mice were randomly divided into three groups: wild-type control mice (WT-Veh), a 3xTg-AD model mouse control group (3xTg-Veh), and a 3xTg-AD model mouse herbal medicine group (3xTg-TQ). Animals were randomly assigned to each group using a randomized block design. The treatment groups received herbal medicine by gavage at the same time each day for 31 days, while the wild-type control group and the 3xTg-AD group received the same volume of water by gavage daily. At five key time points—0 days before treatment, 5 days after treatment, 10 days after treatment, 21 days after treatment, and 31 days after treatment—mice were sacrificed, and hippocampal tissue was collected for RNA sequencing analysis. Sequencing and analysis were performed by Shanghai Weihuan Biotechnology Co., Ltd.

[0033] Principal component analysis (PCA) results showed that, regardless of male mice ( Figure 11 A) or female mouse ( Figure 12 A) PCA results of mice in the 3xTg-TQ group treated with the natural drug compound at 5d, 10d, 21d, and 31d showed small differences between groups, but were significantly different from the control group (0d without treatment). Female mice showed peak upregulation and downregulation of differentially regulated genes 10d after treatment, while male 3xTg-TQ mice had the highest number of upregulation and downregulation differentially regulated genes at 31d, and the number of upregulation differentially regulated genes increased with the extension of intervention time.

[0034] GO enrichment results showed that in the BP of male mice in the 3xTg-TQ group treated with the natural drug combination, significant enrichment of "hematopoietic regulation" was observed at 5 days, followed by enrichment of "cellular response to topologically incorrect proteins," "Wnt signaling pathway," "interleukin-1β production," and cell cycle-related pathways after 10 days. At 21 days of drug treatment, significant enrichment of the "extrinsic apoptosis signaling pathway" was observed, and at 31 days, enrichment was associated with pathways related to energy metabolism, including "negative regulation of immune system processes," "glycoprotein biosynthesis," and "energy acquisition through the oxidation of organic compounds." Figure 11 C). In the enrichment of MF, the main enrichment is related to GTPase and ubiquitination (C). Figure 11 D). In CC enrichment, membrane rafts were found to be significantly enriched at 5, 10, 21, and 31 days after drug treatment. Figure 11 E).

[0035] Enrichment of blood protein (BP) in female mice in the 3xTg-TQ group treated with a natural drug combination was observed at 5 days, showing an enrichment in response to unfolded or misfolded proteins. At 10 days of treatment, differentially expressed gene enrichment was associated with "endometrial system organization," "nucleotide phosphate metabolism," "lymphocyte differentiation," and "hematopoietic regulation." At 21 days of treatment, enrichment was observed in "negative regulation of immune system processes," "protein stability," and cell cycle-related factors. At 31 days of treatment, enrichment was primarily associated with the "Wnt signaling pathway." Figure 12 C). In MF enrichment, GTPase-related enrichment was observed at 5d, 10d, and 21d, while protein kinase-related regulatory changes were observed at 10d, 21d, and 31d. Figure 12 D). In the enrichment of CC, collagen-containing extracellular matrix and related enrichment of membrane-containing components were found at 5d, 10d, and 31d. Figure 12 E).

[0036] Protein metabolism network results showed that male mice in the 3xTg-TQ group treated with the natural drug combination ( Figure 13 , Figure 14 The first stage of treatment involves assisting the body in initiating its defense mechanisms. The drug utilizes the bidirectional regulatory function of the immune system to remodel and stabilize the immune system, promoting NK cell proliferation while downregulating T cell immune regulation, assisting the body in immune surveillance and upregulating immune clearance. In the second stage (5-21 days), the drug assists the body in transition and repair, preventing the gradual collapse of multi-system homeostasis caused by decompensation. During this stage, progressive suppression of innate immunity occurs after drug treatment, and the drug's regulation of IL-1β plays a positive role in promoting anti-inflammatory phenotypic transformation and alleviating AD pathology. In the third stage (21-31 days), the body's homeostasis is restored after drug treatment. The drug's regulatory effect on the acute inflammatory response is further enhanced, accompanied by a significant enrichment of negative regulation of immune system processes. The drug promotes the return of the immune system from a pathological chronic activated state to homeostasis, effectively controlling inflammation by enhancing endogenous negative feedback mechanisms.

[0037] Female mice in the 3xTg-TQ group treated with natural drug compound ( Figure 15 , Figure 16The first phase (0-5 days) of the treatment involves a protective defense, during which the drug works synergistically with the body to perform dual immune regulation, suppressing the inflammatory response and initiating a controlled immune response, driving immune cells to migrate to specific regions. The second phase (5-21 days) follows drug treatment, during which immune remodeling occurs, transforming the pro-inflammatory immune environment into an anti-inflammatory and pro-repair mode. This is achieved by downregulating MHC class I antigens and inducing T cell immune tolerance to prevent excessive immune responses that could damage neurons, and by restraining Th1 responses to reduce IFN-γ production, thereby suppressing the pro-inflammatory environment. The third phase (21-31 days) after drug treatment promotes the body's transition to homeostasis. The establishment of immune homeostasis is manifested in the sustained suppression of the inflammatory response and the potential role of NK cells as an immune surveillance mechanism.

[0038] Example 4 The formula used in the acute toxicity test was as follows: Rehmannia glutinosa (10 parts), Achyranthes bidentata (10 parts), Lycium barbarum (10 parts), Gastrodia elata (3 parts), Polygala tenuifolia (3 parts), Curcuma longa (3 parts), Ligusticum chuanxiong (2 parts), Paeonia lactiflora (3 parts), Ziziphus jujuba var. spinosa (6 parts), Uncaria rhynchophylla (3 parts), Zaocys dhumnades (15 parts), Carthamus tinctorius (1 part), Atractylodes macrocephala (10 parts), Poria cocos (6 parts), Ophiopogon japonicus (10 parts), Angelica sinensis (3 parts), Glycyrrhiza uralensis (2 parts), Panax ginseng (10 parts), and Salvia miltiorrhiza (2 parts).

[0039] The test sample was prepared in whole-number doses (112 doses / dose). Each formulation granule in each dose was weighed according to the formulation ratio. The required number of formulation granules was weighed, with a deviation range of ±0.1 g. The test sample was placed in a beaker, and Watson's drinking water was added and stirred. After transferring to a graduated cylinder, Watson's drinking water was added to the final volume, and then transferred back to a beaker. The mixture was continuously stirred with a magnetic stirrer until a homogeneous formulation was visually observed. The concentrations for the low, medium, and high dose groups were 125 mg / ml, 250 mg / ml, and 500 mg / ml, respectively.

[0040] The acute toxicity test of this invention was conducted entirely by Suzhou Xishan Zhongke Pharmaceutical Research and Development Co., Ltd. Forty SD rats (half male and half female) were selected and randomly divided into four groups (n=10 each) using the Provantis 10.5.0.4 system. The four groups served as a negative control group (Watson's drinking water), and low, medium, and high dose groups of Tianqi granules (7.5, 15, and 30 g / kg / day). Animals were administered the drug orally via gavage three times a day, with each dose being 20 mL / kg, administered 4 h ± 1 h apart. Observations were conducted for 14 consecutive days after administration, with dissection performed on day 15 (D15). The day of administration was defined as day 1 (D1), the day before administration as (D-1), and the first week of the experiment as W1. Observations were taken once before the first administration on the first day of administration, and every hour for 3 hours after each administration, with data recorded. Thereafter, observations were taken twice daily, once in the morning and once in the afternoon, for 14 consecutive days. All animals were weighed once before administration (D-1), and again on D1, D2, D7, and D14; food intake was measured weekly. After 14 days of observation, a gross necropsy was performed on D15, and the lesions were recorded.

[0041] Under the conditions of this experiment, SD rats were administered Tianqi granules via single oral gavage at doses of 7.5, 15, and 30 g / kg / day (approximately 4, 8, and 16 times the clinical dose, respectively), and observed for 14 consecutive days. No animals died or were near death. At doses of 15 and 30 g / kg / day, female animals showed faster weight gain and increased food intake; no other significant abnormalities were observed. Gross necropsy also revealed no significant abnormalities in any organs. The maximum tolerated dose (MTD) was 30 g / kg / day. Observing the toxic reactions or mortality of SD rats after a single oral gavage administration of Tianqi granules provides reference information for evaluating the safety of clinical use.

[0042] In summary, the natural drug compound of this invention delays the pathological process of 3xTg-AD mice through multiple levels and targets. It systematically improves the pathogenesis of the hippocampus by alleviating neuroinflammation, improving protein quality control, regulating nucleotides to restore the cell cycle, stabilizing lipid and glucose metabolism, and enhancing antioxidant capacity, from defense and protection through repair and remodeling to homeostasis.

[0043] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A natural drug mixture for treating Alzheimer's disease, characterized in that, The ingredients are composed of the following parts by weight: Gastrodia elata 3-6 parts, Polygala tenuifolia 3-6 parts, Curcuma longa 3-6 parts, Uncaria rhynchophylla 3-6 parts, Rehmannia glutinosa 9-15 parts, Achyranthes bidentata 9-15 parts, Lycium barbarum 6-10 parts, Ziziphus jujuba var. spinosa 6-9 parts, Ligusticum chuanxiong 2-5 parts, Paeonia lactiflora 3-6 parts, Zaocys dhumnades 9-15 parts, Carthamus tinctorius 1-5 parts, Atractylodes macrocephala 6-10 parts, Poria cocos 6-9 parts, Ophiopogon japonicus 9-15 parts, Angelica sinensis 3-9 parts, Panax ginseng 9-15 parts, Salvia miltiorrhiza 2-5 parts, Glycyrrhiza uralensis 2-5 parts.

2. The natural drug mixture for treating Alzheimer's disease according to claim 1, characterized in that, This includes pharmaceutically acceptable excipients in effective doses.

3. A natural drug mixture for treating Alzheimer's disease according to claim 1, characterized in that, The raw materials are prepared slices of Chinese medicinal herbs, extracts of Chinese medicinal herbs, or effective parts of Chinese medicinal herbs.

4. A natural drug mixture for treating Alzheimer's disease according to claim 1, characterized in that, The drug combination can be formulated as any one of the following: oral liquid, granules, powder, tablets, capsules, or pills.

5. A natural drug mixture for treating Alzheimer's disease according to claim 1, characterized in that, The preparation is in the form of granules.

6. The use of the natural drug compound according to any one of claims 1-5 in the preparation of a medicament for the prevention and / or treatment of Alzheimer's disease.

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