Application of traditional Chinese medicine monomer in treatment of vascular dementia

Abstract

The invention particularly relates to application of a traditional Chinese medicine monomer in treatment of vascular dementia. The component of the traditional Chinese medicine monomer is astragaloside III. Animal experiments prove that the astragaloside III can improve the problem of poor spatial learning ability and memory ability of a mouse with vascular dementia, and also has a treatment effect on a brain function lesion region caused by vascular dementia. A cell hypoxia and sugar deficiency damage model is utilized to verify that the protection effect of the astragaloside III is better than that of astragaloside I and astragaloside IV, and it is verified that the astragaloside III has a synergistic effect in common administration with a positive drug donepezil.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicine. Specifically, it relates to the application of a traditional Chinese medicine monomer in the treatment of vascular dementia. Background Art

[0002] Vascular Dementia (VD) is a common type of dementia in clinical practice and is a cognitive impairment syndrome caused by cerebrovascular diseases. Exploring its pathogenesis and seeking effective therapeutic drugs are one of the urgent public health problems to be solved in countries around the world. VD is currently the only type of dementia that can be effectively treated through intervention (Biesbroek JM, Biessels GJ. Diagnosing vascular cognitive impairment: current challenges and future perspectives. International Journal of Stroke 2023, 18(1): 36-43.). However, there is no approved specific drug for the treatment of VD globally. Clinically, dihydroergot alkaloids, calcium antagonists, acetylcholinesterase inhibitors, etc. are often used for treatment, but these drugs can only mildly improve or stabilize the condition and will also cause side effects or adverse reactions to varying degrees (Liu Cong et al. Pathogenic mechanism and drug intervention of vascular dementia. Chinese Journal of Gerontology, 2023, 43(14): 3580-3583; Liu AK et al. Effect of collaborative dementia care on potentially inappropriate medication use: outcomes from the CareEcosystem randomized clinical trial. Alzheimer’s & Dementia 2023, 19(5): 1865-1875.).

[0003] Astragalus membranaceus (Fisch.) Bunge var. mongholicus (Bunge) Hsiao or Astragalus membranaceus (Fisch.) Bunge of the Leguminosae family, the dried roots of which have a long application history, were first recorded in "Shennong Ben Cao Jing" and listed as top-grade herbs, and they pertain to the spleen and lung meridians. Currently, preparations such as Guiqi Congzhi Decoction, Yiqi Congming Decoction, and Astragalus membranaceus injection with Astragalus membranaceus as the monarch drug are widely used in the clinical treatment of VD, and can effectively improve the cognitive dysfunction and mental state of patients (Zhang Ruihua, et al. Pharmacological effects and current status of clinical applications of Astragalus membranaceus and its active components. Shaanxi Journal of Traditional Chinese Medicine, 2021, 42(8): 1138-1146.). Through the analysis of 207 selected literature on the application of traditional Chinese medicine in the clinical treatment of VD, it was found that there were 38 traditional Chinese medicines with a frequency distribution of more than 70%, and Astragalus membranaceus was the single herb with the highest frequency of use and was regarded as one of the most promising candidate traditional Chinese medicines (Han Dejun, et al. Analysis of the treatment methods and medication rules of randomized controlled literature on traditional Chinese medicine for treating dementia. Journal of Traditional Chinese Medicine, 2014, 55(12): 1051-1054.). Modern pharmacological studies have also proven that Astragalus membranaceus has a significant effect in treating VD, can improve the learning and memory function of dementia rats, and reduce apoptosis and damage in the hippocampal region (Setel DD, et al. Astragalus membranaceus treatment combined with caloric restriction may enhance gene sis factors and decrease apoptosis in the hippocampus of rats. Archives of Gerontology and Geriatrics, 2022, 98: 104584.). So far, more than 90 triterpenoid saponins and 35 polysaccharide components have been reported to be isolated from Astragalus membranaceus, including 13 new compounds and 1 novel skeleton, and these two types of components have obvious advantages in the treatment of VD (alehi B, et al. Astragalus species: insights on its chemical composition toward pharmacological applications. Phytotherapy Research, 2020, 35(5): 2445-2476.).Astragaloside can significantly inhibit apoptosis and neuroinflammation of ischemic brain cells caused by VD, reduce the accumulation of senescent cells in the brain, and also has excellent neuroprotective, antioxidant and anti-cholinesterase activities (Xu L, et al. Adjuvant therapy with Astragalus membranaceus for post-stroke fatigue: a systematic review. Metabolic Brain Disease 2020, 35(1): 83-93; Salehi B, et al. Astragalus species: insights on its chemical composition toward pharmacological applications. Phytotherapy Research 2020, 35(5): 2445-476.). Summary of the Invention

[0004] The object of the present invention is to provide a Chinese medicine monomer component, astragaloside III, for the treatment of vascular dementia, which can significantly improve the cognitive dysfunction of vascular dementia. In addition, the combined use of astragaloside III and donepezil can also produce a synergistic therapeutic effect.

[0005] In one aspect, the present invention provides the use of astragaloside III or a pharmaceutically acceptable salt thereof in the preparation of a drug for the prevention and treatment of dementia.

[0006] In a second aspect, the present invention provides a pharmaceutical composition comprising astragaloside III or a pharmaceutically acceptable salt thereof and donepezil.

[0007] In one embodiment, the pharmaceutical composition is used for the prevention and treatment of dementia.

[0008] In one embodiment, the molar ratio of astragaloside III to donepezil is 0.5-2:1, preferably 0.8-1.2:1, more preferably 1:1.

[0009] In a third aspect, the present invention also provides the use of the pharmaceutical composition in the preparation of a drug for the prevention and treatment of dementia.

[0010] In one embodiment, the dementia described in the present invention includes vascular dementia and Alzheimer's disease, preferably vascular dementia.

[0011] In one embodiment, the "pharmaceutically acceptable salts" of the present invention include salts formed by astragaloside III and pharmaceutically acceptable acids or pharmaceutically acceptable bases. The pharmaceutically acceptable salts include salts with inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, sulfuric acid, phosphoric acid, etc., salts with organic acids such as acetic acid, trifluoroacetic acid, oxalic acid, malonic acid, fumaric acid, maleic acid, succinic acid, citric acid, tartaric acid, adipic acid, gluconic acid, glucuronic acid, benzoic acid, phthalic acid, terephthalic acid, lactic acid, hippuric acid, glutamic acid, aspartic acid, 1,2-ethanedisulfonic acid, hydroxyethanesulfonic acid, lactobionic acid, oleic acid, pectic acid, stearic acid, tannic acid, methanesulfonic acid, ethanesulfonic acid, trifluoromethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, dodecyl sulfate, methyl sulfate, naphthalenesulfonic acid, sulfosalicylic acid, etc., and also include lithium salts, sodium salts, potassium salts, calcium salts, magnesium salts, aluminum salts, iron salts, zinc salts, ammonium salts.

[0012] In the present invention, the drug or pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0013] Any conventional carrier or excipient can be used in the drug or pharmaceutical composition of the present invention. The choice of a particular carrier or excipient or combination of carriers or excipients will depend on the mode of administration for treating a particular patient or the type of medical condition or disease condition. In this regard, the preparation of a pharmaceutical composition suitable for a particular mode of administration is entirely within the skill of those in the pharmaceutical art. Additionally, the carriers or excipients used in the pharmaceutical compositions of the present invention are commercially available. By way of further illustration, conventional formulation techniques are described in the following references: Remington: The Science and Practice of Pharmacy, 20th Edition, Lippincott Williams & White, Baltimore, Maryland (2000); and H. C. Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Edition, Lippincott Williams & White, Baltimore, Maryland (1999).

[0014] The drug or pharmaceutical composition according to the present invention is suitable for oral administration. The drug or pharmaceutical composition suitable for oral administration can be in the form of capsules, tablets, pills, lozenges, cachets, troches, powders, granules; or in the form of solutions or suspensions in aqueous or non-aqueous liquids; or as water-in-oil or oil-in-water liquid emulsions, etc.

[0015] Drugs or pharmaceutical compositions intended for oral administration in solid dosage forms (i.e., in the form of capsules, tablets, pills, etc.) will generally contain an active ingredient and one or more pharmaceutically acceptable carriers, such as sodium citrate or dibasic calcium phosphate. These solid dosage forms may also optionally or alternatively include: fillers or bulking agents, such as starch, microcrystalline cellulose, lactose, sucrose, glucose, mannitol, and / or silica; binders, such as carboxymethylcellulose, alginate, gelatin, poly(vinylpyrrolidone), sucrose, and / or acacia; humectants such as glycerol; disintegrants, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and / or sodium carbonate; dissolution inhibitors, such as paraffin; absorption promoters, such as quaternary ammonium compounds; wetting agents, such as cetyl alcohol and / or glycerol monostearate; absorbents, such as kaolin and / or bentonite; lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and / or mixtures thereof; coloring substances, and buffering agents.

[0016] The drugs or pharmaceutical compositions of the present invention may also contain release agents, wetting agents, coating agents, sweetening agents, flavoring agents, fragrances, preservatives, antioxidants, and the like. Examples of pharmaceutically acceptable antioxidants include: water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, lecithin, propyl gallate, α-tocopherol, etc.; metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid, sorbitol, tartaric acid, phosphoric acid, etc. Coating agents for tablets, capsules, pills, etc., including those for enteric coatings, such as cellulose acetate phthalate, ethyl cellulose acetate phthalate, hydroxypropylmethyl cellulose phthalate, methacrylic acid-methacrylate copolymer, cellulose acetate-tricarboxylate, carboxymethylethyl cellulose, hydroxypropylmethyl cellulose acetate-succinate cellulose, etc. Coating agents also include talc, polyethylene glycol, hypromellose, and titanium dioxide.

[0017] The drugs or pharmaceutical compositions may also be formulated in such a way, for example, using variable proportions of hydroxypropylmethyl cellulose, to slowly release or control the active ingredient. Or other polymeric matrices, liposomes, and / or microspheres. In addition, the drugs or pharmaceutical compositions may optionally contain opacifying agents and may be formulated such that they release the active ingredient only in the gastrointestinal tract or preferably in certain parts thereof and optionally in a delayed manner. Examples of available embedding compositions include polymeric substances and waxes. If convenient, the active ingredient may also be in the form of microcapsules together with one or more of the above excipients.

[0018] Suitable oral liquid dosage forms include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, etc. Liquid dosage forms usually contain an active ingredient and an inert diluent, such as water or other solvents, solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzoic acid esters, 1,3 - butanediol, oils (especially cottonseed oil, peanut oil, corn oil, wheat germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and sorbitan fatty acid esters and mixtures thereof. In addition to the active ingredient, suspensions may also contain suspending agents, such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum hydroxide, bentonite, agar and tragacanth and mixtures thereof.

[0019] The drugs or pharmaceutical compositions of the present invention can also be administered parenterally (e.g., by intravenous, subcutaneous, intramuscular or intraperitoneal injection). For parenteral administration, the active ingredient is usually mixed with a vehicle suitable for parenteral administration, which includes, for example, sterile aqueous solutions, saline, low molecular weight alcohols (such as propylene glycol, polyethylene glycol), vegetable oils, gelatin, fatty acid esters (such as ethyl oleate), etc. Parenteral formulations may also contain one or more antioxidants, solubilizers, stabilizers, preservatives, wetting agents, emulsifiers, buffers or dispersants. These formulations can be made sterile by using sterile injectable media, bactericides, filtration, irradiation or heating.

[0020] The drugs or pharmaceutical compositions can also be formulated for administration by inhalation. Pharmaceutical compositions suitable for administration by inhalation are usually in the form of aerosols or powders. The drugs or pharmaceutical compositions are generally administered using well - known delivery devices (such as metered - dose inhalers, dry - powder inhalers, nebulizers or similar delivery devices).

[0021] When administered by inhalation using a pressurized container, the drugs or pharmaceutical compositions of the present invention usually contain an active ingredient and a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gases. Additionally, the drugs or pharmaceutical compositions can be in the form of capsules or cartridges (e.g., made of gelatin), which contain the compounds of the present invention and a powder suitable for a powder inhaler. Suitable powder matrices include, for example, lactose or starch.

[0022] Finally, known transdermal delivery systems and excipients can also be used to administer the drugs or pharmaceutical compositions of the present invention transdermally. For example, the active ingredient can be mixed with a permeation enhancer (such as propylene glycol, polyethylene glycol monolaurate, azacycloalkan - 2 - one, etc.) and incorporated into a patch or similar delivery system. If necessary, other excipients can also be used in the transdermal composition, including gelling agents, emulsifiers and buffers.

[0023] The dosage of the drug or pharmaceutical composition of the present invention can be appropriately varied according to the dosage form, the severity of symptoms, age, body weight of the patient to be administered, and the judgment of the physician. In the case of oral administration, generally for adults, the active ingredient can be administered once or in several divided doses at 0.01 - 5000 mg, preferably 0.1 - 2500 mg, more preferably 0.5 - 1000 mg per day. In the case of an injection, generally for adults, 0.0001 - 2000 mg can be administered once or in several divided doses. The optimal dosage can be established using conventional tests and procedures well known in the art.

[0024] The present invention confirmed the improvement effect of astragaloside Ⅲ on the spatial learning ability and memory ability of mice through the Morris maze experiment, and confirmed its therapeutic effect on cognitive dysfunction in vascular dementia. The present invention also performed pathological examinations on mouse brain sections, and the pathological examination results more intuitively reflected the therapeutic effect of astragaloside Ⅲ on related functional regions of the brain. In order to further study the therapeutic effect of astragaloside Ⅲ on vascular dementia, the present invention selected HT22 cells to establish a vascular dementia model, and compared the effects of astragaloside Ⅲ, astragaloside Ⅰ, and astragaloside Ⅳ on the active proliferation of a cell hypoxia and hypoglycemia injury model, and found that astragaloside Ⅲ had a significantly better effect. At the same time, the present invention also co-administered astragaloside Ⅲ and the positive drug donepezil, and confirmed that the two could play a synergistic effect in the treatment of vascular dementia. Description of the Drawings

[0025] Figure 1 It is a graph showing the detection results of cerebral blood flow by Doppler for each group (Control, Model, Astragaloside Ⅲ);

[0026] Figure 2 It is a graph showing the results of maze latency for each group (Control, Model, Astragaloside Ⅲ). * P < 0.1, ** P < 0.01, NS indicates no statistical difference;

[0027] Figure 3 It is a graph showing the maze paths of mice for each group (Control, Model, Astragaloside Ⅲ);

[0028] Figure 4 It is a schematic diagram of the hippocampal region of mouse brain sections for each group (Control, Model, Astragaloside Ⅲ);

[0029] Figure 5Cell viability results graphs for different drug administration groups (n = 8, Control group, Model group, Astragaloside I administration group, Astragaloside Ⅲ administration group, Astragaloside Ⅳ administration group, Donepezil administration group). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, NS indicates no statistical difference;

[0030] Figure 6 Cell viability results graphs for different drug administration groups (n = 8, Control group, Model group, Astragaloside Ⅲ administration group, Donepezil administration group, Astragaloside Ⅲ + Donepezil administration group). * P < 0.05, *** P < 0.001, **** P < 0.0001, NS indicates no statistical difference. Detailed implementation manners

[0031] The present invention will be further described below through examples. It should be understood that these examples are only for illustrative purposes and in no way limit the protection scope of the present invention.

[0032] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0033] Unless otherwise specified, the materials, reagents, etc. used in the following examples can all be obtained from commercial channels.

[0034] Example 1: Pharmacodynamic effects and mechanism of action of a traditional Chinese medicine monomer for preventing and treating vascular dementia on vascular dementia mice

[0035] I. Experimental materials and methods

[0036] 1. Experimental drugs and reagents

[0037] The experimental drug (Astragaloside Ⅲ) was purchased from Chengdu Mansite Biotechnology Co., Ltd.

[0038] 2. Experimental methods

[0039] (1) In this experiment, 30 male C57BL / 6 mice aged 6 - 8 weeks, SPF grade, with a mass of (20 ± 5) g were purchased from Liaoning Changsheng Biotechnology Co., Ltd. The animals were housed in the School of Pharmacy, Harbin Medical University.

[0040] (2) Animal model establishment: After adaptively feeding C57BL / 6 mice for one week, the method of intermittent permanent ligation of the left common carotid artery was used. The mice were anesthetized by intraperitoneal injection of avertin (1 g / kg body weight), fixed in the supine position on the operating table, the skin and subcutaneous tissue were incised in the middle of the neck, the subcutaneous connective tissue and the anterior cervical muscles were bluntly dissected, the left common carotid artery was exposed by avoiding the vagus nerve, the proximal and distal ends of the left common carotid artery were ligated, cut in the middle, and the subcutaneous connective tissue and skin were sutured layer by layer.

[0041] (3) Animal grouping and drug dosage: After adaptively feeding C57BL / 6 mice for one week, they were randomly divided into 3 groups, namely the control group (Control), the model group (Model), and the astragaloside III administration group (AstragalosideⅢ), with 10 mice in each group. A 1 ml syringe was used for gavage, and gavage was performed daily for 21 consecutive days. The dosage of the traditional Chinese medicine monomer was calculated according to the gavage dose in the relevant saponin drug literature for mice, and the administration concentration was 40 mg / kg. The control group was gavaged with deionized water.

[0042] (4) Doppler blood flow measurement: Before and 21 days after drug administration, the mice in each group were anesthetized, fixed in the prone position on the operating board, the head hair was removed with depilatory cream, the scalp was cut open to separate the muscle tissue to expose the skull, the transparent reagent S1 in the living skull transparency kit of Javis was applied on the skull for 10 minutes, then the transparent reagent S2 was evenly smeared on the mouse skull, and the mouse was placed under the probe of the Doppler blood flow detector to detect the blood flow distribution and velocity change in the mouse brain, and the corresponding data were recorded.

[0043] (5) The Morris water maze experiment is a classic method for evaluating the spatial learning and memory abilities of rodents, and can objectively reflect changes in the spatial memory ability of animals. The Morris experimental system mainly consists of three parts: a water maze device, an image acquisition system, and a software automatic analysis system. The water maze device consists of a circular pool and a platform with adjustable position and height. The test of the water maze in this experiment is divided into two parts: the place navigation experiment is carried out for the first 5 days, and the spatial exploration experiment is carried out on the 6th day. The place navigation experiment is mainly used to test the spatial learning ability of mice. At the beginning of each time, the mouse is gently placed into the water facing the pool wall in a selected quadrant, and each experimental mouse is given 60 seconds to swim to find the hidden platform. If the mouse successfully finds the platform within the given time, the mouse can rest on the platform for 10 seconds; if it fails to find the platform within the given time, the experimenter helps the mouse to guide it to the platform, and the mouse can also rest on the platform for 10 seconds. After each training session, the mouse is dried in time with a dry towel to avoid the influence of hypothermia on the mouse. After the place navigation experiment is over, the platform is removed, and the spatial exploration experiment is carried out the next day. The mouse is placed into the water at the same location in a selected quadrant, the swimming trajectory of the mouse within 60 seconds is recorded, and at the same time, the residence time of the mouse in the quadrant of the hidden platform and the number of times the hidden platform is crossed are recorded to observe the memory ability of the test mouse during spatial exploration. Mark the position of the hidden platform with blue dots on the computer so that the number of times passing through the position where the hidden platform is located can be counted.

[0044] (6) After obtaining the experimental data, the heart, liver, spleen, lungs, kidneys, and brain tissues of the mice are taken out for preservation for future use.

[0045] (7) The samples to be sent for inspection are fixed with 4% paraformaldehyde. After being in good fixation state, they are trimmed, dehydrated, embedded, sectioned, stained, coverslipped, and finally examined under the microscope according to the SOP procedure of the pathological experiment of this unit to obtain qualified slides.

[0046] (8) Browse the sections or digital sections under the microscope, and observe the tissue sections in detail at different magnifications. Describe in words the basic pathological changes in the sections such as congestion, stasis, hemorrhage, edema, degeneration, necrosis, hyperplasia, fibrosis, organization, granulation tissue, inflammatory changes, etc., and reflect the differences between the sections.

[0047] (9) Perform statistical processing on the results:

[0048] ① Before administration, measure the cerebral Doppler blood flow of mice in different groups respectively to determine whether the model is successfully constructed.

[0049] ② The Morris water maze test results were recorded to determine the spatial learning and memory abilities of mice. Graphpad prism 9.0 software was used for data statistical analysis. Measurement data were expressed as mean ± standard deviation (x±s); one-way analysis of variance (One-way ANOVA) was used for comparison among multiple groups, and t-test was used for comparison between groups; P<0.05 was considered statistically significant. Charts were drawn and statistical analysis was performed.

[0050] Example 2: Effects of different astragaloside monomers on cell viability in a cell hypoxia and glucose deprivation injury model

[0051] I. Experimental materials and methods

[0052] 1. Experimental drugs and reagents

[0053] The experimental drugs (astragaloside I, astragaloside III, astragaloside IV) were purchased from Chengdu Mansite Biotechnology Co., Ltd., and the positive drug donepezil was purchased from Eisai (China) Pharmaceutical Co., Ltd.

[0054] 2. Experimental methods

[0055] (1) Cell culture: HT22 cells were cultured in basal DMEM containing 10% FBS in a cell culture incubator at 37°C with 95% O2 and 5% CO2. The culture medium was changed every 2 days.

[0056] (2) Establishment of a cell hypoxia and glucose deprivation injury model by inducing HT22 cells with Na2S2O4: The cells were incubated with 1 mmol / L Na2S2O4 for 10 min, then washed twice with phosphate buffer (PBS), and the cells were cultured in low-glucose DMEM for subsequent studies.

[0057] (3) Cell grouping and drug administration: The experiment was divided into a Control group, a Model group, an astragaloside I (Astragaloside I) administration group, an astragaloside III (Astragaloside III) administration group, an astragaloside IV (Astragaloside IV) administration group, and a positive drug donepezil (Donepezil) administration group. The cells in the Control group were not treated with hypoxia and glucose deprivation injury or drug administration, while the cells in the Model group, the astragaloside I (Astragaloside I) administration group, the astragaloside III (Astragaloside III) administration group, the astragaloside IV (Astragaloside IV) administration group, and the positive drug donepezil (Donepezil) administration group all needed to be cultured after hypoxia and glucose deprivation injury treatment. After the model was established, the corresponding drugs were added respectively, and the cell drug dosage was 200 μmol / L for all groups. After culturing for 24 h, subsequent experiments were carried out.

[0058] (4) Detection by CCK-8 method: After the administration time ended, the cells were taken out of the incubator, the supernatant was removed, 100 μL of fresh medium was added to each well, and then 10 μL of CCK-8 was added to each well. The cells were put back into the cell incubator and incubated for 1 h, and then the absorbance was measured with an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 450 nm. Graphpad prism 9.0 software was used for data statistical analysis. Measurement data were expressed as mean ± standard deviation (x±s); one-way analysis of variance (One-way ANOVA) was used for comparison among multiple groups, and the t-test was used for comparison between groups; P<0.05 was considered statistically significant. Draw charts and perform statistical analysis.

[0059] Example 3: Effects of Astragaloside Ⅲ and the positive drug Donepezil on cell viability in a cell model of hypoxia and hypoglycemia injury

[0060] II. Experimental materials and methods

[0061] 1. Experimental drugs and reagents

[0062] The experimental drug (Astragaloside Ⅲ) was purchased from Chengdu Mansite Biotechnology Co., Ltd., and the positive drug Donepezil was purchased from Eisai (China) Pharmaceutical Co., Ltd.

[0063] 2. Experimental methods

[0064] (1) Cell culture: HT22 cells were cultured in basal DMEM containing 10% FBS and placed in a cell incubator at 37 °C with 95% O2 and 5% CO2. The medium was changed every 2 days.

[0065] (2) Establishment of a hypoxia and hypoglycemia injury model in HT22 cells induced by Na2S2O4: The cells were incubated with 1 mmol / L Na2S2O4 for 10 min, then washed twice with phosphate buffer (PBS), and the cells were cultured in low-glucose DMEM for subsequent studies.

[0066] (3) Cell grouping and administration: The experiment was divided into a Control group, a Model group, an Astragaloside Ⅲ administration group, a positive drug Donepezil administration group, and an Astragaloside Ⅲ + Donepezil administration group. The cells in the Control group were not subjected to oxygen-glucose deprivation injury treatment or drug administration, while the cells in the Model group, Astragaloside Ⅲ (Astragaloside Ⅲ) administration group, positive drug Donepezil (Donepezil) administration group, and Astragaloside Ⅲ + Donepezil (Astragaloside Ⅲ + Donepezil) administration group all needed to be cultured after being treated in an oxygen-glucose deprivation injury environment. After modeling, the corresponding drugs were added respectively, and the cell drug dosage was 200 μmol / L. The molar ratio of Astragaloside Ⅲ to Donepezil in the Astragaloside Ⅲ + Donepezil (Astragaloside Ⅲ + Donepezil) administration group was 1:1. After culturing for 24 h, subsequent experiments were carried out.

[0067] (4) Detection by CCK-8 method: After the administration time ended, the cells were taken out of the incubator, the supernatant was removed, 100 μL of fresh medium was added to each well, and then 10 μL of CCK-8 was added to each well. Then it was put back into the cell incubator and incubated for 1 h, and then the absorbance was measured with an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 450 nm. Graphpad prism 9.0 software was used for data statistical analysis. Measurement data were expressed as mean ± standard deviation (x ± s); one-way analysis of variance (One-way ANOVA) was used for comparison among multiple groups, and t-test was used for comparison between groups; P < 0.05 was considered statistically significant. Draw charts and conduct statistical analysis.

[0068] Experimental results:

[0069] According to Figure 1 the results, after administering drugs to mice, the cerebral blood flow changes in each group of mice were monitored by a Doppler blood flow detector, which proved that the vascular dementia model was successfully constructed and the cerebral blood flow in vascular dementia mice was improved after administration of Astragaloside Ⅲ.

[0070] According to Figure 2 the results, after training, compared with the Model group, the maze latency time in the Astragaloside Ⅲ administration group was significantly shortened, and the cognitive dysfunction of mice was improved (P < 0.01).

[0071] According to Figure 3The results showed that the route taken by the Model group mice to find the target platform was complex, indicating that VD mice had decreased spatial learning and memory abilities and cognitive dysfunction. Compared with the maze trajectory of the Model group mice, the maze trajectory of the Astragaloside III-treated mice was significantly clearer, and they were able to find the target hole in a shorter distance.

[0072] according to Figure 4 The results showed that the number of neurons in the hippocampus of the control group was abundant and tightly arranged, the neuron morphology and structure were complete and clear, the nucleus and cytoplasm were clearly divided, the nucleolus was obvious, and no obvious inflammation was observed; the hippocampus of the model group showed sparse cells, sparse structure, thinning or even missing cell layers, deformed neuronal cell bodies, and dark staining and condensation of the nuclei (black arrow); the nuclei of some neurons in the hippocampus of the astragaloside III group were darkly stained and condensed, and the cell bodies showed polygonal changes, but the neurons were arranged more neatly, and the structure was clearer and thicker. Compared with the model group, the pathological damage of neurons in the hippocampus was significantly improved (black arrow).

[0073] according to Figure 5 The results showed that compared with the Control group, the cell proliferation activity in the Model group was significantly decreased (P<0.0001), and the vascular hypoxia-glucose deprivation injury model in the Model group was successfully established. After 24 hours of administration, it was found that Astragaloside III promoted the proliferation of HT22 cells, but its effect on cell proliferation was slightly lower than that of the positive drug donepezil, but significantly higher than Astragaloside I and Astragaloside IV (P<0.01).

[0074] according to Figure 6 The results showed that compared with the Control group, the cell proliferation activity of the Model group was significantly decreased (P<0.0001), and the hypoxia-glucose deprivation injury model of the Model group was successfully established. The synergistic administration of donepezil and astragaloside III at the same dose was better than that of astragaloside III and donepezil alone (P<0.001 and P<0.05), showing a synergistic effect.

[0075] In summary, the Chinese medicine monomer of the present invention can improve cognitive dysfunction and cerebrovascular lesions in mice with vascular dementia, and also has a certain therapeutic effect on the relevant brain functional areas of mice with cognitive dysfunction due to vascular dementia, and has explorable clinical medical prospects.

[0076] The above describes the preferred embodiments of the present invention, but it is not intended to limit the present invention. Those skilled in the art may make improvements and changes to the embodiments disclosed herein without departing from the scope and spirit of the present invention.

Claims

1. Use of astragaloside III or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing and treating dementia.

2. The use according to claim 1, characterized in that: The dementia includes vascular dementia and Alzheimer's disease, preferably vascular dementia.

3. The use according to claim 1 or 2, characterized in that The medicament further comprises a pharmaceutically acceptable carrier or excipient.

4. A pharmaceutical composition comprising astragaloside III or a pharmaceutically acceptable salt thereof and donepezil.

5. The pharmaceutical composition according to claim 4, characterized in that The pharmaceutical composition is used for preventing and treating dementia.

6. The pharmaceutical composition according to claim 5, characterized in that The dementia includes vascular dementia and Alzheimer's disease, preferably vascular dementia.

7. The pharmaceutical composition according to any one of claims 4 to 6, characterized in that The molar ratio of astragaloside III to donepezil is 0.5-2:1, preferably 0.8-1.2:1, and more preferably 1:

1.

8. The pharmaceutical composition according to any one of claims 4 to 7, characterized in that The pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

9. Use of the pharmaceutical composition according to any one of claims 4 to 8 in the preparation of a medicament for preventing and treating dementia.

10. The use according to claim 1, characterized in that: The dementia includes vascular dementia and Alzheimer's disease, preferably vascular dementia.

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