CN101181285A - Application of astragaloside IV in the preparation of drugs for treating neurodegenerative diseases - Google Patents

Abstract

The invention belongs to the technical field of medicines, and specifically relates to the application of astragaloside IV in the preparation of medicines for treating neurodegenerative diseases. Astragaloside IV is one of the main active components of Astragalus membranaceus. Its molecular formula is C ₄₁ H ₆₈ O ₁₄ , and it is a white powder. Experiments have proved that astragaloside IV has a protective effect on brain damage in mice. Therefore, it can be used as a medicine for treating neurodegenerative diseases, especially senile dementia, and can be made into various dosage forms with pharmaceutically acceptable carriers.

Description

Application of astragaloside IV in the preparation of drugs for treating neurodegenerative diseases

technical field

The invention belongs to the technical field of medicine, and specifically relates to the application of astragaloside IV as a medicine for preventing and treating various neurodegenerative diseases (especially senile dementia).

Background technique

A highly developed brain is one of the most striking signs that distinguish humans from other creatures in the world. Human memory, cognition, language, personality and emotions are all closely related to normal brain function. Once the human brain has pathological changes, these functions are severely impaired. Due to the different damaged parts and pathological mechanisms in the brain, patients can manifest different neurological symptoms and form different neurological diseases. Neurodegenerative diseases (neurode2generative diseases) are a class of chronic, progressive neurological diseases. Although the lesion sites and causes of these diseases are different, neurodegeneration is their common denominator. Such diseases mainly include Alzheimer's disease (Alzheimerqs disease, AD), Parkinson's disease (Pakinsonqs disease, PD), Huntington's disease (Huntington disease), different types of spinocerebellar ataxias (spinal cerebellar ataxias), dentate Nucleus pallidus subthalamic nuclear atrophy (dentatorubropallidoluysianatrophy), amyotrophic lateral sclerosis (amyotrophic lateral sclerosis) and spinal muscular atrophy (spinal muscular atrophy), etc. AD and PD mainly occur in middle-aged and elderly people. With the aging of the population, the incidence of AD and PD is increasing. At present, there are 4 million people in the United States suffering from AD, about 100,000 people die from AD every year, and the annual medical expenses are as high as 60 billion US dollars. The epidemiological research on AD in our country is not yet perfect. It is generally believed that the prevalence of dementia in people over 65 years old is about 4%, and the annual incidence rate is 0.16-112%. The prevalence of PD is second only to AD, and mainly occurs in people over middle age, with a prevalence rate of 2% among people over 65 years old. In addition, Huntington's chorea, different types of spinocerebellar ataxia, amyotrophic lateral sclerosis and spinal muscular atrophy can occur at different ages.

In my country, with the aging of the population, the incidence of neurodegenerative diseases, especially senile dementia, is increasing day by day, which has become one of the major problems of human public health. Treatment of such disorders has been difficult for many years due to the complexity of brain function. In the past ten years, with the rapid development of molecular biologists, neurobiology, behavioral science and other disciplines of knowledge and research methods, many new discoveries have been made in the study of the pathological mechanism of neurodegenerative diseases. Various therapeutic drugs are also emerging one after another. At present, the drugs for the prevention and treatment of Alzheimer's disease include cholinergic precursors, cholinesterase inhibitors, cholinergic agonists, catecholamines, and serotonergic drugs. , various types of neurotransmitters, neuropeptides, opioid antagonists (such as naloxone), antioxidant and neuroprotective agents, neurotrophic substances, hormones (such as tacrine, estrogen), metabolic enhancers, Biofilm regulators, anti-inflammatory drugs, antidotes, anti-amyloid drugs, etc., active ingredients of traditional Chinese medicine such as huperzine A, ginkgo biloba extract, catechin, apigenin A, ginsenosides, gypenosides, etc. But so far, no medicine, any therapy has achieved satisfactory curative effect. Therefore, it is an urgent task to find new effective treatments or drugs for the prevention and treatment of Alzheimer's disease.

Independent development of innovative drugs is an urgent task in my country at present. Finding effective active ingredients from traditional Chinese medicine is an effective way, and it is also the advantage of my country's innovative drug development. my country has a long history of traditional Chinese medicine, and has accumulated rich experience in treating various neurodegenerative diseases with Chinese herbal medicine. Among the active ingredients of these plants, it is speculated that there are substances that can prevent and treat various neurodegenerative diseases. Therefore, It is an ideal way to screen lead compounds with the activity of preventing and treating various neurodegenerative diseases from natural products, and finally find drugs with clinical application prospects.

Contents of the invention

The object of the present invention is to propose an application of astragaloside IV as a medicine for preventing and treating various neurodegenerative diseases.

Radix Astragali (Radix Astragali) is the dried root of Astragalus membranaceus (Fisch) Bge.Var.mongholicus (Bge.) Hsiao or Astragalus membranaceus (Fisch) Bge. of the leguminous plant Astragalus membranaceus (Fisch) Bge. Mild in nature, traditional medicine believes that it has the effects of invigorating qi and strengthening the surface, diuretic and detoxification, astringing sores and promoting granulation, and has a long history of medicinal use. Modern studies have shown that astragalus has the effects of enhancing immunity, metabolism, lowering blood pressure and diuresis, among which astragalosides, especially astragaloside IV, are its main physiologically active components.

Astragaloside IV (Astragaloside IV) is one of the main active ingredients of Astragalus membranaceus, which can anti-inflammatory, lower blood pressure, analgesic and sedative, and promote regeneration of liver DNA levels. Modern pharmacological studies have shown that astragaloside IV has pharmacological effects such as improving the deformability of white blood cells, improving myocardial contraction and relaxation, promoting insulin secretion and scavenging free radicals. After literature search and follow-up, there have been no research reports on the activity of astragaloside IV monomer components in the prevention and treatment of various neurodegenerative diseases so far.

The molecular formula of Astragaloside IV is C ₄₁ H ₆₈ O ₁₄ , and the system name is β-D-Glucopyranoside, (3β, 6α, 16β, 20R, 24S)-20, 24-epoxy-16, 25-dihydroxy-3 -(β-D-xylopyranosyloxy)-9,19-cyclolanstan-6-yl. It is a white powder, melting point: 309-310°C, slightly soluble in methanol, insoluble in ethyl acetate, acetone, and water, dissolved in ethanol when heated, and precipitated after cooling. The structural formula is as follows:

Extraction and identification of astragaloside IV:

1 kg of medicinal material Astragalus membranaceus, crushed into 20 mesh coarse powder, soaked in 12 liters of water for 60 minutes, decocted for 120 minutes, three times in total, combined the extracts, filtered to remove the precipitate, and the supernatant was passed through a macroporous adsorption resin column (chemical plant of Nankai University, Tianjin, AB- 8 styrene type macroporous adsorption resin) (Φ:h=1:10). First elute with 10 liters of deionized water to remove water-soluble impurities. Carry out elution with 5 liters of 0.2% aqueous sodium hydroxide solution, followed by elution with 5 liters of 40% ethanol, and then elution with 5 liters of 75% ethanol, collect the part, evaporate the ethanol to 100ml at low temperature, and centrifuge to obtain Astragalus The crude product of glycoside IV was recrystallized from ethanol to obtain a white powder. According to the spectral data ( ¹ H-NMR, ¹³ C-NMR, DEPT, MS, UV), this monomeric compound was identified as Astragaloside IV (Astragaloside IV). HPLC-ELSD analysis showed that the purity was above 98%.

The astragaloside IV proposed by the present invention is used as a pharmaceutical composition for preventing and treating various neurodegenerative diseases, and the pharmaceutical composition includes the active ingredient astragaloside IV and a pharmaceutically acceptable carrier.

The present invention also proposes a method for preparing the composition of the above-mentioned medicine, the method comprising mixing or dissolving astragaloside IV and a pharmaceutically acceptable carrier.

Astragaloside IV can be mixed or dissolved with any pharmaceutically acceptable carrier, such as the pharmaceutically acceptable carrier for transdermal, mucous membrane, enteral and parenteral administration. The drug is used in the form of conventional pharmaceutical preparations, such as tablets, granules, powders, capsules, cachets or suppositories in solid form, or ointment or ointment in semi-solid form, or crystalline powder injection, Or liquid form of injection, suspension, syrup, emulsion or liniment. Pharmaceutically acceptable excipients and additives are used in the drug, and these pharmaceutically acceptable excipients and additives include non-toxic compatible fillers, binders, disintegrants, buffers, preservatives, antioxidants , lubricants, flavoring agents, thickeners, colorants, emulsifiers or stabilizers. Such as lactose, citric acid, stearic acid, magnesium stearate gypsum powder, sucrose, corn starch, talc, gelatin, agar, pectin, peanut oil, cocoa butter, ethylene glycol, glucose, procaine hydrochloride, hydrochloric acid Lidocaine and ascorbic acid etc. The medicine can be prepared according to the conventional techniques of various formulations.

The contents and results of the experimental research are listed below, as evidence that astragaloside IV can be used to prepare drugs for the prevention and treatment of neurodegenerative diseases (especially senile dementia).

1. The protective effect of Astragaloside IV on monosodium glutamate (MSG)-induced hippocampal neuron injury.

Materials and methods

Drug: Astragaloside IV, MSG.

Culture medium for hippocampal neurons: the culture medium (planting solution) was Duibecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum, 10% horse serum, and 1% glutamine. The culture medium for feeding hippocampal neurons was DMEM supplemented with 10% horse serum, N-21%, B-272%, and glutamine 1%.

Isolation and culture of hippocampal neurons: take newborn Wistar rats on the same day, peel off the hippocampus, cut into 1-2mm tissue pieces, digest with dissecting solution containing 0.25% trypsin at 37-C for 30 minutes, blow and beat to make them evenly dispersed, Make a cell suspension. Add an appropriate amount of planting solution, inoculate the cells at a density of 1×10 ⁵ /ml in a 35mm culture dish coated with poly-lysine, and culture in an incubator at 36°C and 10% CO ₂ . After 24 hours, the planting solution was replaced with 2 mL feeding solution. Afterwards, the liquid was changed once every 3 days. To inhibit excessive proliferation of non-neuronal cells, 3 μg·mL ^-1 of cytarabine was added to the medium on the third day of culture.

Toxicity of glutamic acid to neurons and protective effect of astragaloside IV: After digesting the cells in the logarithmic growth phase, the cell suspension was made into a 96-well culture plate, 90 pl per well (containing 2×10 ⁴ cells). After culturing for 24 hours, 10 μL of astragaloside IV with different concentrations was added every 1 hour before and after, and the same volume of neuron culture medium was added to the control group, and 4 parallel wells were set up for each group. After fully mixing, continue to culture for 24, 48, 72h. MTT method, fluorescence microscopy (PI-Hoechst double staining method), acridine orange/ethidium bromide (A0/EB) fluorescence staining, flow cytometry, DNA fragment analysis, etc. to determine cell survival and cell apoptosis .

Results: Astragaloside IV could partially, mostly or even almost completely counteract the toxic damage of MSG to neurons and the induced apoptosis; this protective effect depends on the dose and the time of action.

2. The protective effect of Astragaloside IV on β-amyloid-induced injury of rat PC 12 cells.

Materials and methods

Drugs: MSG, astragaloside IV, β-amyloid (Aβ), Aβ 25-35. Aβ stock solution: prepared as 10mg/ml solution with 0.1% trifluoroacetic acid for preservation.

PC 12 cell line: originated from rat pheochromocytoma, can differentiate into sympathetic nerve-like cells, which are close to neurons in terms of morphology, physiology and biochemistry. PC 12 cells have the properties of neurosecretory cells and neurons, and are widely used in the study of neuron death and neurotoxicity.

Culture of PC 12 cells: Routine culture in RPMI-CM medium. RPMI-CM is RPMI-1640 supplemented with 10% fetal bovine serum, 5% horse serum, 1% glutamine. After cultured in RPMI-CA containing 100ng/ml nerve growth factor (mNGF 2.5s) for 6 days, PC 12 cells differentiated into cells with neuronal phenotype. The full length (Aβ1-40) of β-amyloid (Aβ) and its cytotoxic sequence Aβ25-35 between amino acid residues 25-35 were added.

Toxicity of Aβ25-35 to neurons and protective effect of astragaloside IV: Differentiated PC 12 cells were made into cell suspension and inoculated in 96-well plates, and treated with different concentrations of Aβ1-40 or Aβ25-35 and astragaloside IV for different time , MTT method, fluorescence microscopy (PI-Hochest double staining method), flow cytometry, DNA fragment analysis, etc. to judge cell survival and cell apoptosis.

Results: Astragaloside IV could partially, mostly or even almost completely counteract the damage of Aβ25-35 to differentiated PC 12 cells, and the protective effect of astragaloside IV depended on its action time and dose.

3. Protective effect of Astragaloside IV on MSG-induced brain damage in adult mice

Materials and methods

Animals: Kunming mice, male and female, 8 weeks old.

Drugs: MSG, astragaloside IV.

Instruments: Y-maze, DMS-Z Morris water maze instrument, XDB-Z mouse hole plate tester, ZIL-Z mouse automatic activity program automatic control instrument, all of the above instruments were purchased from the Institute of Materia Medica, Chinese Academy of Medical Sciences.

Methods: Mice were randomly divided into normal control group, MSG group, drug control group and experimental group, with 14 animals in each group. The MSG group was intragastrically administered with MSG 3mg/kg body weight, the drug control group was intraperitoneally injected with astragaloside IV at 5 mg/kg body weight, and the experimental group was intragastrically injected with 5 mg/kg body weight of astragaloside IV at the same time as MSG 3mg/kg body weight. Treatment was once a day for a total of 10 days. 12 hours after the last injection, 4 animals were randomly selected from each group, anesthetized by intraperitoneal injection of pentobarbital (0.06 mg/kg body weight), and sacrificed by cardiac perfusion with 10% formalin solution. The brains were decapitated and fixed in formalin solution of the same concentration for 1 week. Embed in paraffin, make 10m serial sections, and take 1 piece every 100m. Tar violet staining, light microscopy. The remaining mice in each group were subjected to 4 behavioral examinations on the 1st day, 31st day, 61st day and 91st day after the last injection, to check the learning and memory ability of the mice, autonomous activities and their types, and to explore Behavior, rope climbing ability, etc.

Results: 1. Behavioral examination results showed that there was no significant difference between the normal control group and the drug control group in the learning and memory and rope climbing abilities, autonomous activities and exploration behaviors of the 4 inspections; the learning and memory and rope climbing abilities of the mice in the MSG group And exploration behavior, compared with the control group, the first three inspections were significantly lower, the difference was very significant (P<0.05), and the fourth inspection (that is, the 91st day) had a certain increase, but the difference was still significant (P<0.05). On the contrary, the autonomic activity of MSG mice was significantly increased in the first three inspections, the difference was very significant (P<0.01), and it was close to normal in the fourth (ie 91 days) inspection (P>0.05). It is worth noting that the results of the four behavioral examinations all showed that the various behavioral examination indexes of the experimental group (astragaloside IV) mice were not significantly different from those of the normal control mice (P>0.05). 2. The results of morphological examination showed that there was no significant difference in the morphology of nerve cells in the hippocampus of the mice in the drug control group and the normal control group. The neurons in the hippocampus of the mice in the MS6 group were significantly degenerated and necrotic, while the experimental group treated with MS6+astragaloside IV was smaller. The cell morphology of rat hippocampus was similar to that of normal group mice, without significant difference.

4. Protective effect of Astragaloside IV on Aβ 25-35-induced brain injury in mice

Material Animals: Kunming male mice, 28±2g.

Drugs: MSG; AB 25-35 (Sigma Company), 1 mg dissolved in 1 ml of normal saline; astragaloside IV.

Instrument: DM5-Z Morris water maze apparatus, manufactured by Institute of Materia Medica, Chinese Academy of Medical Sciences.

Methods: Aβ25-35 was aged in a 37°C incubator for 96 hours to make it into a toxic gel-state Aβ. Animals were randomly divided into 4 groups: sham operation group, model group (Aβ25-35 group), three doses (1 mg/kg, 5 mg/kg, 25 mg/kg) astragaloside IV group. The mice in the sham operation group were injected with 3 μL of normal saline in the lateral ventricle, and the mice in the other four groups were injected with condensed Aβ25-353 μL in the lateral ventricle. The astragaloside IV group started intraperitoneal injection of different doses of astragaloside IV solution 1 hour after the injection of Aβ25-35, and continued the administration for 20 days. On the 16th day after mice were injected with Aβ25-35 in the lateral ventricle, the mice were subjected to the Morris water maze test for 5 consecutive days, with two time frames per day. During the experiment, the temperature was kept at 24-25°C. Add 2 catties of milk powder into the Morris water maze, wash it with hot water, and then add water to 1cm above the safety platform, so that the safety platform cannot be seen in the milk, and keep the water temperature at 22°C.

Table 1 Protective effect of astragaloside IV on Aβ 25-35-induced directional learning and memory impairment in mice

group Dosing mg/kg × days of administration Number of animals (only)   Latency (S, mean±S.D) P ^*   Control Aβ25-35 Astragaloside IV 1×205×2025×20   1010101010 50.1±3.983.5±4.864.2±5.757.2±5.152.6±5.5 <0.05<0.01<0.01

^* Compared to Aβ25-35.

Results: The directional learning and memory ability of mice was measured by Morris water maze, that is, the latency to find a safe platform. It was found that during the training period of 1 to 4 days, there was no significant difference between the drug treatment group, the sham operation group, and the model group, and the difference was quite obvious after the 5th day. Intraperitoneal injection of astragaloside IV showed a significant protective effect on the damage of Aβ 25-35 to the brain of mice, and this protective effect was dose-dependent.

5. Protective effect of Astragaloside IV on MSG+Aβ-induced brain injury in adult mice

Animals: Kunming mice, male and female, 8 weeks old.

Drugs: Same as 4.

Instrument: DM5-Z Mortis water maze apparatus, manufactured by Institute of Materia Medica, Chinese Academy of Medical Sciences.

Methods: Animals were randomly divided into 4 groups: sham operation group, Aβ+MSG group, Aβ+MSG+astragaloside (2mg/kg) group, Aβ+MSG+astragaloside (10mg/kg) group. The mice in the sham operation group were injected with 3 μL of normal saline into the lateral ventricle, and the mice in the other four groups were injected with 3 μL of condensed Aβ 25-35 in the lateral brain. MSG + astragaloside IV group was intraperitoneally injected with astragaloside IV solution (2 mg/kg, 10 mg/kg) 1 hour after MSG administration. Aβ 25-35 was only administered once, and MSG and astragaloside IV were administered for 8 days, once a day. On the 9th day after the administration of Aβ 25-35, the mice were subjected to the Morris water maze test, and the conditions were the same as in 4.

Result: It was found that there was no significant difference between the administration group and the sham operation group and the model group in the training period of 1 to 4 days, and the difference after the 5th day was quite obvious. damage, and the effect was dose-dependent.

6. Effects of Astragaloside IV on Learning, Memory and Memory Acquisition in Mice with Cerebral Ischemia

Animal: Kunming mouse, 28±2g.

Drugs: Astragaloside IV.

Instrument: Program-controlled mouse dark avoidance tester.

Method: Mice were randomly divided into sham operation group, operation group and experiment (operation+administration) group. Block the bilateral common carotid arteries for 5 minutes in the wakeful state of the mice (causing global cerebral ischemia), and inject astragaloside IV intraperitoneally at 5 mg/kg in the experimental group 5 minutes before blocking the common carotid arteries, the operation group and the sham operation group The same volume of normal saline was injected intraperitoneally, and then once a day, for a total of 10 injections. The first training was carried out 24 hours after the last injection. During the training, put the mouse face away from the hole and put it into the bright room, and start the timer at the same time. Animals enter the dark room through the hole and receive electric shock (AC, 36V), and the timing stops automatically. The mice were taken out, and the time required for each mouse to encounter the electric shock from the light room to the dark room was recorded, which was the incubation period. The test was repeated after 24 hours, and the number of animals entering the dark room, the incubation period and the number of shocks received within 5 minutes were recorded.

Results: The learning and memory acquisition functions of the sham operation group were not significantly different from the normal ones; the memory acquisition dysfunction of the operation group was very obvious, manifested as a short latency period and a large number of errors; The number of times decreased and was close to that of the sham operation group.

7. Preliminary clinical observation of Astragaloside IV in the treatment of Alzheimer's disease

Drug: Astragaloside IV Capsules (50mg/capsule).

Diagnostic criteria: Cases were diagnosed according to the diagnostic criteria of the American Diagnostic and Statistical Manual of Mental Disorders, Version IV (DSM-IV) (1994), and dementia (mild, moderate, heavy) severity diagnosis.

Observation objects: Han nationality, 10 males and 10 females, aged 60-70 years, 12 with mild dementia and 8 with moderate dementia.

Test method: The test is carried out according to the double-blind method. The above-mentioned confirmed patients were divided into two groups according to their conditions. One group took placebo, and the other group took astragaloside IV capsules (50mg/capsule), 1 capsule each time, 3 times a day, for 1 month continuously, and after stopping the drug for 1 week, Continue to the next course of treatment. All patients were treated for 3 courses of treatment, and the whole course of observation and treatment lasted for 6 months. Before and after treatment, the cognitive function test was carried out according to the Hasegawa Scale, the living ability test was carried out according to the daily life function level scale (ADL), and the depression level was checked according to the Hamilton Depression Scale.

Results: In the medication group, except for a few patients (1/10) whose condition remained at the original level, the condition of the rest (9/10) patients were improved to varying degrees: cognitive function test scores increased, living ability enhanced, and sleep improved. No adverse drug reactions were found. Patients in the placebo group showed no signs of improvement.

Detailed ways

The formulations below illustrate dosage forms of the invention.

Example 1:

Preparation of Astragaloside IV Sodium Chloride Injection:

Take 15g of astragaloside IV, add 2L of ethanol and 2L of propylene glycol in turn, stir to dissolve, add 900g of sodium chloride, add water for injection to 100L, mix well, filter and sterilize, subpackage, 100ml per bottle, potting, and sterilize Astragalus Sodium chloride infusion solution. For the treatment of various neurodegenerative diseases.

Example 2:

Preparation of Astragaloside IV Pills:

Put 23.46g of polyethylene glycol (6000) and 6.20g of poloxamer (188) in a beaker, heat on a water bath at 90°C to melt, stir evenly, add 12.5g of astragaloside IV, stir evenly, add 10ml of medicinal Water and ethanol, stir fully to dissolve astragaloside IV, continue stirring to volatilize ethanol, keep warm for later use; preheat the dropping pill machine, keep the temperature of the liquid storage room at 92°C ± 2°C, and pre-cool the coolant to 10-12°C ℃, adjust the distance between the drip nozzle and the cooling liquid surface, pour the liquid medicine into the liquid storage chamber, start the valve, and drop the medicine liquid into the coolant drop by drop at a rate of about 28 drops/min; collect the dripping pills at the collection port , absorb the surface coolant with gauze, freeze in the freezer of the refrigerator (-18°C) for 28 hours, put it in a vacuum drying oven, and dry it under vacuum at room temperature to obtain astragaloside IV pills.

Example 3:

Preparation of Astragaloside IV Capsules:

Take 50 g of the main drug astragaloside IV, 60 g of auxiliary materials microcrystalline cellulose, and 140 g of lactose, mix the main drug and various auxiliary materials according to the method of equal addition, pass through a 40-mesh sieve, and mix evenly. The uniformly mixed material is put into an appropriate container, and an appropriate concentration of povidone K30 prepared by 75% alcohol is added to make a soft material, and then squeezed through a 20-mesh sieve to make wet granules. Spread the wet granules flat, dry them in an oven at 60°C, sieve through a 18-mesh sieve, add 0.2% magnesium stearate, mix them evenly, fill capsules, and pack to obtain astragaloside IV capsules, which have obvious effects in treating senile dementia.

Claims (3)

1. the application of astragaloside in preparation treatment neurodegenerative diseases medicine.

2. one kind is the prevention of active ingredient and the pharmaceutical composition of treatment neurodegenerative diseases with the astragaloside.

3. pharmaceutical composition according to claim 2, it is characterized in that dosage form has tablet, granule, powder, capsule, the cachet of solid form or fastens agent, or the ointment of semi-solid form or ointment, or crystalline powder shape injection, or the injection of liquid form, suspending agent, syrup, Emulsion or liniment.