Application of tranquilizing composition in preparation of medicine for preventing or treating Alzheimer disease

Abstract

The invention belongs to the field of traditional Chinese medicine, and particularly relates to application of a tranquilizing composition in preparation of a medicine for preventing or treating Alzheimer's disease, the tranquilizing composition has a remarkable treatment effect on CL4176 nematodes, and has a remarkable inhibition effect on paralysis phenotypes caused by intramuscular A beta overexpression of the CL4176 nematodes. The nerve soothing composition can obviously delay the paralysis phenotype of the Alzheimer's disease caenorhabditis elegans, compared with a positive drug memantine hydrochloride, the paralysis inhibiting drug effect is obviously improved, and the nerve soothing composition has an obvious treatment effect on CL2355 nematodes, has an obvious recovery effect on chemotactic dysfunction of CL2355, has an obvious nerve protection effect, and can be used for treating the CL2355 nematodes. The medicinal value is higher.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of traditional Chinese medicine, and particularly relates to application of an anshen composition in preparation of a medicine for preventing or treating Alzheimer's disease. BACKGROUND

[0002] Alzheimer's disease (AD) is a neurodegenerative disease mainly characterized by progressive cognitive impairment and memory impairment, and is the most common form of senile dementia. Clinically, it is characterized by memory impairment, aphasia, agnosia, visual-spatial skill impairment, executive function disorder, and personality and behavior changes, and is manifested as overall dementia. It accounts for more than 75% of dementia patients. With the aggravation of the aging degree in China, AD will become one of the major diseases affecting the health of the elderly, and effective prevention and treatment of AD has become another major issue before us.

[0003] Amyloid deposition (Aβ protein) and neurofibrillary tangles are two major pathological features of AD. Modern medicine has conducted research on the pathogenesis of AD for many years, but due to the complexity of the causes, the pathogenesis of the disease is still unclear. The "Aβ cascade hypothesis" is one of the mechanisms widely accepted by the scientific community. The theory believes that the abnormal deposition of Aβ in the brain of patients through a series of cascade reactions such as free radical reaction, mitochondrial oxidative damage and inflammatory reaction, directly or indirectly acts on neurons and glial cells, eventually leads to abnormal function or death of neurons, causes cognitive impairment and memory loss, and eventually causes dementia. At present, Aβ amyloid protein is one of the most recognized targets for screening anti-AD drugs.

[0004] AD has complex causes, a long course, and many links in the pathogenesis, and needs long-term medication. Currently marketed anti-Alzheimer's disease drugs include acetylcholinesterase inhibitors (such as galantamine) and N-methyl-D-aspartate receptor (NMDA receptor) antagonists (such as memantine), but the above-mentioned drugs are expensive, and have significant side effects such as hallucinations, confusion, dizziness, headache and fatigue after taking the drugs, and the above-mentioned drugs are only aimed at the disease, not the cause. Therefore, the anti-AD drugs currently used in clinical practice can only control but cannot reverse the patient's condition. In this case, it is urgent to find more effective anti-AD drugs.

[0005] In view of the above technical problems, the inventors accidentally found that the Tibetan medicine Anshen Pill can dose-dependently slow down the AD pathological characteristics of Caenorhabditis elegans, and can be used for preparing a medicine for preventing or treating Alzheimer's disease, thereby providing a new use of the Anshen Pill. SUMMARY

[0006] The primary object of the present application is to provide the use of a nerve-calming composition in the preparation of a medicament for preventing neurodegenerative diseases, wherein the nerve-calming composition is prepared from the following raw medicinal materials: areca nut, eaglewood, clove, mace, costus, Chinese gall, pinnateleaf rhododendron leaf, long pepper, black pepper, ironwood, purple sand, asafetida, rabbit heart, bison heart, and brown sugar.

[0007] The second object of the present application is to provide the use of a nerve-calming composition in the preparation of a medicament for treating neurodegenerative diseases, wherein the nerve-calming composition is prepared from the following raw medicinal materials: areca nut, eaglewood, clove, mace, costus, Chinese gall, pinnateleaf rhododendron leaf, long pepper, black pepper, ironwood, purple sand, asafetida, rabbit heart, bison heart, and brown sugar.

[0008] Preferably, the neurodegenerative disease is Alzheimer's disease.

[0009] Preferably, the Alzheimer's disease is selected from one or more of the following: early-onset Alzheimer's disease, late-onset Alzheimer's disease, and familial Alzheimer's disease.

[0010] Preferably, the nerve-calming composition is added with a pharmaceutically acceptable excipient to prepare any one of the following dosage forms: cream, capsule, tablet, pill, granule, solution, powder, and suspension.

[0011] Preferably, the dosage form is a pill or a powder.

[0012] The present application provides the use of a nerve-calming composition in the preparation of a medicament for preventing or treating Alzheimer's disease, wherein the nerve-calming composition has a significant therapeutic effect on CL4176 nematodes, which is manifested in a significant inhibition of the paralysis phenotype of CL4176 nematodes caused by intramuscular overexpression of Aβ, i.e., the paralysis phenotype of Caenorhabditis elegans with Alzheimer's disease is significantly delayed, and the inhibitory effect on paralysis is significantly improved compared with the positive drug memantine hydrochloride, which has a higher medicinal value. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 Pharmaceutical outer packaging

[0014] Figure 2 Effect of the medicament on the paralysis phenotype of the Caenorhabditis elegans model of Alzheimer's disease

[0015] Figure 3 Toxicity evaluation of the medicament on Caenorhabditis elegans

[0016] Figure 4 Evaluation of the neuroprotective effect of the medicament on Caenorhabditis elegans DETAILED DESCRIPTION

[0017] The technical solutions of the present application are described in further detail below with reference to specific embodiments, but the scope of protection of the present application is not limited to the following description.

[0018] It should be noted that, unless otherwise specified, the methods described in the following embodiments are all conventional methods, and the reagents described are all commercially available.

[0019] This calming composition is prepared from fifteen traditional Tibetan medicinal herbs, including areca nut (processed), agarwood, cloves, nutmeg, costus root, jujube, galangal, long pepper, black pepper, iron rod, purple alum, asafoetida, rabbit heart, wild yak heart, and brown sugar. This Tibetan medicine, refined using traditional pharmaceutical techniques, is also known as Sanda Anshen San or Anshen Wan. Its main functions are to nourish the heart and calm the mind, suppress wind, and is used for wind entering the vital energy channels due to imbalances in the body, neurosis, delirium, excessive dreaming and whispering, palpitations, tremors, mania, and aphasia. The national drug approval number is Z63020048, and its packaging is as follows. Figure 1 As shown. The sedative composition of this invention was specifically used in experiments with sedative pills, but preparing the drug from sedative pills into other dosage forms, such as sedative powder, also has the same effect. Therefore, this invention protects the drug composition and is not limited to a specific dosage form.

[0020] Example 1: An Shen Wan (a traditional Chinese medicine) delays muscle paralysis in Caenorhabditis elegans (AD) worms.

[0021] 1. Biomaterials

[0022] (1) The AD nematode strain CL4176 was purchased from the Caenorhabditis Genetics Center (CGC); it is a transgenic strain that specifically expresses human Aβ in muscle under temperature induction at 25℃. 1-42 , Aβ 1-42 Accumulation in muscle tissue eventually leads to nematode paralysis. In this embodiment, the Caenorhabditis elegans CL4176 strain was used as a pathological model for screening anti-AD drugs.

[0023] (2) Escherichia coli OP50 (uracil leak mutant), purchased from Caenorhabditis Genetics Center (CGC), was used as food for Caenorhabditis elegans.

[0024] 2. Reagents

[0025] (1) An Shen Wan: National Medicine Approval Number Z63020048.

[0026] (2) Memantine hydrochloride (MEM), chemical name: 1-amino-3,5-dimethyladamantaneamine hydrochloride, molecular formula: C 12 H 22ClN, purchased from MCE (MedChemExpress), CAS: 41100-52-1. Memantine hydrochloride is a non-competitive MDA receptor antagonist used to treat moderate to severe Alzheimer's dementia. Literature reports that memantine (21.58 μg / ml) can significantly inhibit the paralytic phenotype of the transgenic nematode strain CL4176. In this example, it is used as a positive control drug.

[0027] (3) Composition and preparation of solid NGM (Nematode Growth Medium) culture medium (taking 1 liter as an example):

[0028] Ingredients Amount NaCl 3.00g K2HPO4 2.34g KH2PO4 17.23g 0.50g 2.50g Proteose peptone 17.00g Supplement H2O to 5.00g

[0029] After preparing the solid NGM medium, autoclave it at 121°C for 20 minutes. Under aseptic conditions, add 1 mL of 5 mg / mL cholesterol, 1 mL of 1 M MgSO4, and 1 mL of 1 M CaCl2, and mix well. While still hot, pour the mixture into sterilized 9 cm culture plates, approximately 20 mL per plate. Let it stand until the medium solidifies, then set aside for later use.

[0030] (4) M9 liquid formulation

[0031] Agar 20.00g Na2HPO4 6.00g KH2PO4 3.00g 1000mL 5.00g 1 M MgSO4 Ingredients Supplement H2O to Amount

[0032] (5) Preparation of lysis buffer: 6.4% NaClO solution and 1M NaOH solution are mixed at a volume ratio of 1:1.

[0033] 3. Prepare NGM tablets containing the drug.

[0034] (1) Preparation of NGM tablets containing An Shen Wan (a traditional Chinese medicine):

[0035] Grind Anshenwan into powder using a mortar and pestle. Weigh 1.00g of Anshenwan and add 100ml of water to prepare a 1% Anshenwan aqueous solution. Sonicate at room temperature for 30min, then centrifuge at 10000rpm for 10min. Collect the supernatant, filter through a 0.22μm aqueous filter, and store at 4℃ for later use. Add the Anshenwan aqueous solution to melted NGM medium to prepare NGM medium containing Anshenwan at final concentrations of 1mg / ml, 0.1mg / ml, and 0.01mg / ml. Allow the medium to solidify. Spread Escherichia coli OP50 evenly on the medium as food for nematodes.

[0036] (2) Preparation of NGM tablets containing memantine hydrochloride:

[0037] Memantine hydrochloride was dissolved in sterile water to prepare a stock solution of 2.158 mg / ml. The memantine stock solution was added to melted NGM medium to prepare NGM medium with a final concentration of 0.022 mg / ml memantine hydrochloride. The medium was allowed to solidify. E. coli OP50 was then evenly spread onto the medium as food for the nematodes.

[0038] 4. Implementation Steps

[0039] (1) Cultivation of nematodes

[0040] Nematodes were inoculated onto solid NGM plates coated with E. coli OP50 and then cultured in an incubator at 15°C. When the nematodes grew to adulthood, they were synchronized.

[0041] (2) Nematode synchronization

[0042] Select NGM medium containing a large number of adult nematodes and some hatched nematode eggs. Rinse the nematodes off the medium with M9 solution and transfer them to centrifuge tubes. Allow the nematodes to settle freely to the bottom of the tubes, and discard the supernatant. Add lysis buffer to the centrifuge tubes according to the amount of nematodes, and vortex for 5-7 minutes until all nematodes break down. Stop vortexing and aliquot into 1.5 mL centrifuge tubes. Wash the nematode eggs three times with M9 solution.

[0043] (3) Effects of Anshenwan on Caenorhabditis elegans CL4176

[0044] After synchronization, the nematode eggs were aliquoted into centrifuge tubes and transferred to NGM culture dishes coated with OP50 and mixed with different concentrations of An Shen Wan and memantine hydrochloride, and NGM culture dishes coated with OP50 and containing an equal volume of sterile water (blank control). Each culture dish contained 60 nematodes, and three culture dishes were used as parallels for each drug concentration. The nematodes were cultured at 15°C for 3 days to reach the L3 stage.

[0045] To induce Aβ expression in nematodes, L3-stage nematodes were induced at 25°C. After 34 hours, the number of paralyzed nematodes was counted. Counts were made every two hours until all nematodes were paralyzed, meaning that only their heads could move when mechanically stimulated with platinum wire. Results are shown below. NaCl .

[0046] The experimental results show that water-soluble Anshenwan at concentrations of 1 mg / ml, 0.1 mg / ml, and 0.01 mg / ml can significantly inhibit paralysis caused by Aβ overexpression in transgenic nematodes. In particular, 1 mg / ml Anshenwan significantly inhibited the paralysis phenotype of nematodes induced by Aβ (p<0.001), and all the above groups showed a certain dose-response relationship.

[0047] Example 2: Toxicity evaluation of Anshenwan against Caenorhabditis elegans (AD)

[0048] 1. The biological materials are the same as in Example 1.

[0049] 2. The reagents are the same as in Example 1.

[0050] 3. Prepare NGM tablets containing the drug as in Example 1.

[0051] 4. Implementation steps (1) The nematode culture is the same as in Example 1.

[0052] (2) The nematode synchronization is the same as in Example 1.

[0053] (3) Effect of Anshenwan on Caenorhabditis elegans CL4176: Synchronized nematode eggs aliquoted into centrifuge tubes were transferred to NGM culture dishes coated with OP50 and containing different concentrations of Anshenwan and memantine hydrochloride, and NGM culture dishes coated with OP50 and containing an equal volume of sterile water (blank control). Each culture dish contained 90 nematodes, and three culture dishes were used as parallels for each drug concentration. The dishes were incubated at 15°C for 96 hours. The nematode body length was measured under a trinocular stereomicroscope, with 30 nematodes measured in each group.

[0054] See results 1.00g It can be seen that the body length of nematodes at the three concentrations of 1 mg / ml, 0.1 mg / ml and 0.01 mg / ml of An Shen Wan aqueous extract was not significantly different from that of the blank control. However, the body length of nematodes at 0.022 mg / ml of memantine hydrochloride was significantly different from that of the blank control (p<0.001), and it showed an inhibitory effect on the growth and development of nematodes.

[0055] Example 3: Neuroprotective effect of Anshen Pill on Caenorhabditis elegans (AD)

[0056] 1. Biomaterials

[0057] (1) The AD nematode strain CL2355 was purchased from Caenorhabditis Genetics Center (CGC): it is a transgenic strain whose neurons express human Aβ protein and serve as a model for Aβ accumulation. This strain of nematodes showed chemotactic dysfunction in chemosensory behavior testing. In this example, the Caenorhabditis elegans strain CL2355 was used as the object of chemotactic testing, and CL2122 was used as its control strain. The neurons of CL2122 do not express human Aβ protein and do not have chemotactic dysfunction.

[0058] (2) Escherichia coli OP50 (uracil leakage mutant) is the same as in Example 1.

[0059] 2. Reagents

[0060] (1) An Shen Wan is the same as in Example 1;

[0061] (2) The composition and preparation of solid NGM (Nematode Growth Medium) culture medium (taking 1 liter as an example) are the same as in Example 1;

[0062] (3) The formulation of M9 liquid is the same as in Example 1;

[0063] (5) The preparation of the lysis buffer is the same as in Example 1;

[0064] (6) Preparation of chemotactic odor attractant: 0.1% benzaldehyde solution and 1% NaN3 solution were mixed at a volume ratio of 1:1; the control was made by mixing sterile water and 1% NaN3 solution at a volume ratio of 1:1.

[0065] 3. Preparation of NGM tablets containing sedative pills

[0066] Grind Anshenwan into powder using a mortar and pestle. Weigh 1.00g of Anshenwan and add 100ml of water to prepare a 1% Anshenwan aqueous solution. Sonicate at room temperature for 30min, then centrifuge at 10000rpm for 10min. Collect the supernatant, filter through a 0.22μm aqueous filter, and store at 4℃ for later use. Add the Anshenwan aqueous solution to melted NGM medium to prepare an NGM medium containing a final Anshenwan concentration of 1mg / ml. Allow the medium to solidify. Spread Escherichia coli OP50 evenly on the medium as food for nematodes.

[0067] 4. Preparation and labeling of chemotactic plates

[0068] Using a 90cm NGM plate, mark four equally sized quadrants and a 0.3cm diameter circle in the center. Label each quadrant as "T" (test group) or "C" (control group), ensuring that all positions are equidistant and at least 2cm from the origin. Add 2μL of 0.1% benzaldehyde solution and 1% NaN3 solution to the test quadrant (T), and add 2μL of sterile water and 1% NaN3 solution to the control quadrant (C).

[0069] Specific distribution: the upper left and lower right quadrants are the test quadrants, and the upper right and lower left quadrants are the control quadrants.

[0070] 5. Implementation steps (1) The nematode culture is the same as in Example 1;

[0071] (2) The nematode synchronization was the same as in Example 1;

[0072] (3) Effects of Anshenwan on Caenorhabditis elegans CL2355

[0073] The synchronized CL2355 and CL2122 nematode eggs, aliquoted into centrifuge tubes, were transferred to NGM culture dishes (blank control) coated with OP50 and mixed with different concentrations of An Shen Wan (a traditional Chinese medicine), and coated with OP50 and containing an equal volume of sterile water as the drug. Each culture dish contained 500 nematodes, and three culture dishes were used as parallels. The dishes were incubated at 15°C for 48 hours, and then transferred to 25°C for 24 hours to induce Aβ expression and observe the neuroprotective effect of the compounds.

[0074] After induction of expression, the nematodes were rinsed with M9 solution, and each washed nematode was placed in a single drop of buffer solution within the center circle of a labeled chemotactic plate. The plates were incubated in the dark at 25°C for 1 hour to induce a chemotactic response in the nematodes to the attractant. After 1 hour of incubation, the number of nematodes in each quadrant was counted. The chemotactic index was calculated using the following formula, based on the established experimental protocol:

[0075] CI = (Total number of nematodes in the test quadrant - Total number of nematodes in the control quadrant) / (Total number of nematodes in the test quadrant + Total number of nematodes in the control quadrant); CI values ​​range from -1 to 1, and higher values ​​indicate stronger chemotaxis.

[0076] See results Proteose peptone 10.00g Agar 40.00g 1000mL Figure 2 Figure 3 Figure 4 It can be seen that 1 mg / ml of Anshenwan can significantly enhance the chemotactic index of Aβ transgenic nematode elegans (p<0.001), which means that 1 mg / ml of Anshenwan can partially restore the chemotactic function defect of CL2355 strain and has obvious neuroprotective effect.

[0077] In summary, this invention provides the application of the Sandar Anshen composition in the preparation of drugs for the prevention or treatment of Alzheimer's disease. The Anshen pills described therein exhibit significant therapeutic effects on CL2355 nematodes, demonstrating a significant recovery effect on chemotactic dysfunction of CL2355 and a significant inhibitory effect on the paralytic phenotype caused by Aβ overexpression in the muscles of CL4176 nematodes. This means it can significantly delay the paralytic phenotype of *C. elegans* in Alzheimer's disease, and compared with the positive control drug memantine hydrochloride, its inhibitory efficacy on paralysis is significantly improved. Within the concentration range where the Sandar Anshen composition has a significant therapeutic effect on CL4176 nematodes, it does not show any inhibitory effect on the growth and development of nematodes, and compared with the positive control drug memantine hydrochloride, it has higher safety and higher pharmaceutical value.

Claims

1. Use of a soothing composition for the manufacture of a medicament for the prevention of neurodegenerative diseases, characterized in that, The tranquilizing composition is made of the following raw medicinal materials: areca nut, eaglewood, clove, mace, costus, zanthoxylum, long pepper, black pepper, ironwood, purple sand, asafetida, rabbit heart, bison heart and brown sugar.

2. Use of a soothing composition for the manufacture of a medicament for the treatment of a neurodegenerative disease, characterized in that, The tranquilizing composition is made of the following raw medicinal materials: areca nut, eaglewood, clove, mace, costus, zanthoxylum, long pepper, black pepper, ironwood, purple sand, asafetida, rabbit heart, bison heart and brown sugar.

3. Use according to claim 1 or 2, characterized in that, The neurodegenerative disease is Alzheimer's disease.

4. The use according to claim 3, wherein the compound is ###0002### The Alzheimer's disease is selected from one or more of the following: early-onset Alzheimer's disease, late-onset Alzheimer's disease and familial Alzheimer's disease.

5. Use according to claim 1 or 2, wherein the compound is ###0002### The tranquilizing composition is added with pharmaceutically acceptable adjuvants to prepare any one of the following dosage forms: cream, capsule, tablet, pill, granule, solution, powder and suspension.

6. Use according to claim 5, wherein The dosage form is a pill or a powder.

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