Traditional Chinese medicine composition for treating Alzheimer's disease and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a traditional Chinese medicine composition for treating Alzheimer's disease and application thereof.The traditional Chinese medicine composition is prepared from radix puerariae, radix astragali, radix rehmanniae, rhizoma dioscoreae, corn stigma, radix trichosanthis and kadsura longepedunculata. After the traditional Chinese medicine composition is applied to Abeta transgenic nematodes CL4176 and CL2355, compared with a Control group, different doses of quenching thirst decoction formula can obviously prolong half paralysis time of the CL4176 nematodes and inhibit CL2355 nematode paralysis induced by exogenous 5-HT, so that the quenching thirst decoction formula slows down the paralysis process of the nematodes caused by Abeta, reduces the hypersensitivity of the exogenous 5-HT, and can be used for treating the Abeta transgenic nematodes. It is shown that the Xiaoke decoction formula has an obvious treatment effect on the phenotype of nematode paralysis caused by A beta.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a traditional Chinese medicine composition for treating Alzheimer's disease and application thereof. Background Art

[0002] Alzheimer's disease (AD) is a progressive neurodegenerative disorder and the most common form of dementia (accounting for 60%-80%). It primarily affects memory, thinking, behavior, and daily functioning, ultimately leading to the loss of independence. Early symptoms include recent memory loss, difficulty finding words, and disorientation in time and space. In the middle stages, personality changes, irritability, depression, decreased executive function, and the need for assistance with daily living occur. In the later stages, speech loss, motor deficits, and swallowing difficulties can occur, ultimately requiring full-time care. Current research has identified AD's pathogenesis as including β-amyloid plaques, neurofibrillary tangles, and neuronal death, and drug research is targeting these mechanisms. Developed therapeutic agents primarily include symptom-modifying drugs and disease-modifying therapies (DMTs), such as cholinesterase inhibitors (ChEIs), NMDA receptor antagonists, anti-β-amyloid monoclonal antibodies, and tau-targeted drugs. However, a cure remains elusive.

[0003] With the in-depth study of traditional Chinese medicine, it has been found that some Chinese herbal ingredients show certain potential in improving cognitive function and slowing disease progression. Many researchers have already invested in related research. For example, invention patent CN114306543B discloses a Chinese herbal composition for treating Alzheimer's disease, its preparation method, and its use. The Chinese herbal composition can improve learning ability, expression levels of β-amyloid precursor protein, interleukin-1β expression levels, and expression levels of acetylcholine metabolizing enzymes in Kunming mice with β-amyloid protein-induced Alzheimer's disease, while having no effect on the mice's major organ indices and body weight, indicating that the Chinese herbal composition is effective in treating Alzheimer's disease and has no side effects. Invention patent CN105311279B discloses the use of a Chinese herbal composition in the preparation of a drug for the prevention or treatment of Alzheimer's disease. The disclosed Chinese herbal composition has a significant inhibitory effect on the paralysis phenotype in an animal pathological model of Alzheimer's disease, indicating that the disclosed Chinese herbal composition has the potential to prevent or treat Alzheimer's disease and can be used in the preparation of a drug for the prevention or treatment of Alzheimer's disease.

[0004] Xiaoke Pills, a compound Chinese and Western medicine preparation, are a tonic that nourishes the kidneys and yin, invigorates qi and promotes fluid production. They are primarily used to treat diabetes mellitus caused by qi and yin deficiency, characterized by polydipsia, polyuria, polyphagia, weight loss, fatigue, poor sleep, and low back pain; they are also used for type 2 diabetes patients with these symptoms. The original formula consists of 265g of Pueraria root, 53g of Astragalus root, 159g of Rehmannia root, 26.5g of Chinese yam, 265g of corn silk, 265g of Radix Trichosanthis, 53g of Schisandra chinensis, and 0.25g of glibenclamide. During their research, the inventors unexpectedly discovered that Xiaoke Pills are effective in treating Alzheimer's disease, and that removing glibenclamide from the pills significantly enhances the therapeutic effect, achieving unexpected benefits. Summary of the Invention

[0005] The primary purpose of the present invention is to provide a traditional Chinese medicine composition for treating Alzheimer's disease, which comprises kudzu root, astragalus root, rehmannia root, yam, corn silk, trichosanthes root and schisandra chinensis.

[0006] Preferably, the traditional Chinese medicine composition comprises the following raw materials in parts by weight: 10-40 parts of Pueraria root, 2-8 parts of Astragalus, 6-24 parts of Rehmannia root, 1-4 parts of Chinese yam, 10-40 parts of corn silk, 10-40 parts of Radix Trichosanthis and 2-8 parts of Schisandrae Chinensis.

[0007] Preferably, the traditional Chinese medicine composition is composed of the following raw materials in parts by weight: 10-40 parts of Pueraria root, 2-8 parts of Astragalus, 6-24 parts of Rehmannia root, 1-4 parts of Chinese yam, 10-40 parts of corn silk, 10-40 parts of Radix Trichosanthis and 2-8 parts of Schisandrae Chinensis.

[0008] Preferably, the traditional Chinese medicine composition is composed of the following raw materials in parts by weight: 10 parts of Pueraria root, 2 parts of Astragalus, 6 parts of Rehmannia root, 1 part of Chinese yam, 10 parts of corn silk, 10 parts of Radix Trichosanthis and 2 parts of Schisandrae Chinensis.

[0009] The second object of the present invention is to provide a preparation method of the traditional Chinese medicine composition, comprising the following steps: weighing the medicine in proportion, adding 10 times distilled water, soaking, boiling and then simmering over low heat, filtering with gauze and collecting the filter residue, adding 5 times distilled water for decocting, boiling and then simmering over low heat, decocting the filter residue twice, combining the filtrates three times and then centrifuging or filtering, collecting the filtrates and concentrating, and freeze-drying when the extract is concentrated to a density of 1.25 to obtain the product.

[0010] The third object of the present invention is to provide the use of the traditional Chinese medicine composition in the preparation of drugs for treating Alzheimer's disease.

[0011] The present invention has the beneficial effects of: the present invention relates to the field of biotechnology, and specifically to a traditional Chinese medicine composition for treating Alzheimer's disease and its use. The traditional Chinese medicine composition comprises kudzu root, astragalus root, rehmannia root, yam, corn silk, trichosanthes root, and schisandra chinensis. The traditional Chinese medicine composition is improved from Xiaoke Pills. When the traditional Chinese medicine composition is applied to Aβ-transgenic nematodes CL4176 and CL2355, different doses of Xiaoke Decoction significantly prolong the half-life of paralysis in CL4176 nematodes compared to the control group, and inhibit exogenous 5-HT-induced paralysis in CL2355 nematodes. This indicates that Xiaoke Decoction slows the progression of Aβ-induced paralysis in nematodes and reduces exogenous 5-HT hypersensitivity, indicating that Xiaoke Decoction has a significant therapeutic effect on the paralysis phenotype of nematodes caused by Aβ. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0013] Figure 1 (A) Effects of 5-HT hypersensitivity in C. elegans; (B) Paralysis rate and (C) PT50 in C. elegans.

[0014] Note: Concentration gradient: crude drug amount L = 0.5 mg / mL; M = 5 mg / mL; H = 50 mg / mL; *P < 0.05, **P < 0.01, ***P < 0.001. DETAILED DESCRIPTION

[0015] The following describes embodiments of the present invention in detail. Examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended only to explain the present invention and are not to be construed as limiting the present invention.

[0016] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. All reagents or instruments used without specifying the manufacturer are commercially available conventional products.

[0017] The original recipe of Xiaoke Decoction consists of eight medicinal herbs: Pueraria root: 265g, Astragalus: 53g, Rehmannia root: 159g, Chinese yam: 26.5g, Corn silk: 265g, Radix Trichosanthis: 265g, Schisandra chinensis: 53g, and Glibenclamide 0.25g.

[0018] Preparation method: Add water to the above eight ingredients, Pueraria root, Rehmannia root, Corn silk, and Radix Trichosanthis to boil for 5 hours, filter, and concentrate the filtrate to an appropriate amount; grind Astragalus, Schisandra chinensis, and Chinese yam into fine powder, mix well with the above-mentioned concentrated liquid, dry, grind, sieve, and mix well, make pills with the remaining concentrated liquid, dry, add glibenclamide, and coat with a dextrin solution of black iron oxide and talcum powder to make 1000 pills.

[0019] Dosage and Administration: Oral. Take 5-10 pills at a time, 2-3 times a day. Take with warm water before meals. Or as directed by a physician.

[0020] According to the above method and usage and dosage, the dosage of the drug is calculated, and the obtained Chinese medicine composition and its dosage are as follows: Example 1, Chinese medicine composition 1

[0021] Chinese medicine composition 1: Pueraria root: 5.3g, Astragalus root: 1.06g, Rehmannia root: 3.18g, Chinese yam: 0.53g, Corn silk: 5.3g, Radix Trichosanthis: 5.3g, Schisandra chinensis: 1.06g;

[0022] Weigh the medicinal components of the compound and add 10 times the amount of distilled water according to the dosage. Soak for 30 minutes. Bring to a boil, then simmer over low heat for 30 minutes. Filter through gauze, collect the residue, and add 5 times the amount of distilled water for decoction. Bring to a boil, then simmer over low heat for 30 minutes. Decoction the residue twice, combine the filtrates, and centrifuge or filter. Collect the filtrates and concentrate them. Once the extract has a density of 1.25, freeze-dry it. Prepare the sample, calculate the yield of freeze-dried powder of Xiaoke Decoction, and set aside.

[0023] Example 2, Chinese medicine composition 2

[0024] Chinese medicine composition 2: Pueraria root: 10.6g, Astragalus root: 4.24g, Rehmannia root: 12.72g, Chinese yam: 1.06g, Corn silk: 21.2g, Radix Trichosanthis: 21.2g, Schisandra chinensis: 4.24g;

[0025] Weigh the medicinal components of the compound and add 10 times the amount of distilled water according to the dosage. Soak for 30 minutes. Bring to a boil, then simmer over low heat for 30 minutes. Filter through gauze, collect the residue, and add 5 times the amount of distilled water for decoction. Bring to a boil, then simmer over low heat for 30 minutes. Decoction the residue twice, combine the filtrates, and centrifuge or filter. Collect the filtrates and concentrate them. Once the extract has a density of 1.25, freeze-dry it. Prepare the sample, calculate the yield of freeze-dried powder of Xiaoke Decoction, and set aside.

[0026] Example 3, Chinese medicine composition 3

[0027] Chinese medicine composition 3: Pueraria root: 21.2g, Astragalus root: 2.12g, Rehmannia root: 6.36g, Chinese yam: 2.12g, Corn silk: 10.6g, Radix Trichosanthis: 21.2g, Schisandra chinensis: 2.12g;

[0028] Weigh the medicinal components of the compound and add 10 times the amount of distilled water according to the dosage. Soak for 30 minutes. Bring to a boil, then simmer over low heat for 30 minutes. Filter through gauze, collect the residue, and add 5 times the amount of distilled water for decoction. Bring to a boil, then simmer over low heat for 30 minutes. Decoction the residue twice, combine the filtrates, and centrifuge or filter. Collect the filtrates and concentrate them. Once the extract has a density of 1.25, freeze-dry it. Prepare the sample, calculate the yield of freeze-dried powder of Xiaoke Decoction, and set aside.

[0029] After preliminary experiments, it was found that Chinese medicine composition 1 had better effects, the lowest toxicity, and the best overall effects. Therefore, Chinese medicine composition 1 described in Example 1 was used in the following examples.

[0030] Example 4: Application of Chinese medicine composition in treating Alzheimer's disease

[0031] 1. Materials and Reagents

[0032] 1.1 Nematodes

[0033] Transgenic AD model Caenorhabditis elegans strains

[0034] CL2355(dvIs50[pCL45(snb-1::Abeta1–42::3UTR(long)+mtl-2::GFP]I);

[0035] CL2122(dvIs15[(pPD30.38)unc-54(vector)+(pCL26)mtl-2::GFP]);

[0036] CL4176(dvIs27[myo-3p::Abeta1–42::let-8513′UTR)+rol-6(su1006)]X);

[0037] All were purchased from the Caenorhabditis elegans Genetics Center (CGC).

[0038] 1.2 Reagents

[0039] 5-Hydroxytryptamine (5-HT): Product No. S31021, purchased from Shanghai Yuanye Biotechnology Co., Ltd., with a working solution concentration of 5 mg / mL, prepared and used immediately.

[0040] 2. Methods

[0041] 2.1 Preparation of drugs

[0042] The Chinese medicine composition described in Example 1 was prepared according to the following method:

[0043] Weigh the medicinal components of the compound and add 10 times the amount of distilled water according to the dosage. Soak for 30 minutes. Bring to a boil, then simmer over low heat for 30 minutes. Filter through gauze, collect the residue, and add 5 times the amount of distilled water for decoction. Bring to a boil, then simmer over low heat for 30 minutes. Decoction the residue twice, combine the filtrates, and centrifuge or filter. Collect the filtrates and concentrate them. Once the extract has a density of 1.25, freeze-dry it. Prepare the sample, calculate the yield of freeze-dried powder of Xiaoke Decoction, and set aside.

[0044] 2.2 Administration and Dosage Concentration

[0045] Prepare drug-containing NGM culture medium. Preparation method: After wet heat sterilization, cool the NGM to approximately 70°C. Add the appropriately diluted traditional Chinese medicine formula in a clean bench, mix thoroughly, and pour into glass culture dishes. The final concentrations of the drug-containing NGM plates are as follows: low-dose group (L: 0.5 mg / mL of crude drug), medium-dose group (M: 5 mg / mL of crude drug), and high-dose group (H: 50 mg / mL of crude drug). The original formula dosage is calculated as 20 pills per day. After the NGM is allowed to cool and solidify, add 450 μL of OP50 to each dish as nematode food and spread evenly. After the OP50 is air-dried, incubate it in a 37°C incubator overnight. Remove it and store it in a constant-temperature incubator below 15°C until needed.

[0046] 2.3 Cultivation and synchronization of Caenorhabditis elegans

[0047] Use a pick to streak OP50 culture onto a solid LB plate. Incubate at 37°C for 12-14 hours. Pick a single OP50 colony and transfer it to liquid LB medium. Incubate at 37°C on a shaker for 12-14 hours. Store at 4°C until ready to use. After solidification, evenly coat NGM medium with the OP50 culture. Incubate at 37°C for 12-14 hours. Store at 15°C until ready to use.

[0048] Remove the frozen nematodes from the refrigerator and quickly thaw them in a 37°C water bath. Pour the nematodes into a new centrifuge tube, rinse with M9 to remove the cryoprotectant solution, and let it rest for 1 hour. Remove the pellet from the bottom of the centrifuge tube and add it to a lawn of NGM culture medium seeded with OP50 E. coli. Incubate at 16°C until ready to use.

[0049] Synchronize nematodes using the lysis method. Collect nematodes cultured in NGM medium using M9 buffer, add lysis buffer, and vortex for 5-7 minutes. Once the adult worms are completely lysed, centrifuge at 5000 rpm for 1 minute, discard the supernatant, and wash twice with M9 buffer to obtain eggs. Incubate at an appropriate temperature based on the culture conditions of the nematode strain to obtain synchronized nematodes.

[0050] 2.4 5-HT sensitivity assay in CL2355 and CL2122 nematodes

[0051] The groups were Blank group (CL2122 type nematodes), Control group (CL2355 type nematodes), Xiaoke Decoction original formula (low, medium and high), and Xiaoke Decoction reduced formula (low, medium and high).

[0052] CL2355 nematodes are 5-HT hyposensitive and develop 5-HT sensitivity after temperature induction. Exogenous 5-HT paralyzes them. CL2122 nematodes do not express Aβ and are often used as a control for CL2355 nematodes. Synchronized CL2355 and CL2122 strains of C. elegans were obtained using a lysis method. The resulting eggs were placed in M9 buffer and incubated at 15°C for 24-48 hours until they reached the L1 stage. Approximately 800 nematodes were transferred to each drug-containing NGM dish and incubated in a 15°C incubator for 72 hours. The worms were then transferred to a 25°C incubator and incubated for an additional 38 hours. The worms were harvested with M9 buffer and centrifuged at 3000 rpm for 1 minute. The supernatant was discarded and repeated three times. Prepare a 5 mg / mL 5-HT solution in M9. Add nematodes (50 μl) and 5-HT solution (200-300 μl) to a 96-well plate. Wait 5 minutes and count under a microscope. Nematodes are considered paralyzed if they remain motionless or only move their heads for 5 seconds. Calculate the percentage of paralyzed nematodes in each well. Repeat three times for each group.

[0053] 2.5 Paralysis experiment of CL4176 nematodes

[0054] Groups: Control group, original Xiaoke Decoction (low, medium, high), and reduced Xiaoke Decoction (low, medium, high).

[0055] C. elegans strain CL4176 was synchronized. The resulting eggs were placed in M9 buffer and incubated at 15°C for 24-48 hours to mature into L1 larvae. Approximately 60 worms were transferred to each drug-containing NGM dish and incubated in a 15°C incubator for 84 hours. The eggs were then transferred to a 25°C incubator for further incubation. Paralyzed worms were counted in all groups starting after paralysis-like pathological signs appeared in the control group. Counts were stopped every two hours until paralyzed worms in the control group accounted for at least 80% of the total number of worms tested. Three replicates were performed for each group.

[0056] 3. Experimental Results

[0057] To investigate whether Xiaoke Decoction can alleviate the toxicity of Aβ protein accumulation in neurons, we used the CL2355 strain of AD nematodes to conduct 5-HT paralysis experiments ( Figure 1A). The CL2122 strain of nematodes is a blank transgenic control (blank group) for the CL2355 strain. The CL2122 strain of nematodes does not express Aβ protein in its neurons. The control group consists of CL2355 nematodes transfected with the Aβ gene. The results showed that, compared with the control group, different doses of the reduced Xiaoke Decoction significantly alleviated exogenous 5-HT-induced paralysis in CL2355 nematodes, indicating that the reduced Xiaoke Decoction reduced CL2355 nematodes' hypersensitivity to exogenous 5-HT. Treatment with the original Xiaoke Decoction also reduced CL2355 nematodes' hypersensitivity to exogenous 5-HT, with the medium-dose group showing the most pronounced effect. In summary, these data demonstrate that Xiaoke Decoction can effectively alleviate AD-like phenotypes in C. elegans.

[0058] Next, paralysis experiments were conducted using the transgenic Caenorhabditis elegans CL4176 strain, which had human Aβ transfected downstream of the myo-3p promoter. 1-42 gene, Aβ when cultured at 15℃ 1-42 The gene will not be expressed. When transferred to 25℃ for culture, Aβ 1-42 Gene expression is high, Aβ 1-42 Protein accumulates in large quantities in the muscles of nematodes, which can lead to muscle cell dysfunction over time and the development of AD-like pathological features of paralysis phenotype in nematodes. Nematode paralysis experiments showed that different concentrations of Xiaoke Decoction (original and reduced) can effectively slow down nematode paralysis and significantly prolong the half-life of paralysis ( Figure 1 BC), indicating that Xiaoke decoction has a significant therapeutic effect on the paralysis phenotype of nematodes caused by Aβ deposition.

[0059] In summary, the present invention provides a traditional Chinese medicine composition for treating Alzheimer's disease. This composition is derived from Xiaoke Pills. When applied to Aβ-transgenic nematodes CL4176 and CL2355, the composition significantly prolonged the half-life of paralysis in CL4176 nematodes compared to the control group at different doses, and inhibited exogenous 5-HT-induced paralysis in CL2355 nematodes. This indicates that Xiaoke Pills mitigates the progression of Aβ-induced paralysis in nematodes and reduces exogenous 5-HT hypersensitivity, demonstrating that Xiaoke Pills have a significant therapeutic effect on the Aβ-induced paralysis phenotype in nematodes.

Claims

1. A Chinese medicine composition for treating Alzheimer's disease, characterized in that: The traditional Chinese medicine composition comprises kudzu root, astragalus root, rehmannia root, yam, corn silk, trichosanthes root and schisandra chinensis.

2. The Chinese medicine composition according to claim 1, wherein The traditional Chinese medicine composition comprises the following raw materials in parts by weight: 10-40 parts of kudzu root, 2-8 parts of astragalus, 6-24 parts of rehmannia, 1-4 parts of yam, 10-40 parts of corn silk, 10-40 parts of trichosanthes and 2-8 parts of schisandra chinensis.

3. The Chinese medicine composition according to claim 2, wherein The traditional Chinese medicine composition is composed of the following raw materials in parts by weight: 10-40 parts of kudzu root, 2-8 parts of astragalus, 6-24 parts of rehmannia, 1-4 parts of yam, 10-40 parts of corn silk, 10-40 parts of trichosanthes and 2-8 parts of schisandra chinensis.

4. The Chinese medicine composition according to claim 3, wherein The traditional Chinese medicine composition is composed of the following raw materials in parts by weight: 10 parts of kudzu root, 2 parts of astragalus, 6 parts of rehmannia, 1 part of yam, 10 parts of corn silk, 10 parts of trichosanthes and 2 parts of schisandra chinensis.

5. The method for preparing the Chinese medicine composition according to any one of claims 1 to 4, wherein: The method comprises the following steps: weighing medicines in proportion, adding 10 times distilled water, soaking, boiling and then simmering over low heat, filtering with gauze and collecting filter residues, adding 5 times distilled water for decocting, boiling and then simmering over low heat, decocting the filter residues twice, combining the filtrates three times and then centrifuging or filtering, collecting the filtrates and concentrating, and freeze-drying when the filtrates are concentrated to an extract with a density of 1.25 to obtain the extract.

6. Use of the Chinese medicine composition according to any one of claims 1 to 4 in the preparation of a drug for treating Alzheimer's disease.

Images