Traditional Chinese medicine composition for treating Alzheimer disease as well as preparation method and application of traditional Chinese medicine composition

Abstract

The invention relates to the technical field of preparation and application of traditional Chinese medicine compositions, and particularly discloses a traditional Chinese medicine composition for treating Alzheimer's disease and a preparation method and application thereof. The traditional Chinese medicine composition is prepared from the following traditional Chinese medicine raw materials in parts by weight: 9-30 parts of astragalus membranaceus, 9-30 parts of codonopsis pilosula, 6-10 parts of herba epimedii, 9-15 parts of rhizoma polygonati, 3-10 parts of rhizoma acori graminei, 3-10 parts of polygala tenuifolia, 6-12 parts of dendrobe, 3-6 parts of rhodiola rosea, 3-10 parts of radix curcumae, 9-15 parts of cortex lycii radicis, 3-9 parts of Hubei fritillary bulb, 3-10 parts of radix angelicae, 3-10 parts of pericarpium citri reticulatae and 3-10 parts of fresh ginger. The traditional Chinese medicine composition provided by the invention accords with the traditional Chinese medicine treatment concept of tonifying deficiency and filling marrow, inducing resuscitation and soothing nerves, dredging collaterals and eliminating evil, and tonifying spleen and assisting transportation, not only can relieve damage of neurons, but also can reverse alzheimer disease related disorder signal channels in neuronal cells, microglial cells and astrocytes.

Description

Technical Field

[0001] This application relates to the field of traditional Chinese medicine composition preparation and application technology, and more specifically, it relates to a traditional Chinese medicine composition for treating Alzheimer's disease, its preparation method and uses. Background Technology

[0002] Alzheimer's disease (AD) is a degenerative disease of the central nervous system and the most common type of dementia in the elderly. Its main characteristics include progressive cognitive impairment and behavioral disturbances. Mild dementia patients experience memory loss, disorientation, and personality changes; moderate dementia patients lose language abilities and are unable to live independently; while severe dementia patients are completely dependent on others for care, lose basic physiological functions, and severely impact their quality of life.

[0003] The pathological mechanisms of Alzheimer's disease are complex, primarily involving abnormal deposition of β-amyloid protein, hyperphosphorylation of tau protein leading to neurofibrillary tangles, and also neuroinflammation, oxidative stress, and synaptic dysfunction. These pathological changes directly or indirectly cause neuronal damage. Specifically, Aβ deposition can trigger excessive activation of microglia, releasing inflammatory factors, inducing neuroinflammation, and damaging neurons; neurofibrillary tangles affect the normal structure and function of neurons, leading to neuronal degeneration and death; and synaptic dysfunction disrupts signal transmission between neurons, thereby affecting cognitive function. In Alzheimer's disease, neuronal damage is a key factor leading to core symptoms such as cognitive decline. Once neurons are damaged or even die, it is often irreversible and a significant reason for the continued progression of Alzheimer's disease. Therefore, alleviating neuronal damage is a core focus of Alzheimer's disease treatment.

[0004] Western medicine commonly uses cholinesterase inhibitors and NMDA receptor antagonists to relieve symptoms, but these only provide temporary relief and cannot stop disease progression. Due to the complexity of the pathological mechanisms, single-target drugs have limited efficacy, and long-term use may lead to drug resistance and side effects, such as gastrointestinal reactions and liver damage. Existing traditional Chinese medicines mostly focus on regulating single targets or single cell types, resulting in relatively limited efficacy.

[0005] Therefore, this application is hereby submitted. Summary of the Invention

[0006] To address the limitations of existing technologies in treating Alzheimer's disease with traditional Chinese medicine, this application provides a traditional Chinese medicine composition for treating Alzheimer's disease, its preparation method, and its uses. This composition focuses on alleviating neuronal damage and reversing disease signaling pathways in Alzheimer's disease-related cells. It integrates Western medicine theories of neuronal cell damage and signaling pathway regulation with traditional Chinese medicine theories such as "the brain is the residence of the spirit," thus possessing significant meaning and application value for the treatment of Alzheimer's disease.

[0007] Specifically, in order to achieve the purpose of this application, the following technical solution is proposed:

[0008] In a first aspect, this application provides a traditional Chinese medicine composition for treating Alzheimer's disease, prepared from the following parts by weight of traditional Chinese medicine raw materials:

[0009] Astragalus membranaceus 9-30 parts, Codonopsis pilosula 9-30 parts, Epimedium brevicornu 6-10 parts, Polygonatum sibiricum 9-15 parts, Acorus tatarinowii 3-10 parts, Polygala tenuifolia 3-10 parts, Dendrobium nobile 6-12 parts, Rhodiola rosea 3-6 parts, Curcuma longa 3-10 parts, Lycium chinense root bark 9-15 parts, Fritillaria cirrhosa 3-9 parts, Angelica dahurica 3-10 parts, Citrus reticulata peel 3-10 parts, Zingiber officinale 3-10 parts.

[0010] In some embodiments, it is prepared from the following parts by weight of traditional Chinese medicine raw materials:

[0011] Astragalus membranaceus 10-25 parts, Codonopsis pilosula 10-25 parts, Epimedium brevicornu 6-10 parts, Polygonatum sibiricum 9-12 parts, Acorus tatarinowii 3-9 parts, Polygala tenuifolia 5-10 parts, Dendrobium nobile 6-12 parts, Rhodiola rosea 3-6 parts, Curcuma longa 3-9 parts, Lycium chinense root bark 10-15 parts, Fritillaria cirrhosa 5-9 parts, Angelica dahurica 3-9 parts, Citrus reticulata peel 3-9 parts, Zingiber officinale 3-9 parts.

[0012] In some embodiments, it is prepared from the following parts by weight of traditional Chinese medicine raw materials:

[0013] Astragalus membranaceus 10-20 parts, Codonopsis pilosula 10-20 parts, Epimedium 7-10 parts, Polygonatum sibiricum 9-10 parts, Acorus tatarinowii 3-6 parts, Polygala tenuifolia 6-10 parts, Dendrobium nobile 8-12 parts, Rhodiola rosea 5-6 parts, Curcuma longa 5-7 parts, Lycium chinense root bark 12-15 parts, Fritillaria cirrhosa 5-7 parts, Angelica dahurica 3-7 parts, Citrus reticulata peel 5-7 parts, Zingiber officinale 3-5 parts.

[0014] Secondly, this application also provides a method for preparing the above-mentioned traditional Chinese medicine composition for treating Alzheimer's disease, comprising the following:

[0015] Weigh each Chinese herbal raw material according to the weight proportions to obtain a Chinese herbal mixture;

[0016] The obtained mixture of traditional Chinese medicines was extracted using solvent extraction to obtain an extract.

[0017] The obtained extract was concentrated under reduced pressure to obtain a paste;

[0018] The obtained extract was spray-dried to obtain a dry powder, which is the traditional Chinese medicine composition for treating Alzheimer's disease.

[0019] In some embodiments, the extraction of the obtained traditional Chinese medicine mixture using solvent extraction specifically includes:

[0020] The resulting mixture of Chinese medicinal materials was subjected to a first extraction using water at 10-15 times the total weight of the raw materials. After extraction at 100℃ for 60-90 minutes, the first extract and residue were obtained by filtration.

[0021] The residue was subjected to a second extraction using 8-12 times the total weight of the Chinese herbal raw materials in water. The extraction was carried out at 100℃ for 60-90 minutes, and then filtered to obtain the second extract.

[0022] The first and second extracts were mixed and combined to obtain the final extract.

[0023] In some embodiments, the concentration of the obtained extract under reduced pressure specifically includes:

[0024] The obtained extract was concentrated under reduced pressure at 70-85℃ to obtain a concentrate with a relative density of 1.1-1.2.

[0025] The obtained concentrate was centrifuged and the centrifuged liquid was filtered to obtain ultrafiltrate;

[0026] The obtained ultrafiltrate was concentrated under reduced pressure at 70-85℃ to obtain an extract with a relative density of 1.1-1.2.

[0027] Thirdly, this application also provides the use of the above-described traditional Chinese medicine composition in the preparation of a medicament for treating or preventing Alzheimer's disease.

[0028] In some embodiments, pharmaceutically acceptable excipients are added to the traditional Chinese medicine composition to prepare an oral preparation, the dosage form of which includes tablets, capsules, granules, pills, or oral liquid preparations.

[0029] In some embodiments, the treatment of Alzheimer's disease includes slowing the progression of Alzheimer's disease, and the traditional Chinese medicine composition improves memory impairment and / or disorientation caused by Alzheimer's disease by alleviating neuronal damage and reversing Alzheimer's signaling pathways, including neuroinflammation and neuronal activity-related signaling pathways.

[0030] In some embodiments, the neuroinflammatory pathways include at least one of the following: proteasome, peroxisome, autophagy, antigen processing and presentation; and / or,

[0031] The signaling pathways related to neuronal activity include at least one of mitochondria, autophagy, endoplasmic reticulum protein processing, GABAergic synapses, endocytosis, dopaminergic synapses, oxidative phosphorylation, and thermogenicity.

[0032] Furthermore, the efficacy and effects of the traditional Chinese medicine components in this application are described as follows:

[0033] Dried tangerine peel is the dried, mature peel of Citrus reticulata Blanco and its cultivated varieties, belonging to the Rutaceae family. It has the effects of regulating qi and strengthening the spleen, drying dampness and resolving phlegm, and is an important medicine for regulating qi stagnation in the spleen and stomach and treating cough with phlegm and dampness.

[0034] Acorus tatarinowii Schott, a plant in the Araceae family, is a dried rhizome that can open the orifices and resolve phlegm, refresh the mind and improve intelligence, resolve dampness and harmonize the stomach. It is particularly good at opening the heart orifices, improving mental abnormalities and dampness obstructing the middle jiao syndrome.

[0035] Lycium bark is the dried root bark of Lycium chinense Mill. or Lycium barbarum L., both belonging to the Solanaceae family. It can cool the blood and eliminate steaming heat, clear the lungs and reduce fire. It is a commonly used medicine for reducing deficiency heat and clearing lung heat, and it can also cool the blood and stop bleeding.

[0036] Yuanzhi is the dried root of Polygala tenuifolia Willd. or Polygalasibirica L., plants of the Polygalaceae family. It has the effects of calming the mind and improving intelligence, harmonizing the heart and kidneys, resolving phlegm and opening the orifices, and dissipating carbuncles and swellings. It is good at treating insomnia, forgetfulness and phlegm obstruction of the orifices.

[0037] Rhodiola crenulata (Hook.f.et Thoms.) H.Ohba, a plant in the Crassulaceae family, is known for its dried roots and rhizomes. It is believed to invigorate qi and blood circulation, clear the meridians and relieve asthma, and is suitable for qi deficiency and blood stasis syndromes as well as qi deficiency cough and asthma. It also has a significant anti-hypoxia effect.

[0038] Astragalus is the dried root of Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao or Astragalus membranaceus (Fisch.) Bge., belonging to the legume family. It can replenish qi and raise yang, strengthen the exterior and stop sweating, promote diuresis and reduce swelling, and generate body fluids and nourish blood. It is an important medicine for replenishing qi, strengthening the spleen, and strengthening the exterior.

[0039] Epimedium is the dried leaf of Epimedium brevicornu Maxim. and Epimedium sagittatum (Sieb. et Zucc.) Maxim., plants of the Berberidaceae family. It has the effects of tonifying kidney yang, strengthening tendons and bones, and dispelling wind and dampness. It is mainly used to treat kidney yang deficiency, weakness of tendons and bones, and wind-cold-dampness arthralgia.

[0040] Dendrobium is the dried stem of Dendrobium nobile Lindl. and Dendrobium huoshanense CZTang et SJCheng, which belong to the Orchidaceae family. It can benefit the stomach and promote the production of body fluids, nourish yin and clear heat. It is good at nourishing the yin of the stomach and kidneys and can treat the syndrome of insufficient stomach yin and deficiency of kidney yin.

[0041] Polygonatum is the dried rhizome of Polygonatum sibiricum Red. and Polygonatum kingianum Coll. et Hemsl., plants of the Liliaceae family. It has the effects of replenishing qi and nourishing yin, strengthening the spleen, moistening the lungs, and benefiting the kidneys. It is a nourishing product that gently tonifies the spleen, lungs, and kidneys.

[0042] Fritillaria hupehensis Hsiao et KCHsia, a plant in the Liliaceae family, is a dried bulb that can clear heat and resolve phlegm, stop cough, and dissipate nodules. It is mainly used to treat cough due to lung heat, thick yellow phlegm, and scrofula and carbuncles caused by phlegm-fire stagnation.

[0043] Turmeric is the dried tuberous root of plants such as Curcuma wenyujin YHChen et C.Ling and Curcuma longa L. in the ginger family. It can promote blood circulation and relieve pain, regulate qi and relieve depression, clear the heart and cool the blood, and promote bile secretion and reduce jaundice. It is a "qi medicine in blood" and is good at treating qi stagnation and blood stasis syndrome.

[0044] Ginger is the fresh rhizome of Zingiber officinale Rosc., a plant in the ginger family. It can relieve exterior syndromes and dispel cold, warm the middle jiao and stop vomiting, and warm the lungs and stop coughing. It is a commonly used medicine for treating colds caused by wind and cold and vomiting due to stomach cold. It can also detoxify fish and crab poisoning.

[0045] By adopting the above technical solution, this application has the following beneficial effects compared with the prior art.

[0046] 1. The traditional Chinese medicine composition provided in this application uses Astragalus membranaceus and Codonopsis pilosula as the principal herbs, which play a role in invigorating qi and strengthening the spleen, targeting the core pathogenesis of Alzheimer's disease of "deficiency of the root and excess of the branch"; it uses Epimedium, Polygonatum sibiricum, Polygala tenuifolia, and Acorus tatarinowii as the assistant herbs, which play a role in tonifying the kidney and replenishing essence, resolving phlegm and opening the orifices; it uses Dendrobium nobile, Rhodiola rosea, Curcuma longa, Lycium chinense root bark, Fritillaria cirrhosa, and Angelica dahurica as adjuvant herbs, which play a role in promoting blood circulation, regulating qi, clearing heat and resolving phlegm; and it uses Citrus reticulata peel and Zingiber officinale as guiding herbs, which harmonize the other herbs and guide them to the appropriate meridians. The above-mentioned traditional Chinese medicines are rationally combined to achieve synergistic regulation among the components, which is in line with the traditional Chinese medicine treatment concept of invigorating qi and strengthening the spleen, tonifying the kidney and replenishing essence, resolving phlegm and opening the orifices, and promoting blood circulation and unblocking the meridians.

[0047] 2. The traditional Chinese medicine composition provided in this application can not only alleviate neuronal damage, but also reverse the disordered signaling pathways related to Alzheimer's disease in neurons, microglia, and astrocytes, and has excellent effects on the treatment of Alzheimer's disease.

[0048] 3. The traditional Chinese medicine composition provided in this application can significantly improve the responsiveness and behavioral impairment of zebrafish in the AlCl3-induced AD model group, making the cognitive function and behavioral level of the zebrafish in the AD model group close to that of normal fish, indicating that the traditional Chinese medicine composition can effectively improve Alzheimer's disease caused by neuronal damage. Attached Figure Description

[0049] Figure 1 These are heatmaps of pathways involved in the regulation of neuroinflammation in Alzheimer's disease by various single drugs and prescriptions;

[0050] Figure 2 This is a heatmap of pathways related to the regulation of astrocyte inflammation in Alzheimer's disease by various single drugs and combinations;

[0051] Figure 3 These are heatmaps of pathways involved in the regulation of neurons in Alzheimer's disease by various single drugs and combinations;

[0052] Figure 4 These are heatmaps of relevant pathways in various embodiments and comparative models of Alzheimer's disease neuroinflammation.

[0053] Figure 5 These are heatmaps of relevant pathways in various embodiments and comparative models of Alzheimer's disease astrocyte inflammation.

[0054] Figure 6 These are heatmaps of relevant pathways in various embodiments and comparative models of Alzheimer's disease neuronal regulation;

[0055] Figure 7 This is a schematic diagram of the behavioral testing procedure for verifying the efficacy of zebrafish.

[0056] Figure 8 These are time-distance movement curves of different groups of zebrafish under light and dark stimuli;

[0057] Figure 9 This is a statistical analysis chart of the swimming distance and maximum speed of different groups of bright field juvenile fish;

[0058] Figure 10 This is a statistical analysis chart showing the swimming distance and maximum speed of different groups of juvenile fish in dark fields. Detailed Implementation

[0059] This application discloses traditional Chinese medicine compositions, their preparation methods, and their applications.

[0060] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0061] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0062] It should be understood that the order of steps or the sequence of actions is not important as long as this application remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0063] The use of any and all instances or exemplary language such as “e.g.” or “including” herein is merely intended to better illustrate the application and does not constitute a limitation on the scope of the application unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of this application.

[0064] Furthermore, the numerical ranges and parameters used to define this application are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise explicitly stated, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately." Here, "approximately" generally means an actual value within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range.

[0065] Example

[0066] Table 1 Examples and Comparative Parts of Traditional Chinese Medicine Compositions

[0067]

[0068]

[0069] Preparation method of traditional Chinese medicine composition for treating Alzheimer's disease:

[0070] Weigh each Chinese herbal raw material according to the corresponding weight proportions to obtain a Chinese herbal mixture.

[0071] The first extraction was performed on the obtained Chinese herbal medicine mixture using 10 times the total weight of the raw materials in water. After boiling over high heat, the mixture was simmered over low heat for 90 minutes, and then filtered through 200-mesh gauze to obtain the first extract and the residue. The second extraction was performed on the residue using 8 times the total weight of the raw materials in water. After boiling over high heat, the mixture was simmered over low heat for 60 minutes, and then filtered through 200-mesh gauze to obtain the second extract. The first and second extracts were then mixed and combined to obtain the final extract.

[0072] The combined extracts were concentrated under reduced pressure for the first time by rotary evaporation at 80°C to a paste with a relative density of 1.1–1.2. The paste was centrifuged at 10,000 rpm for 10 min to remove impurities, filtered through 200-mesh nylon gauze, and the ultrafiltrate was collected. The ultrafiltrate was concentrated under reduced pressure for the second time by rotary evaporation at 80°C to a paste with a relative density of 1.1–1.2.

[0073] The extract was stirred evenly and preheated in water at 60°C. The spray dryer was set with a fan frequency of 40Hz, an air inlet temperature of 130°C, a needle blowing time of 3 seconds, an atomization pressure of 0.2MPa, and a peristaltic pump speed of 18rpm for spray drying to obtain a water extract freeze-dried powder, which is the traditional Chinese medicine composition for treating Alzheimer's disease.

[0074] Validation Example 1: Transcriptome sequencing and analysis of drug administration in neural cell models (SY5Y, U251, HMC3)

[0075] 1) Single drug extraction

[0076] The traditional Chinese medicine compositions in Examples 1 to 3 and Comparative Examples 1 to 3 comprise the following 14 single herbs: Astragalus membranaceus, Codonopsis pilosula, Epimedium brevicornu, Polygonatum sibiricum, Acorus tatarinowii, Polygala tenuifolia, Dendrobium nobile, Rhodiola rosea, Curcuma longa, Lycium chinense root bark, Fritillaria cirrhosa, Angelica dahurica, Citrus reticulata peel, and Zingiber officinale. The herbal raw materials were extracted with water using ultrasonic or Soxhlet methods and then freeze-dried into lyophilized powder. An appropriate amount of the lyophilized powder was weighed and dissolved in PBS to prepare an extract stock solution with a concentration of 50 mg / mL.

[0077] 2) Preparation of mother liquor for traditional Chinese medicine composition:

[0078] An appropriate amount of the lyophilized water extract powder from the examples and comparative examples was weighed and dissolved in PBS to prepare a stock solution of extract with a concentration of 50 mg / mL for later use.

[0079] 3) Cell plating and drug administration

[0080] The study involved three types of adherent cell lines. The specific steps for constructing neuronal cell models (SY5Y cell model), astrocyte inflammation model (U251 cell model), and neuronal model (HMC3 cell model) included:

[0081] For adherent cells, at the logarithmic growth phase, remove the cell culture flask from the incubator, add 1 mL of trypsin, gently shake to ensure the entire cell surface is covered with trypsin, and place in the incubator to digest until some cells float. Add 2 mL of complete culture medium to stop digestion, use a Pasteur pipette to detach the cells, centrifuge, discard the supernatant, add 2 mL of culture medium, gently pipette to mix, and count using a cell counting chamber. For suspension cells, at the logarithmic growth phase, collect the cells by centrifugation, add 2 mL of culture medium, gently pipette to mix, and count using a cell counting chamber. Seed HMC3 cells at a density of 3000 / well in 384-well plates. After overnight adhesion, stimulate with 5 ng / mL IL-1β for 24 h to establish a neuroinflammation model, and then treat with 200 μg / mL of drug extract for 24 h. SY5Y and U251 were inoculated into 384-well plates at a density of 8000 in RPMI 1640 complete medium containing 100 ng / mL PMA. After directional induction for 24-48 h, the plates were treated with 200 μg / mL of the drug extract for 24 h.

[0082] 4) Transcriptome sequencing and analysis

[0083] Cell lysis, reverse transcription, and library construction for drug screening were performed according to the instructions of the relevant kits. Samples were sequenced using an Illumina HiSeq X10, with 2 M reads per sample. The data were then analyzed. Gene set enrichment analysis (GSEA) was used to enrich signaling pathways; an FDR < 0.25 was considered to indicate significant pathway regulation.

[0084] Depend on Figures 1 to 3The results showed the regulatory effects of 14 single drugs on dysregulated pathways in Alzheimer's disease neuroinflammation models (HMC3), astrocyte models (U251), and neuron models (SY5Y). Red indicates significant upregulation of the pathway, blue indicates significant downregulation, and white indicates no significant effect. This demonstrates that any single drug in the traditional Chinese medicine composition can regulate at least one of the signaling pathways related to Alzheimer's disease neuroinflammation. In the neuroinflammatory model (HMC3) and the astrocyte model (U251), the traditional Chinese medicine composition primarily inhibits overactivated neuroinflammatory and metabolic signaling pathways, including neuroinflammatory-related signaling pathways such as proteasomes, peroxisomes, autophagy, and antigen processing and presentation; various pathological pathways of neurodegenerative diseases and disease-related signaling pathways such as Alzheimer's disease; and metabolic-related signaling pathways such as valine / leucine / isoleucine degradation, fatty acid metabolism, RNA degradation, unsaturated fatty acid biosynthesis, ketone body synthesis and degradation, fructose and mannose metabolism, and alanine / aspartate / glutamate metabolism. Simultaneously, in the neuronal model (SY5Y), the composition exerts its effects by activating signaling pathways related to neuronal activity, such as mitochondria, autophagy, endoplasmic reticulum protein processing, GABAergic synapses, endocytosis, dopaminergic synapses, oxidative phosphorylation, and thermogenesis.

[0085] Depend on Figures 4 to 6 The results demonstrate the regulation of dysregulated pathways in the examples, comparative examples, donepezil, and GV971 in the Alzheimer's disease neuroinflammation model (HMC3), the astrocyte inflammation model (U251), and the neuronal model (SY5Y). Red indicates significant upregulation of the pathway, blue indicates significant downregulation, and white indicates no significant effect. Therefore, compared to the traditional Chinese medicine compositions in comparative examples 1 to 3, the traditional Chinese medicine compositions in examples 1 to 3 participate in the regulation of more disease-related signaling pathways. Especially in the neuroinflammation and glial cell models, the traditional Chinese medicine compositions can better regulate neuroinflammation-related signaling pathways such as the phagosome signaling pathway and the calcium signaling pathway, as well as neuronal activity-related signaling pathways such as the cell cycle signaling pathway and protein output, thus contributing to the treatment of Alzheimer's disease.

[0086] Verification Example 2: Analysis of Zebrafish Efficacy Verification

[0087] 1) Construction of the Zebrafish AD Model

[0088] Adult zebrafish of the TU strain were raised in a recirculating aquatic environment at 28.5±0.5℃ with a light / dark cycle of 14 hours of light / 10 hours of darkness. They were fed twice daily, morning and evening, with brine shrimp and pelleted fish food. Zebrafish embryos used in the experiment were produced through hybridization of adult fish. Parent zebrafish were placed in hybridization rearing boxes at a female-to-male ratio of 1:1 or 1:2. The resulting embryos were cultured in E3 medium (0.03% sea salt, 0.00014% methylene blue reverse osmosis aqueous solution) and placed in an incubator at 28.5±0.5℃. During the culture process, dead embryos were removed daily, and 30% of the medium was replaced.

[0089] 2) Toxicity testing

[0090] For 3 days per fertile period (dpf), normally developed zebrafish were placed in 6-well plates for drug treatment, with treatment continued for up to 5 dpf, at a rate of 30 juveniles per well, and 2 mL of culture medium added to each well. The experiment was divided into a solvent control group (E3 medium containing 0.5% DMSO), an AD model group (150 μM AlCl3), a positive control group (150 μM AlCl3 + 10 μM Donepezil), and a test drug group (150 μM AlCl3 + a specific concentration of the test drug). Four traditional Chinese medicine compound drugs and one traditional Chinese medicine control were used for 5-plate toxicity testing. Each of the five drugs had six concentration gradients (1, 50, 100, 500, 1000, and 2000 mg / mL). During treatment, the survival of the juveniles was observed and recorded every 24 hours, and the corresponding fresh culture medium was replaced. Concentrations that were neither lethal nor affected the viability of the juveniles were selected for the experiment.

[0091] 3) Behavioral efficacy assessment

[0092] The test was conducted on a 96-well plate, with n=12 per group. The test procedure is as follows: Figure 7 As shown:

[0093] Juvenile zebrafish with a 5 dpf were selected and placed one fish per well in a 96-well plate. After placing the 96-well plate in the DanioVision (Noldus) zebrafish behavior tracking system, the juveniles were first allowed to acclimatize to the dark environment for 20 minutes. Then, a light-dark stimulation test was conducted, providing the juvenile zebrafish with dark and light stimulation for four cycles. Each cycle consisted of 5 minutes of darkness and 5 minutes of light (100% system illumination). The behavioral trajectory data of the juveniles were collected.

[0094] Depend on Figures 8 to 10The results showed that by analyzing the behavioral trajectory data of zebrafish in different groups, the average swimming distance of juvenile fish in each group, and the rate of change in speed ΔS caused by light and dark alternation, the differences in behavioral impairment and reaction ability of zebrafish in the control group, model group, western medicine control group (Model + Donepezil), traditional Chinese medicine control group (Model + GV971), and composition experimental group (Model + Drug) were evaluated. The test results showed that the model group significantly reduced the swimming distance of zebrafish, causing behavioral impairment in juvenile zebrafish, and the reduced change in swimming speed under light and dark stimuli indicated a decrease in reaction ability in the model group. Compared with the marketed western medicine donepezil and traditional Chinese medicine GV971, the traditional Chinese medicine composition significantly improved the reaction ability and behavioral impairment of AD model zebrafish, indicating that this traditional Chinese medicine composition can effectively improve the clinical symptoms of AD cognitive impairment and behavioral abnormalities caused by neuronal damage.

[0095] In summary, the foregoing results indicate that the traditional Chinese medicine composition prepared in this application can not only alleviate neuronal damage, but also reverse Alzheimer's disease-related disordered signaling pathways in neurons, microglia, and astrocytes, significantly improving the responsiveness and behavioral impairments of AlCl3-induced AD model group zebrafish, making the cognitive function and behavioral level of the AD model group zebrafish close to that of normal fish. This demonstrates that the traditional Chinese medicine composition can effectively improve Alzheimer's disease caused by neuronal damage.

[0096] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A traditional Chinese medicine composition for treating Alzheimer's disease, characterized in that, It is prepared from the following parts by weight of traditional Chinese medicine raw materials: Astragalus membranaceus 9-30 parts, Codonopsis pilosula 9-30 parts, Epimedium brevicornu 6-10 parts, Polygonatum sibiricum 9-15 parts, Acorus tatarinowii 3-10 parts, Polygala tenuifolia 3-10 parts, Dendrobium nobile 6-12 parts, Rhodiola rosea 3-6 parts, Curcuma longa 3-10 parts, Lycium chinense root bark 9-15 parts, Fritillaria cirrhosa 3-9 parts, Angelica dahurica 3-10 parts, Citrus reticulata peel 3-10 parts, Zingiber officinale 3-10 parts.

2. The traditional Chinese medicine composition for treating Alzheimer's disease according to claim 1, characterized in that, It is prepared from the following parts by weight of traditional Chinese medicine raw materials: Astragalus membranaceus 10-25 parts, Codonopsis pilosula 10-25 parts, Epimedium brevicornu 6-10 parts, Polygonatum sibiricum 9-12 parts, Acorus tatarinowii 3-9 parts, Polygala tenuifolia 5-10 parts, Dendrobium nobile 6-12 parts, Rhodiola rosea 3-6 parts, Curcuma longa 3-9 parts, Lycium chinense root bark 10-15 parts, Fritillaria cirrhosa 5-9 parts, Angelica dahurica 3-9 parts, Citrus reticulata peel 3-9 parts, Zingiber officinale 3-9 parts.

3. The traditional Chinese medicine composition for treating Alzheimer's disease according to claim 2, characterized in that, It is prepared from the following parts by weight of traditional Chinese medicine raw materials: Astragalus membranaceus 10-20 parts, Codonopsis pilosula 10-20 parts, Epimedium 7-10 parts, Polygonatum sibiricum 9-10 parts, Acorus tatarinowii 3-6 parts, Polygala tenuifolia 6-10 parts, Dendrobium nobile 8-12 parts, Rhodiola rosea 5-6 parts, Curcuma longa 5-7 parts, Lycium chinense root bark 12-15 parts, Fritillaria cirrhosa 5-7 parts, Angelica dahurica 3-7 parts, Citrus reticulata peel 5-7 parts, Zingiber officinale 3-5 parts.

4. A method for preparing a traditional Chinese medicine composition for treating Alzheimer's disease according to any one of claims 1-3, characterized in that, Including the following: Weigh each Chinese herbal raw material according to the weight proportions to obtain a Chinese herbal mixture; The obtained mixture of traditional Chinese medicines was extracted using solvent extraction to obtain an extract. The obtained extract was concentrated under reduced pressure to obtain a paste; The obtained extract was spray-dried to obtain a dry powder, which is the traditional Chinese medicine composition for treating Alzheimer's disease.

5. The method for preparing the traditional Chinese medicine composition for treating metabolic-related fatty liver disease according to claim 4, characterized in that, The extraction of the obtained traditional Chinese medicine mixture using solvent extraction specifically includes: The resulting mixture of Chinese medicinal materials was subjected to a first extraction using water at 10-15 times the total weight of the raw materials. After extraction at 100℃ for 60-90 minutes, the first extract and residue were obtained by filtration. The residue was subjected to a second extraction using 8-12 times the total weight of the Chinese herbal raw materials in water. The extraction was carried out at 100℃ for 60-90 minutes, and then filtered to obtain the second extract. The first and second extracts were mixed and combined to obtain the final extract.

6. The method for preparing the traditional Chinese medicine composition for treating metabolic-related fatty liver disease according to claim 4, characterized in that, The concentration of the obtained extract under reduced pressure specifically includes: The obtained extract was concentrated under reduced pressure at 70-85℃ to obtain a concentrate with a relative density of 1.1-1.

2. The obtained concentrate was centrifuged and the centrifuged liquid was filtered to obtain ultrafiltrate; The obtained ultrafiltrate was concentrated under reduced pressure at 70-85℃ to obtain an extract with a relative density of 1.1-1.

2.

7. The use of a traditional Chinese medicine composition as described in any one of claims 1-3 in the preparation of a medicament for treating or preventing Alzheimer's disease.

8. The application as described in claim 7, characterized in that, Pharmaceutically acceptable excipients are added to the traditional Chinese medicine composition to prepare an oral preparation, the dosage form of which includes tablets, capsules, granules, pills or oral liquid preparations.

9. The application as described in claim 7, characterized in that, The treatment of Alzheimer's disease includes slowing the progression of Alzheimer's disease. The traditional Chinese medicine composition improves memory impairment and / or disorientation caused by Alzheimer's disease by alleviating neuronal damage and reversing Alzheimer's signaling pathways, including neuroinflammation and neuronal activity-related signaling pathways.

10. The application as described in claim 9, characterized in that, The neuroinflammatory pathways involved include at least one of the following: proteasome, peroxisome, autophagy, antigen processing and presentation; and / or, The signaling pathways related to neuronal activity include at least one of mitochondria, autophagy, endoplasmic reticulum protein processing, GABAergic synapses, endocytosis, dopaminergic synapses, oxidative phosphorylation, and thermogenicity.

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