A traditional Chinese medicine composition for preventing and / or treating Alzheimer's disease and a preparation method thereof

Abstract

The present application relates to the technical field of traditional Chinese medicine. The present application provides a traditional Chinese medicine composition for preventing and / or treating Alzheimer's disease, which comprises red ginseng and prepared radix falcaliae multiflorae. The present application also provides a preparation method of the traditional Chinese medicine composition for preventing and / or treating Alzheimer's disease. The traditional Chinese medicine composition has a protective effect on HT22 cells induced by A beta 1-42, can improve the anti-aging effect of HT22 cells, can significantly improve the learning and memory impairment of dementia model mice, can prolong the lifespan of N2 wild type Caenorhabditis elegans, can significantly inhibit the production and deposition of beta-amyloid protein closely related to Alzheimer's disease, can significantly activate the expression of hippocampus and cortex SIRT1, can significantly inhibit the expression of hippocampus and cortex aging marker genes of mice, can inhibit brain aging, and thus can effectively prevent and / or treat Alzheimer's disease.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine technology, and in particular to a traditional Chinese medicine composition for the prevention and / or treatment of Alzheimer's disease and its preparation method. Background Technology

[0002] Data surveys have revealed that neurodegenerative diseases, in particular, require serious attention among age-related illnesses. Alzheimer's disease (AD) is a neurodegenerative disease that accompanies aging, leading to a decline in learning and memory function and behavioral impairment. It is the third leading cause of death after cancer and cardiovascular disease.

[0003] Studies have shown that a prominent pathological feature of Alzheimer's disease (AD) patients is the formation of senile plaques due to the deposition of β-amyloid-β (Aβ) protein in the brain, as well as neurofibrillary tangles caused by the hyperphosphorylation of Tau protein. In addition, various cellular changes are involved, including synaptic dysfunction, mitochondrial damage, microglia activation, and neuroinflammation. Aβ is a highly conserved intrinsic membrane protein generated by the hydrolysis of β-amyloid precursor protein (APP). Aβ aggregation inducing cognitive impairment is currently the most widely accepted hypothesis for the pathogenesis of AD. Currently, no drugs with significant therapeutic effects have been found clinically. The main marketed drugs are cholinesterase inhibitors, which can only improve some clinical symptoms of AD and cannot reverse the disease progression. Therefore, it is urgent to find drugs that can combat neuronal aging, prolong lifespan, and improve age-related neurodegenerative diseases. Summary of the Invention

[0004] The purpose of this invention is to provide a traditional Chinese medicine composition for the prevention and / or treatment of Alzheimer's disease, comprising red ginseng and processed Polygonum multiflorum. This traditional Chinese medicine composition has a protective effect on Aβ1-42-induced hippocampal immortalized cells HT22, can enhance the anti-aging effect of HT22 cells, can significantly improve dementia symptoms and learning and memory impairment in Alzheimer's disease model mice, prolong the lifespan of N2 wild-type Caenorhabditis elegans, and can significantly inhibit the production and deposition of β-amyloid protein, a pathological product closely related to the occurrence of Alzheimer's disease, thereby achieving the purpose of effectively preventing and / or treating Alzheimer's disease.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a traditional Chinese medicine composition for the prevention and / or treatment of Alzheimer's disease, comprising the following raw materials in parts by weight: 1-4 parts red ginseng and 1-6 parts processed Polygonum multiflorum.

[0007] As a preferred option, the ingredients include the following parts by weight: 1.5-3 parts red ginseng and 2-5 parts processed Polygonum multiflorum.

[0008] The present invention also provides a method for preparing the aforementioned traditional Chinese medicine composition, comprising the following steps:

[0009] (1) Crush the red ginseng and the prepared Polygonum multiflorum into powder, sieve them, and then take the sieve material for later use;

[0010] (2) After mixing the sieved material, soak it, then percolate and dry it to obtain the Chinese herbal composition.

[0011] Preferably, the soaking in step (2) is carried out using an ethanol aqueous solution with a volume fraction of 60-80%, and the mass-to-volume ratio of the sieved material after mixing to the ethanol aqueous solution is 1g:(1-5)mL.

[0012] Preferably, the soaking time is 20-30 hours.

[0013] Preferably, after soaking in step (2), an ethanol aqueous solution is directly added for percolation, wherein the volume fraction of the ethanol aqueous solution is 60-80%, and the mass-to-volume ratio of the mixed sieve material to the added ethanol aqueous solution is 1g:(15-25)mL.

[0014] Preferably, the mesh size of the sieve in step (1) is 8-12 mesh.

[0015] The present invention also provides the use of the composition in the preparation of drugs that enhance the anti-aging properties of HT22 cells.

[0016] The present invention also provides the use of the composition in the preparation of a medicament for inhibiting the production and deposition of β-amyloid protein associated with Alzheimer's disease.

[0017] By adopting the above technical solution, the present invention has the following beneficial effects:

[0018] 1. This invention provides a traditional Chinese medicine composition prepared using extracts of red ginseng and processed Polygonum multiflorum for the prevention and / or treatment of Alzheimer's disease. The extracts of red ginseng and processed Polygonum multiflorum obtained by percolation with 70% ethanol show the best efficacy, exhibiting a protective effect against Aβ1-42-induced immortalized hippocampal HT22 cells, inhibiting HT-22 cell damage and death, and demonstrating significant anti-aging effects. Simultaneously, this traditional Chinese medicine composition can also extend the average lifespan of wild-type Caenorhabditis elegans to a certain extent.

[0019] 2. The traditional Chinese medicine composition of the present invention can significantly improve the learning and memory impairment of dementia symptoms in Alzheimer's disease model mice, and significantly inhibit the production and deposition of β-amyloid protein, a pathological product closely related to the occurrence of Alzheimer's disease. The examples also show that it can reduce the content of Aβ1-40 and Aβ1-42 in cerebrospinal fluid.

[0020] 3. The herbal composition of the present invention can improve the pathological morphological damage of the hippocampus in aging model mice, and can significantly increase the number of nerve cells, make their arrangement more orderly, increase the number of cell layers, and make their morphology and structure more like normal cells, and significantly alleviate the phenomena of shrinkage and vacuolization.

[0021] 4. The herbal composition of the present invention can significantly activate the expression of SIRT1 in the hippocampus and cortex, and significantly inhibit the expression of p53 and p21 genes, which are aging markers in the mouse hippocampus and cortex, thereby inhibiting brain aging and thus preventing and treating Alzheimer's disease.

[0022] 5. The traditional Chinese medicine composition of the present invention can also improve indicators related to liver function, blood lipids and heart, improve the expression of CD3+CD4+ and CD3+CD8+ in the spleen, and achieve immunomodulatory effects. Attached Figure Description

[0023] Figure 1 The anti-aging effects of different treatment groups on HT22 cells;

[0024] Figure 2 Results of β-galactosidase staining after treatment with deer extract at different concentrations of 70% ethanol;

[0025] Figure 3 The effects of different treatment groups on the lifespan of wild-type N2 Caenorhabditis elegans;

[0026] Figure 4 Results of water maze movement trajectories of mice in different treatment groups ( Figure 4 In this context, A represents the control group, B represents the model group, C represents the low-dose group, D represents the high-dose group, and E represents the positive control group. Compared with the control group, *P<0.05, ***P<0.001; compared with the model group, ##P<0.01, ###P<0.001).

[0027] Figure 5 The results of the time to reach the hidden platform in the water maze test for mice in different treatment groups (compared with the control group, *P<0.05, ***P<0.001; compared with the model group, ##P<0.01, ###P<0.001);

[0028] Figure 6 The results of platform crossover in the water maze test for mice in different treatment groups (compared with the control group, *P<0.05, ***P<0.001; compared with the model group, ##P<0.01, ###P<0.001);

[0029] Figure 7 The levels of Aβ1-40 and Aβ1-42 in different groups of mice were measured. Figure 7 In this context, A represents the content of Aβ1-40. Figure 7In the diagram, B represents the content of Aβ1-42; n = 6.

[0030] Figure 8 HE staining was used to detect pathological morphology in the CA1 and CA3 regions of the hippocampus in different groups of mice. Figure 8 In the diagram, A represents the staining status of the CA1 region. Figure 8 (B in the text represents the staining status of the CA3 region);

[0031] Figure 9 The expression of Sirt1, p53, and p21 genes in the hippocampus of different groups of mice ( Figure 9 In this context, A represents the relative expression level of SIRT1 mRNA. Figure 9 In this context, B represents the relative expression level of p53 mRNA. Figure 9 In this context, C represents the relative expression level of p21 mRNA; n = 4.

[0032] Figure 10 The expression of Sirt1, p53, and p21 genes in the cerebral cortex of different groups of mice ( Figure 10 In this context, A represents the relative expression level of SIRT1 mRNA. Figure 10 In this context, B represents the relative expression level of p53 mRNA. Figure 10 In this context, C represents the relative expression level of p21 mRNA; n = 4.

[0033] Figure 11 The levels of AST, ALT, TG, CHOL, BUN, CREA, LDH, and CK in the blood of different groups of mice were recorded. Figure 11 In this context, A represents the AST content. Figure 11 B in the figure represents the ALT content. Figure 11 The C in the figure represents the TG content. Figure 11 The "D" in the figure represents the CHOL content. Figure 11 The "E" in the figure represents the BUN content. Figure 11 The F in the figure represents the CREA content. Figure 11 The G in the figure represents the LDH content. Figure 11 H in the figure represents the CK content; n = 6);

[0034] Figure 12 The composition of CD3+CD4+, CD3+CD8+, and the ratio of CD3+CD4+ / CD3+CD8+ in the spleen of different groups of mice. Figure 12 In this context, A represents the expression of CD3+CD4+. Figure 12 In this context, B represents the expression of CD3+CD8+. Figure 12 In this context, C represents the ratio of CD3+CD4+ / CD3+CD8+; compared to the control, *** P<0.001, ****P < 0.0001; compared with the model, ### P<0.001; n=6). Detailed Implementation

[0035] This invention provides a traditional Chinese medicine composition for the prevention and / or treatment of Alzheimer's disease, comprising the following raw materials: red ginseng and processed Polygonum multiflorum. In this invention, the red ginseng is preferably 1-4 parts by weight, more preferably 1.5-3 parts, and even more preferably 2 parts; the processed Polygonum multiflorum is preferably 1-6 parts by weight, more preferably 2-5 parts, and even more preferably 3 parts.

[0036] The present invention also provides a method for preparing the traditional Chinese medicine composition, comprising the following steps: (1) crushing and sieving red ginseng and prepared Polygonum multiflorum separately, and taking the sieve material for later use; (2) mixing the sieve material and soaking it, then percolating and drying to obtain the traditional Chinese medicine composition.

[0037] In this invention, red ginseng and processed Polygonum multiflorum are ground and pulverized separately, then sieved, and the sieved material is collected for later use. The preferred sieve mesh size is 8-12 mesh, more preferably 9-11 mesh, and even more preferably 10 mesh. Both the red ginseng and processed Polygonum multiflorum used in this invention are commercially available.

[0038] In this invention, the sieve residue of red ginseng and processed Polygonum multiflorum is mixed evenly and then soaked. The soaking is preferably performed using an ethanol-water solution, with a volume fraction preferably 60-80%, more preferably 65-75%, and even more preferably 70%. The mass-to-volume ratio of the mixed sieve residue to the added ethanol-water solution is preferably 1g:(1-5)mL, more preferably 1g:(1.5-3)mL, and even more preferably 1g:2mL. The soaking time is preferably 20-30 hours, more preferably 22-26 hours, and even more preferably 24 hours.

[0039] In this invention, percolation is performed after soaking. In this invention, an ethanol-water solution is directly added after soaking for percolation. The volume fraction of the ethanol-water solution is preferably 60-80%, more preferably 65-75%, and even more preferably 70%. The mass-to-volume ratio of the mixed sieve undersize to the added ethanol-water solution is preferably 1g:(15-25)mL, more preferably 1g:(18-22)mL, and even more preferably 1g:20mL. The percolation time is preferably 20-30h, more preferably 22-26h, and even more preferably 24h.

[0040] In this invention, after percolation, the obtained solution needs to be dried to obtain a solid. The preferred drying method is water bath drying, with the water bath temperature preferably at 100°C and the water bath time preferably at 72-96 hours, more preferably at 75-92 hours, and even more preferably at 80-90 hours. The water bath drying continues until the solution becomes very viscous and cannot flow. After water bath drying, vacuum drying is preferably performed. The vacuum drying pressure is preferably -0.10 to -0.05 MPa, more preferably -0.09 to -0.07 MPa, and even more preferably -0.08 MPa. The vacuum drying time is preferably 40-55 hours, more preferably 45-50 hours, and even more preferably 48 hours. The vacuum drying temperature is preferably 50-70°C, more preferably 55-65°C, and even more preferably 60°C. In this invention, the yield is preferably 22-28%, more preferably 25-27%, and even more preferably 26.6%. After obtaining the solid, the present invention preferably grinds the solid into powder.

[0041] This invention also provides the application of the aforementioned traditional Chinese medicine composition in the preparation of drugs that enhance the anti-aging effects of HT22 cells. The traditional Chinese medicine composition of this invention can inhibit HT-22 cell damage and death.

[0042] The present invention also provides the application of the traditional Chinese medicine composition in the preparation of a drug that inhibits the production and deposition of β-amyloid protein associated with Alzheimer's disease.

[0043] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0044] Example 1

[0045] Preparation method of traditional Chinese medicine composition for the prevention and / or treatment of Alzheimer's disease:

[0046] (1) Grind 2 parts of red ginseng and 3 parts of prepared Polygonum multiflorum into powder, then pass them through a 10-mesh sieve and take the sieve material for later use;

[0047] (2) Mix the sieve material evenly, and then soak it in a mixture of sieve material and 70% ethanol aqueous solution at a mass-volume ratio of 1g:2mL for 24h.

[0048] (3) After soaking, add 70% ethanol aqueous solution directly according to the mass-volume ratio of 1g:20mL of the mixed sieve material to 70% ethanol aqueous solution for percolation for 24h.

[0049] (4) The solution obtained by percolation was dried in a water bath at 100°C for 80 hours, and then dried under reduced pressure at -0.08 MPa and 60°C for 48 hours to obtain a solid, which was then ground into powder.

[0050] Example 2

[0051] Preparation method of traditional Chinese medicine composition for the prevention and / or treatment of Alzheimer's disease:

[0052] (1) Grind 1 part red ginseng and 6 parts prepared Polygonum multiflorum into powder separately, then pass them through an 8-mesh sieve and take the sieve material for later use;

[0053] (2) Mix the sieve material evenly, and then soak it in water at a ratio of 1g:1mL of the mixed sieve material to 80% ethanol aqueous solution for 20h.

[0054] (3) After soaking, add 80% ethanol aqueous solution directly according to the mass-volume ratio of 1g:15mL of the mixed sieve material to 80% ethanol aqueous solution for percolation for 24h.

[0055] (4) The solution obtained by percolation was dried in a water bath at 100°C for 90 hours, and then dried under reduced pressure at -0.1 MPa and 70°C for 48 hours to obtain a solid, which was then ground into powder.

[0056] Example 3

[0057] Preparation method of traditional Chinese medicine composition for the prevention and / or treatment of Alzheimer's disease:

[0058] (1) Grind 4 parts of red ginseng and 1 part of prepared Polygonum multiflorum into powder, then pass them through a 12-mesh sieve and take the sieve material for later use.

[0059] (2) Mix the sieve material evenly, and then soak it in a 60% ethanol aqueous solution at a mass-volume ratio of 1g:5mL for 30h.

[0060] (3) After soaking, add 60% ethanol aqueous solution directly according to the mass-volume ratio of 1g:25mL of the mixed sieve material to 60% ethanol aqueous solution for percolation for 24h.

[0061] (4) The solution obtained by percolation was dried in a water bath at 100°C for 80 hours, and then dried under reduced pressure at -0.05MPA and 60°C for 48 hours to obtain a solid, which was then ground into powder.

[0062] Protective effect of Experiment 1 on HT22 cells

[0063] (I) Protective effects of extracts from different processes on HT22 cells

[0064] Preparation of Aβ1-42 solution: Beta-Amyloid peptide (1-42) (human) (ab120301) (purchased from Abcam, UK) was dissolved in hexafluoroisopropanol (HFIP) to a concentration of 1 mg / mL, reacted at 4 °C for 2 h, dried, and stored at -80 °C. Before use, it was dissolved in dimethyl sulfoxide (DMSO) to obtain a final Aβ1-42 solution with a concentration of 400 μM. For detailed preparation procedures, please refer to "Study on the effect of nicotine on the aggregation of Aβ protein in transgenic Caenorhabditis elegans" (Lu Xiaoda. Study on the effect of nicotine on the aggregation of Aβ protein in transgenic Caenorhabditis elegans [D]. Changchun: Changchun University of Science and Technology, 2021.).

[0065] Under sterile conditions, mouse hippocampal neuronal HT-22 cells (purchased from the Peking Union Medical College Cell Bank National Sharing Platform) were incubated at 37°C. When the cells adhered and reached 80% confluence with the culture dish, they were digested with trypsin and the incubation was terminated with 5 mL of LDM medium. The cells were resuspended and counted at 1.37 × 10⁻⁶ cells / mL. 6 Cells / mL: Add 292 μL of cell suspension to 10 mL of fresh DMEM, mix well, and then add 100 μL of cell suspension to each well of a 96-well plate for later use.

[0066] 96-well plates containing HT-22 cell suspension were divided into 8 groups, with 3 replicates per group: blank group, model group, positive control group, 30% ethanol reflux extract group, 30% ethanol percolation extract group, 70% ethanol reflux extract group, 70% ethanol percolation extract group, and water extract group. The blank group received no treatment; the model group received 50 μL of 40 μmol / L LAβ1-42 solution; the positive control group received 50 μL of 15 μM apigenin solution + 50 μL of 40 μmol / L LAβ1-42 solution; and the 30% ethanol reflux extract group received 50 μL of 200 μg / mL 30% ethanol reflux extract + 50 μL of 40 μmol / L apigenin solution. Aβ1-42 solution; 50 μL of 200 μg / mL 30% ethanol percolation extract + 50 μL of 40 μmol / L Aβ1-42 solution were added to the 30% ethanol percolation extract group; 50 μL of 200 μg / mL 70% ethanol reflux extract + 50 μL of 40 μmol / L Aβ1-42 solution were added to the 70% ethanol percolation extract group; 50 μL of 200 μg / mL 70% ethanol percolation extract + 50 μL of 40 μmol / L Aβ1-42 solution were added to the 70% ethanol percolation extract group; 50 μL of 200 μg / mL water extract + 50 μL of 40 μmol / L Aβ1-42 solution were added to the water extract group. Among them, the positive control group, the 30% ethanol reflux extract group, the 30% ethanol percolation extract group, the 70% ethanol reflux extract group, the 70% ethanol percolation extract group and the water extract group were added to the solution and incubated in a 37℃ CO2 incubator for 0.5h. Then, Aβ1-42 solution was added and the incubator was placed in a 37℃ CO2 incubator for 24h together with the blank group and the model group.

[0067] The preparation process of the Aricept solution described in this invention is as follows: Take out one Aricept tablet, grind it, weigh 1.2 mg, add 962 μL LDMSO to dissolve it, and prepare a 3 mM stock solution.

[0068] The 70% ethanol percolation extract of this invention was prepared in Example 1;

[0069] The 30% ethanol percolation extract was prepared using the same procedure as in Example 1, except that "70% ethanol" was replaced with "30% ethanol".

[0070] The 30% ethanol reflux extract was prepared using the same procedure as in Example 1, except that “70% ethanol” was replaced with “30% ethanol” and “infiltration for 24 hours” was replaced with “reflux extraction twice, 2 hours each time”.

[0071] The 70% ethanol reflux extract was prepared using the same procedure as in Example 1, except that "infiltration for 24 hours" was replaced with "reflux extraction twice, 2 hours each time";

[0072] The water extract was prepared using the same procedure as in Example 1, except that the 70% ethanol percolation process was replaced with two decoctions in pure water, the first for 2 hours and the second for 1 hour, and the filtrates were combined.

[0073] After culture, 20 μL of fresh MTT solution (purchased from Beijing Solarbio Science & Technology Co., Ltd.) was added to each well, and the cells were cultured for another 4 hours. The supernatant was discarded, and 200 μL of DMSO solution was added to each well in the dark. The absorbance (OD value) was then measured at 570 nm using a microplate reader to calculate cell viability. Data were statistically analyzed using SPSS 26.0 software, and data are expressed as mean ± standard deviation (x±s). One-way ANOVA was used for comparisons among multiple groups (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001). The results are shown in Table 1.

[0074] Cell viability = A in each treatment group 570 Value / Blank Group A 570 Value × 100%

[0075] Table 1. Effects of different treatment groups on HT-22 cell viability.

[0076]

[0077] The results showed that, compared with the model group, the survival rates of HT-22 cells treated with the positive control group and the extracts processed using different methods were significantly different. All cells in the blank group survived; the model group had the lowest survival rate; and some cells died after treatment with Aβ1-42 solution. The group treated with the traditional Chinese medicine composition described in this invention had the highest cell survival rate compared to the other extract groups. This also indicates that the traditional Chinese medicine composition described in this invention has a protective effect on cells and improves cell survival rate.

[0078] (II) Anti-aging efficacy of extracts processed using different techniques

[0079] This experiment consisted of eight groups: a blank control group, a model group, a positive control group, a 500 μg / mL 30% ethanol reflux extract group, a 500 μg / mL 30% ethanol percolation extract group, a 500 μg / mL 70% ethanol reflux extract group, a 500 μg / mL 70% ethanol percolation extract group, and a 500 μg / mL water extract group. The blank control group was supplemented with HT22 cell suspension; the model group was supplemented with HT22 cell suspension + Dye dye to measure naturally senescent cells; and the positive control group was supplemented with 100 μM resveratrol + Dye dye.

[0080] Under aseptic conditions, HT22 cells were grown in a 37°C incubator. When the cells adhered and reached 80% confluence with the culture dish, they were digested with trypsin and the growth was stopped with 5 mL of LDM medium. The cells were then resuspended and counted at 9 × 10⁻⁶ cells / mL. 5Cells were cultured at a density of 1 mL / mL. 1 mL of DMEM and 100 μL of HT22 cell suspension were added to each well of a 12-well plate, mixed, and incubated at 37°C for 24 h. After cell adhesion, 1 mL of fresh DMEM was added to each well, along with 10 μL of different drug solutions. The mixture was then incubated at 37°C for 24 h. After 48 h, fresh DMEM was added to each well, along with 1.5 μL of LDye dye. The mixture was then incubated at 37°C for 2 h. After incubation, the cells were processed according to the Senescence Assay Kit (Beta Galactosidase, Fluorscence) instructions (purchased from abcam, batch number ab228562), selecting the FITC channel. The results are shown in Table 2.

[0081] Calculation formula: Antiaging efficiency (%) = (FITC) 模型组 -FITC 实验组 ) / FITC 模型组 ×100.

[0082] Table 2. Anti-aging effects of different treatment groups on HT22 cells

[0083]

[0084] The results showed that the 30% ethanol reflux extract group, the 30% ethanol deer antler extract group, the 70% ethanol reflux extract group, the 70% ethanol deer antler extract group, and the water extract group all effectively resisted HT22 cell senescence (P<0.0001). Among them, the 70% ethanol deer antler extract group had the strongest anti-aging effect, which was comparable to that of the positive control group (100 μM resveratrol) (see...). Figure 1 ).

[0085] (III) Effects of β-galactosidase staining on HT22 cell senescence

[0086] This experiment consisted of five groups: a blank control group, a 30 μM resveratrol group, a 500 μg / mL 70% ethanol percolated extract group, a 200 μg / mL 70% ethanol percolated extract group, and a 100 μg / mL 70% ethanol percolated extract group. The blank control group only contained HT22 cell suspension.

[0087] Under sterile conditions, HT22 cells were grown in a 37°C incubator. When the cells adhered and reached 80% confluence with the culture dish, they were digested with trypsin and the growth was stopped with 5 mL of LDM medium. The cells were then resuspended and counted at 1.37 × 10⁻⁶ cells. 6 Cells / mL were added to 900 μL of culture medium in a 12-well plate, followed by 100 μL of cell suspension. At this point, each well contained 1.37 × 10⁶ cells / mL. 5Cells were incubated at 37°C for 24 hours. After cell adhesion, different drug solutions were added to the corresponding wells, and the cells were incubated at 37°C for another 24 hours. After 48 hours, cells were fixed and stained according to the instructions of the β-galactosidase staining kit for cell senescence (purchased from Shanghai Beyotime Biotechnology Co., Ltd., C0602), and incubated overnight in a CO2-free 37°C incubator. The β-galactosidase staining was observed under a microscope, and the results are shown below. Figure 2 .

[0088] Microscopic observation after β-galactosidase staining revealed a significant decrease in the number of blue-stained cells in the 70% ethanol-infused deer extract group compared to the blank control group. This reduction was found to be dose-dependent, with cell processes shrinking and becoming more rounded. The 500 μg / mL concentration was more potent than that of 30 μM resveratrol. Figure 2 As shown in the figure. This indicates that 70% ethanol-infused deer extract can inhibit HT-22 cell damage and death.

[0089] Effect of Experiment Example 2 on Life Extension

[0090] (I) Toxicological experiments on wild-type Caenorhabditis elegans N2

[0091] The 70% ethanol percolation extract prepared in Example 1 was dissolved in ddH2O and prepared into a stock solution with a concentration of 200 mg / mL according to the corresponding proportion of the extract. The stock solution was then gradually diluted to seven concentrations (0.00128 mg / mL, 0.0064 mg / mL, 0.032 mg / mL, 0.16 mg / mL, 0.8 mg / mL, 4 mg / mL and 20 mg / mL).

[0092] Commercially available wild-type *C. elegans* nematodes were synchronized and cultured for 3 days at 20℃ until they developed into adults. These adults were then transferred to 24-well plates, and appropriate concentrations of 70% ethanol percolated extract were added. The nematode survival rate was recorded after 6 days. The results showed that none of the above concentrations of 70% ethanol percolated extract were toxic to wild-type *C. elegans* nematodes.

[0093] (II) Lifespan determination of wild-type N2 Caenorhabditis elegans

[0094] Based on the 70% ethanol percolation extract of wild-type *C. elegans* N2, which showed no toxicity to this species, a preliminary experiment was conducted to screen for the effective concentrations that could exert an effect on *C. elegans* N2. The experimental drug concentrations were set at 0.2 mg / mL, 1 mg / mL, and 5 mg / mL.

[0095] The percolation extracts of 70% ethanol at various concentrations and the positive control 50 mM metformin were respectively dissolved in NGM liquid medium (containing FUDR). The blank control group only contained this NGM liquid medium, and then they were aliquoted into 24-well plates. Under the condition of 20 °C, the wild-type nematode N2 was synchronized and cultured for 3 days. After developing into adults, they were picked into the above-mentioned 24-well plates for culture. Starting from the 10th day, dead nematodes were removed, and then the number of dead, lost, and surviving nematodes was counted every 1-2 days. The survival rate of nematodes was statistically analyzed until all nematodes died. The determination of all samples was repeated three times; data analysis was performed using GraphPad, and statistical analysis of the data was carried out using SPSS 26.0 software. One-way ANOVA was used for data comparison among multiple groups, and the data was expressed as mean ± standard deviation. The results are shown in Figure 3 .

[0096] The results showed that the extract group with a drug concentration of 0.2 mg / mL and the positive control group (50 mM metformin) both increased the lifespan of nematodes after culturing them (p < 0.01 and P < 0.001 respectively), as shown in Figure 3 . Therefore, it was proved that the 70% ethanol percolation extract could extend the average lifespan of Caenorhabditis elegans to a certain extent.

[0097] Effect of Test Example 3 on Learning and Memory Impairment in Alzheimer's Disease Model Mice

[0098] (I) Experimental Animals and Grouping

[0099] SAMP8 is a rapidly aging dementia model mouse, which was developed by Professor Toshio Takeda of Japan through 20 years of modern inbred breeding. Its lifespan is about 12 months, and it generally shows characteristics of accelerated aging at 4-6 months. It has a uniform genetic background and stable aging pathological characteristics. Its rapid aging occurs naturally and is very similar to the progressive occurrence of clinical Alzheimer's disease. In particular, it can form Aβ deposits in the brain, and the expression level increases with age. SAMR1 mice are anti-rapid aging mice with normal life processes. Both were purchased from the Peking University Health Science Center.

[0100] In this experiment, 50 6-month-old SAMP8 mice were randomly divided into a model group, a positive control group (donepezil), a high-dose group of the traditional Chinese medicine composition prepared in Example 1, and a low-dose group of the traditional Chinese medicine composition prepared in Example 1, with 10 mice in each group. Another 10 SAMR1 mice were used as a normal control group. The SAMP8 mice and SAMR1 mice were both purchased from the Peking University Health Science Center, and the animal license number is SCXK(Beijing)2021-0013).

[0101] The traditional Chinese medicine composition prepared in Example 1 and donepezil were respectively mixed with sodium carboxymethyl cellulose solution, with the high-dose group being 234 mg / kg, the low-dose group being 117 mg / kg, and the positive control group being 1.30 mg / kg; the normal control group and the model group were given the same volume of sodium carboxymethyl cellulose solution.

[0102] (II) Mouse Learning and Memory Behavior Test

[0103] All mice in the above groups were administered the drug via gavage (ig), with a dosage of 0.1 mL / 10 g, once daily for 60 consecutive days. Learning and memory performance were then assessed.

[0104] The Morris water maze test was used. The water maze was a circular pool with a diameter of 152 cm, a height of 60 cm, and a depth of 30 cm, with the water temperature maintained at 23 ± 1℃. The pool was divided into four quadrants. A circular platform with a diameter of 11 cm and a height of 29 cm (i.e., the platform was 1 cm below the water surface, called the hidden platform) was placed in the center of quadrant 3. A small camera was installed 2 m above the maze and connected to a video recorder and monitor in another room to record the mice's swimming trajectory and swimming time. The results were tested from day 2 to day 6 of training, for a total of 5 days. Each day was divided into morning and afternoon sessions, with each mouse trained 4 times in each session. The entry point for the mice was the midpoint of the pool wall in each quadrant. The mice were placed in the pool facing the pool wall from the four entry points. The time required for the mice to successfully enter the platform within 60 seconds (the time it takes for the mice to find the platform and stay on it for 5 seconds is considered a successful entry) was measured (i.e., the escape latency period). If the mouse fails to successfully enter the platform within 60 seconds, the experimenter guides it onto the platform and allows it to remain there for 10 seconds, recording the escape latency as 60 seconds.

[0105] The results are as follows Figure 4-6 As shown, compared with SAMR1 mice, the platform escape time of SAMP8 mice in the model group was significantly increased (P < 0.001). Compared with the model group, during the water maze test, the time required for mice treated with both high and low doses of the herbal composition prepared in this invention and the positive control group was significantly reduced (P < 0.001). Compared with SAMR1 mice, the number of crossovers in the model group was significantly reduced (P < 0.05), while compared with the model group, the number of crossovers in the high-dose herbal composition prepared in this invention and the positive control group was significantly increased (P < 0.01, P < 0.001). In summary, the intervention of the positive control group and the high-dose herbal composition prepared in this invention (70% ethanol-infused deer extract) significantly enhanced the spatial memory and learning memory behaviors of mice.

[0106] (III) Detection of Aβ1-40 and Aβ1-42 content in mouse cerebrospinal fluid

[0107] After the water maze test to assess animal memory and behavior, mice were anesthetized with ether, placed face down on a triangular rod, and secured with tape to hang at a 45° angle to their body. A 4mm incision was made along the midline from the head to the occipital protuberance, and then 1mm to the shoulder. A sudden dissection was performed, and the muscles from the occipital bone to the atlantoaxial joint were removed to expose the white dura mater. A 2mm puncture was made between the occipital bone and the atlantoaxial joint with a needle, and cerebrospinal fluid was aspirated using a micropipette. The levels of Aβ1-40 and Aβ1-42 in the cerebrospinal fluid were measured using an ELISA kit (purchased from Jianglai Biotechnology Co., Ltd.) according to the instructions.

[0108] The results are as follows Figure 7 As shown, compared with SAMR1 mice, the levels of Aβ1-40 and Aβ1-42 in the cerebrospinal fluid of SAMP8 mice were significantly higher. Both doses of the traditional Chinese medicine composition prepared according to this invention and the positive control drug donepezil altered this phenomenon. Administration of both low and high doses of the traditional Chinese medicine composition prepared according to this invention and donepezil reduced the levels of Aβ1-40 (P<0.01, P<0.0001, P<0.0001) and Aβ1-42 (P<0.05, P<0.0001, P<0.0001).

[0109] (iv) HE staining to detect pathological morphology in the CA1 and CA3 regions of the mouse hippocampus

[0110] After the water maze test to assess animal memory and behavior, mice were euthanized by cervical dislocation, the skull cap was removed, and the entire brain tissue was rapidly extracted under low-temperature sterile conditions. The tissue was fixed with 4% paraformaldehyde, rinsed with tap water, dehydrated and cleared, then embedded in paraffin and stained with hematoxylin and eosin (HE). Sections were dehydrated in ascending alcohol solution, cleared with xylene, and then sealed. Pathological changes in the CA1 and CA3 regions of the hippocampus were examined using a Carl Zeiss Axio.Scope A1 optical microscope, and scanned using a 3D HHI STEC HPannoramic MIDI scanner. Image analysis was performed using Halo software.

[0111] The results are as follows Figure 8 As shown, significant cytopathological changes were observed in neurons of the CA1 and CA3 regions of the hippocampus in the model group mice. Neurons were disordered, thinned, and significantly fewer in number, with obvious cell shrinkage and vacuolation. Compared with the model group, the hippocampal pathological morphology of mice in each treatment group showed varying degrees of improvement, with a significant increase in the number of neurons, a more orderly arrangement, an increase in the number of cell layers, and a morphological structure approaching that of normal cells. Shrinkage and vacuolation were significantly alleviated. Compared with the model, the high-dose group and the Donepezil-positive group showed only minor cytopathological changes in the CA1 and CA3 regions of the hippocampus. This indicates that the herbal composition of this invention has a good effect on improving the pathological morphological damage of the hippocampus in rapidly aging model mice.

[0112] (V) RT-PCR detection of aging gene expression in the hippocampus and cortex of the brain

[0113] SIRT1, known as a "longevity" protein and a marker of cellular rejuvenation, is closely related to the aging process in various organisms. Increasing SIRT1 expression can extend the lifespan of many organisms, including yeast, flies, worms, and mammals. However, the protein and transcriptional levels of SIRT1 in vivo decline with age. Overexpression of SIRT1 in the mouse brain can significantly extend the lifespan of mice. p53 is a transcription factor that plays a crucial role in cellular stress responses. Its activation upon DNA damage leads to cell growth arrest, guiding cellular and organismal aging. p21 is a downstream gene of p53, and its expression is regulated by p53. p21 can inhibit the activity of cyclin-dependent kinases (CDKs), arresting the cell cycle. P53 and p21 are commonly used biomarkers of aging.

[0114] Hippocampal and cortical samples were collected from mouse brains. RNA was extracted using the Trizol method, and cDNA samples were prepared using a reverse transcription kit (ABI-invitrogen, catalog number 11752050). Following the steps listed in the SYBR real-time quantitative PCR kit (ABI-invitrogen, catalog number 4472920), the gene transcription levels of aging markers P53 and P21 and longevity regulator SIRT1 were measured using a real-time quantitative PCR instrument (Applied Biosystems (USA), StepOne Software). GAPDH was used as an internal control, and the relative gene expression levels of each group relative to the control group were calculated.

[0115] The primer sequences used in the experiment included the F sequence SEQ ID NO:1 and R sequence SEQ ID NO:2 of Actin; the F sequence SEQ ID NO:3 and R sequence SEQ ID NO:4 of SIRT1; the F sequence SEQ ID NO:5 and R sequence SEQ ID NO:6 of p53; and the F sequence SEQ ID NO:7 and R sequence SEQ ID NO:8 of p21. See Table 3 for details. The RT-PCR reaction system consisted of: 2 μL cDNA, 10 μL PCR mix, 1 μL primer F, 1 μL primer R, and 6 μL ddH2O. The RT-PCR reaction program was as follows: pre-denaturation, 95℃, 5 min, 1 cycle; denaturation, 95℃, 10 s; annealing, 58℃, 20 s; extension, 72℃, 20 s. Denaturation, annealing, and extension were repeated 40 times. After the amplification reaction, melting curve analysis was performed: 95℃, 15 s; 60℃, 60 s; 95℃, 15 s.

[0116] Table 3 RT-PCR Primer Sequences

[0117]

[0118] like Figure 9-10 As shown, compared with SAMR1 naturally aging mice, SAMP8 rapidly aging mice showed significantly decreased expression of SIRT1 in the hippocampus and cortex (P<0.0001, 0.0001), and significantly upregulated expression of aging markers p53 gene (P<0.0001, 0.0001) and p21 gene (P<0.0001, 0.0001).

[0119] The low- and high-dose groups of the herbal composition of this invention significantly activated the expression of SIRT1 in the hippocampus of SAMP8 rapidly aging mice (P<0.05, 0.001) and the expression of SIRT1 in the cortex (P<0.05, 0.0001). After administration of the low- and high-dose groups of the herbal composition of this invention, the expression of the mouse hippocampal aging markers p53 and p21 genes was significantly inhibited (P<0.01, 0.0001; P<0.001, 0.0001), and the expression of the mouse cortical aging markers p53 and p21 genes was significantly inhibited (P<0.0001, 0.0001; P<0.05, 0.0001). In summary, the herbal composition of this invention has an inhibitory effect on the aging of SAMP8 rapidly aging mice, and by activating SIRT1, it can prevent and treat Alzheimer's disease by combating brain aging.

[0120] (vi) Blood index testing

[0121] This study measured alanine aminotransferase (ALT) and aspartate aminotransferase (AST), which are related to liver function; triglycerides (TG) and cholesterol (CHOL), which are related to blood lipids; creatinine (CREA) and blood urea nitrogen (BUN), which are related to kidney function; and lactate dehydrogenase (LDH) and creatine kinase (CK), which are related to the heart.

[0122] Eight blood biochemical indicators of mice were measured according to the operating procedures of the XR220PIus+ fully automated biochemical analyzer and the instructions for use of the test kit (purchased from Yuzecheng Biotechnology).

[0123] The results are as follows Figure 11As shown, compared with SAMR1 mice, the levels of AST, ALT, TG, CHOL, BUN, CREA, LDH, and CK in the blood of the mice were significantly higher. However, the levels of these indicators decreased after administration of both low and high doses of the traditional Chinese medicine composition prepared in this invention. Specifically, the model group showed a significant increase in AST compared to the control group (P<0.001), and administration of both low and high doses of the traditional Chinese medicine composition prepared in this invention reduced AST levels (P<0.05, P<0.05). The model group showed a significant increase in ALT compared to the control group (P<0.0001), and administration of high doses of the traditional Chinese medicine composition prepared in this invention reduced ALT levels (P<0.01). The model group showed a significant increase in TG compared to the control group (P<0.001), and administration of high doses of the traditional Chinese medicine composition prepared in this invention reduced TG levels (P<0.01). The model group showed a significant increase in CHOL compared to the control group (P<0.0001), and administration of both low and high doses of the traditional Chinese medicine composition prepared in this invention reduced CHOL levels (P<0.05, P<0.0001). The model group showed a significant increase in BUN compared to the control group (P<0.001), and both low and high doses of the traditional Chinese medicine composition prepared in this invention reduced BUN levels (P<0.05, P<0.05). The model group also showed a significant increase in CREA levels compared to the control group (P<0.0001), and both low and high doses of the traditional Chinese medicine composition prepared in this invention reduced CREA levels (P<0.01, P<0.0001). The model group also showed a significant increase in LDH levels compared to the control group (P<0.0001), and both low and high doses of the traditional Chinese medicine composition prepared in this invention reduced LDH levels (P<0.05, P<0.0001). Finally, the model group showed a significant increase in CK levels compared to the control group (P<0.0001), and both low and high doses of the traditional Chinese medicine composition prepared in this invention reduced CK levels (P<0.0001, P<0.0001).

[0124] Compared with SAMR1 mice, the levels of AST, ALT, TG, CHOL, BUN, CREA, LDH, and CK in the blood of these mice were significantly higher. However, the levels of these indicators were reduced after taking low and high doses of the traditional Chinese medicine composition prepared by this invention. The effect of taking high doses of the traditional Chinese medicine composition prepared by this invention was more significant.

[0125] (vii) Spleen data detection

[0126] Mouse spleen tissue was collected and placed in a petri dish, then washed three times with PBS buffer. The tissue was cut into 1-2 mm pieces and washed three more times with PBS buffer. Five volumes of 0.25% trypsin digestion solution were added, and the mixture was digested at 37°C for 40 min, gently agitated every 5 min. The reaction was terminated by adding 2-5 mL of serum-containing culture medium. The mixture was allowed to stand for 3 min, and the supernatant was collected. The supernatant was filtered twice through a 200-mesh filter and collected. The filtrate was centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. The filtrate was then analyzed using a FlowCytometre FCM (BeamCyte-1026) instrument.

[0127] The results showed that, compared with the normal group, the CD4+ / CD8+ ratio in the spleen of SAMP8 mice in the model group was significantly higher than that in the normal group (P<0.001). The CD4+ / CD8+ ratio in the spleen of mice in the low- and high-dose extract groups was also significantly lower than that in the model group (P<0.001, P<0.001), and comparable to that in the normal group, indicating that the extract has immunomodulatory effects (e.g., ...). Figure 12 (As shown).

[0128] As can be seen from the above embodiments and experimental examples, the present invention provides a traditional Chinese medicine composition for the prevention and / or treatment of Alzheimer's disease. This traditional Chinese medicine composition has a protective effect on Aβ1-42-induced hippocampal immortalized cells HT22 cells, enhances the anti-aging effect of HT22 cells, and can also significantly improve dementia symptoms, learning and memory impairment in Alzheimer's disease model mice, prolong the lifespan of N2 wild-type Caenorhabditis elegans, and can also significantly inhibit the production and deposition of β-amyloid protein, a pathological product closely related to the occurrence of Alzheimer's disease; it can also significantly activate SIRT1, inhibit the expression of p53 and p21 genes, thereby inhibiting brain aging; at the same time, it can also effectively achieve immunomodulatory effects, thus achieving the purpose of preventing and / or treating Alzheimer's disease.

[0129] 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 principle 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 traditional Chinese medicine composition for the prevention and / or treatment of Alzheimer's disease, characterized in that, Made from the following ingredients in parts by weight: 1-4 parts red ginseng and 1-6 parts processed Polygonum multiflorum; The preparation method of the traditional Chinese medicine composition includes the following steps: (1) Crush the red ginseng and the prepared Polygonum multiflorum into powder, sieve them, and then take the sieve material for later use; (2) After mixing the sieved material, soak it, then percolate and dry it to obtain the Chinese herbal medicine composition; The soaking in step (2) is carried out using an ethanol aqueous solution with a volume fraction of 60-80%, and the mass-volume ratio of the sieve residue to the ethanol aqueous solution after mixing is 1g:(1-5)mL. The soaking time is 20-30 hours; After soaking in step (2), an ethanol aqueous solution is directly added for percolation. The volume fraction of the ethanol aqueous solution is 60-80%, and the mass-volume ratio of the mixed sieve material to the added ethanol aqueous solution is 1g:(15-25)mL. The sieve used in step (1) has a mesh size of 8-12.

2. The traditional Chinese medicine composition for treating Alzheimer's disease according to claim 1, characterized in that, It is made from the following ingredients in parts by weight: 1.5-3 parts red ginseng and 2-5 parts processed Polygonum multiflorum.

3. The use of the traditional Chinese medicine composition according to claim 1 in the preparation of drugs for the prevention and / or treatment of Alzheimer's disease.

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