Traditional Chinese medicine composition for treating Alzheimer disease dementia as well as preparation and application of traditional Chinese medicine composition

Abstract

The invention relates to a traditional Chinese medicine composition for treating Alzheimer disease dementia. The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 15-45 parts of astragalus membranaceus, 6-18 parts of lucid ganoderma, 6-18 parts of radix polygoni multiflori preparata, 5-15 parts of cistanche, 5-15 parts of dendrobe, 4-12 parts of fructus lycii, 4-12 parts of poria cocos, 3-9 parts of ginseng, 2.5-7.5 parts of rhizoma cimicifugae and 2.5-7.5 parts of radix pseudostellariae. According to the present invention, liver-kidney yin deficiency is adopted as the basic symptom expression, and in the formula, the radix astragali, the ginseng and the radix pseudostellariae are combined to tonify spleen-lung qi; the prepared fleece-flower root, the cistanche and the barbary wolfberry fruit jointly have the effects of nourishing liver and kidney and benefiting essence and blood; ganoderma lucidum is matched with ginseng to tonify qi and tranquilize The rhizoma cimicifugae can lift yang qi, and the poria cocos can promote diuresis and reduce phlegm and can assist qi tonifying medicines (astragalus membranaceus, ginseng and the like) to enable clear qi to reach the brain and reduce foul qi; dendrobium nobile mainly plays a role in nourishing yin and curing dryness. The traditional Chinese medicine is mainly used for tonifying kidney and qi, accords with the treatment principle of tonifying deficiency, has the effects of reducing phlegm, soothing the nerves and the like, can synergistically improve the Alzheimer disease dementia in multiple aspects, and is high in universality and remarkable in curative effect.

Description

Technical Field

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

[0002] Dementia is a syndrome caused by brain lesions, with clinical features of multiple cognitive impairments such as memory, comprehension, judgment, reasoning, calculation and abstract thinking, which may be accompanied by hallucinations, delusions, behavioral disorders and personality changes. Alzheimer's disease refers to various types of dementia occurring after the age of 65, among which Alzheimer's disease (AD) is the most common type of dementia. As the aging of my country's population continues to deepen, the incidence, prevalence and mortality of Alzheimer's disease have also increased significantly, bringing heavy pressure to society.

[0003] Traditional Chinese medicine has unique theories and rich practical experience in the treatment of Alzheimer's disease. Combining the classical theories of traditional Chinese medicine with clinical practice to find effective treatments is of great significance for alleviating the symptoms of cognitive impairment in patients with Alzheimer's disease and improving the quality of life of patients. In the field of traditional Chinese medicine, Alzheimer's disease is classified as a disease syndrome, literary idiocy, forgetfulness, language reversal, madness, depression, etc., with the characteristics of slow onset and diverse clinical manifestations. The traditional Chinese medicine community holds a relatively consistent view in the theoretical research of Alzheimer's disease - dementia is located in the brain, and the disease is a deficiency in the root and excess in the symptoms. The characteristics of the disease are mostly kidney deficiency, involving the four internal organs of the heart, liver, spleen, and lungs. It is a type of disease syndrome with phlegm and blood stasis as the symptoms, and a mixture of deficiency and excess. Therefore, the treatment of Alzheimer's disease should follow the principle of "tonifying the deficiency", with kidney tonification as the main focus, and according to different syndromes, supplemented with prescriptions such as promoting blood circulation and removing blood stasis, awakening the spleen and removing dampness, removing phlegm and opening the orifices, and clearing the heart and purging fire to adjust the rise and fall of the body's qi, blood, yin and yang, reflecting the principle of traditional Chinese medicine treatment of treating both the symptoms and the root.

[0004] The failed experience in the research and development of anti-dementia drugs mainly targeting AD over the years has made researchers at home and abroad realize that the treatment of dementia requires early diagnosis and multi-target treatment. Traditional Chinese medicine has the characteristics of multi-target, multi-pathway and safety, and has inherent advantages in the treatment of Alzheimer's disease mainly caused by AD.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a Chinese medicine composition for treating Alzheimer's dementia and its preparation and use.

[0007] The technical solution adopted by the present invention is:

[0008] In one aspect, the present invention provides a Chinese medicine composition for treating Alzheimer's dementia, comprising the following raw materials in parts by weight:

[0009] Astragalus membranaceus 15 - 45 parts, Ganoderma lucidum 6 - 18 parts, Polygonum multiflorum preparatum 6 - 18 parts, Cistanche deserticola 5 - 15 parts, Dendrobium officinale 5 - 15 parts, Lycium barbarum 4 - 12 parts, Poria cocos 4 - 12 parts, Panax ginseng 3 - 9 parts, Cimicifuga foetida 2.5 - 7.5 parts, Pseudostellaria heterophylla 2.5 - 7.5 parts.

[0010] A traditional Chinese medicine composition for treating Alzheimer's dementia proposed by the present invention has the basic symptom manifestation of liver - kidney yin deficiency. In the formula, Astragalus membranaceus, Panax ginseng, and Pseudostellaria heterophylla are used together to tonify the qi of the spleen and lung; Polygonum multiflorum preparatum, Cistanche deserticola, and Lycium barbarum together play the role of tonifying the liver and kidney and nourishing essence and blood; Ganoderma lucidum cooperates with Panax ginseng to replenish qi and soothe the nerves; Cimicifuga foetida can lift yang qi, and Poria cocos can promote diuresis and resolve phlegm, which can assist the qi - tonifying herbs (such as Astragalus membranaceus and Panax ginseng) to make the clear qi reach the brain and the turbid qi descend; Dendrobium officinale mainly plays a coordinating function of nourishing yin and relieving dryness. The whole formula focuses on tonifying the kidney and replenishing qi, in line with the treatment principle of "treating deficiency with tonics", and at the same time takes into account the effects of resolving phlegm and soothing the nerves, etc. It can synergistically improve Alzheimer's dementia in multiple aspects, with strong generality and remarkable curative effect.

[0011] Specifically, according to traditional Chinese medicine theory, in the formula, Astragalus membranaceus can replenish qi and lift yang, secure the exterior and stop sweating, generate body fluid and nourish blood, being sweet and warm and tonifying the middle - jiao, greatly tonifying the qi of the spleen and lung; Ganoderma lucidum, with a sweet taste and a neutral nature, belongs to the heart, lung, liver, and kidney meridians, and its efficacy is to replenish qi and soothe the nerves; Polygonum multiflorum preparatum can nourish essence and blood, replenish qi and generate marrow, effectively reduce hyperlipidemia, and inhibit the increase of cholesterol, triglyceride, and free cholesterol in plasma, enhancing lipid metabolism; Cistanche deserticola, with a sweet and astringent taste and a warm nature, can tonify the kidney yang and nourish essence and blood; Dendrobium officinale, with a sweet taste and a slightly cold nature, belongs to the stomach and kidney meridians, can benefit the stomach and generate body fluid, nourish yin and clear heat; Lycium barbarum, with a neutral nature and a sweet taste, belongs to the liver and kidney meridians, can nourish the liver and kidney, benefit essence and improve eyesight, and belongs to the yin - nourishing herbs under the classification of tonics for deficiency; Poria cocos, with a sweet and light taste and a neutral nature, enters the heart, spleen, lung, and kidney meridians, and has the effects of promoting diuresis and percolating dampness, benefiting the spleen and stomach, protecting the kidney, calming the nerves, and generating body fluid; Panax ginseng, with a warm nature, sweet, slightly bitter, and slightly warm taste, belongs to the spleen and lung meridians, can replenish qi and generate body fluid, soothe the nerves and improve intelligence; Cimicifuga foetida, with a pungent taste, slightly sweet, and slightly cold nature, belongs to the lung, spleen, stomach, and large intestine meridians, and has the effects of inducing sweating and rash, clearing heat and detoxifying, and lifting yang qi; Pseudostellaria heterophylla, with a neutral nature and a sweet taste, belongs to the spleen and lung meridians, can replenish qi and strengthen the spleen, generate body fluid and moisten the lungs.

[0012] In addition, combined with pharmacodynamic research, it shows that the traditional Chinese medicine composition of the present invention can effectively treat Alzheimer's dementia through multiple targets and multiple mechanisms, such as reducing oxidative damage of brain tissue, inhibiting deposition of amyloid - β protein in brain tissue, and reducing neuronal pathological damage or apoptosis.

[0013] Preferably, it includes raw materials by weight:

[0014] Astragalus membranaceus 30 parts, Ganoderma lucidum 12 parts, Polygonum multiflorum preparatum 12 parts, Cistanche deserticola 10 parts, Dendrobium officinale 10 parts, Lycium barbarum 8 parts, Poria cocos 7 parts, Panax ginseng 6 parts, Cimicifuga foetida 5 parts, Pseudostellaria heterophylla 5 parts.

[0015] On the other hand, the present invention provides a method for preparing a traditional Chinese medicine composition for treating Alzheimer's dementia as described in any one of the above technical solutions, comprising the following steps:

[0016] S1. Weigh the raw materials according to parts by weight, mix them, add water 3 - 6 times the total weight of the raw materials, soak for 30 - 90 min, then add water to 10 times the total weight of the raw materials, decoct for 45 min to obtain a first decoction and medicinal residues, and then add water 10 times the total weight of the raw materials to the medicinal residues, decoct for 45 min to obtain a second decoction;

[0017] S2. Combine the first decoction and the second decoction to obtain a water decoction of the traditional Chinese medicine composition for treating Alzheimer's dementia.

[0018] The method for preparing the traditional Chinese medicine composition of the present invention is simple and has a low cost, and is suitable for industrial production.

[0019] Preferably, after step S2, it further includes filtering the combined decoction, concentrating the filtrate under reduced pressure to a thick paste, and drying to obtain a dry paste of the traditional Chinese medicine composition for treating Alzheimer's dementia.

[0020] Preferably, after step S2, it further includes subjecting the combined decoction to freeze - drying treatment to obtain a freeze - dried powder of the traditional Chinese medicine composition for treating Alzheimer's dementia.

[0021] More preferably, the freeze - drying treatment includes the following steps: pre - cooling, vacuum pumping, primary sublimation, and analytical drying.

[0022] On yet another aspect, the present invention provides an application of the traditional Chinese medicine composition as described in any one of the above technical solutions in the preparation of a drug for treating Alzheimer's dementia.

[0023] The traditional Chinese medicine composition of the present invention can be applied to the preparation of drugs for preventing or significantly improving Alzheimer's dementia, and has great clinical application value.

[0024] Preferably, pharmaceutically acceptable excipients are added to the traditional Chinese medicine composition to prepare an oral preparation, and the dosage forms of the oral preparation include tablets, capsules, granules, pills or liquid oral preparations. Description of the Drawings

[0025] Figure 1 It is a diagram showing the influence of the traditional Chinese medicine composition of the present invention on the paralysis rate and median paralysis time of CL4176 nematodes in Example 3. Among them, A is the influence of different concentrations of A17 - 3 on the paralysis rate of CL4176 nematodes; B is the influence of different concentrations of A17 - 3 on the median paralysis time of CL4176 nematodes;

[0026] Figure 2 Effect diagram of the traditional Chinese medicine composition of the present invention on the chemotaxis of CL2355 and CL2122 nematodes in Example 3;

[0027] Figure 3 Effect of the traditional Chinese medicine composition of the present invention on the antioxidant stress level of N2 nematodes in Example 3. Among them, A is the effect of different concentrations of A17-3 on the survival rate of N2 nematodes under the oxidative stress conditions induced by paraquat; B is the median survival time of N2 nematodes under the oxidative stress conditions induced by paraquat at different concentrations of A17-3. Taking S-com as the Control, ***P<0.001;

[0028] Figure 4 Comparison diagram of the exploration ability and learning and memory ability of mice in each group in the novel object recognition experiment in Example 3. Taking ddH 2 O as the Control, n≥6, *P<0.05, **P<0.01, ****P<0.0001;

[0029] Figure 5 Comparison diagram of the motor ability of mice in each group in the water maze experiment in Example 3. Among them, A is the movement speed of each group of mice during the experiment; B is the total movement distance of each group of mice during the experiment. Taking ddH 2 O as the Control, n≥6, ns represents no significant difference;

[0030] Figure 6 Comparison diagram of the average latency of mice in each group in the place navigation experiment of the water maze in Example 3. Among them, A is the average latency of each group of mice during the experiment (5 training days); B is the average latency of each group of mice on the 5th day during the experiment. Taking ddH 2 O as the Control, n≥6, *P<0.05;

[0031] Figure 7 Comparison diagram of the typical swimming paths of mice in each group in the spatial exploration experiment of the water maze in Example 3;

[0032] Figure 8 Comparison diagram of the residence time in the quadrant where the platform is located in the spatial exploration experiment of the water maze of mice in each group in Example 3. Taking ddH 2 O as the Control, n≥6, **P<0.01, ***P<0.001, ****P<0.0001;

[0033] Figure 9 Pathological section diagram of the hippocampal region of the brain tissue of mice in each group in Example 3 (CA3 region, HE staining, ×20);

[0034] Figure 10It is the pathological section diagram of the hippocampal region of the brain tissue of mice in each group in Example 3 (immunohistochemistry method, ×20);

[0035] Figure 11 It is the comparison diagram of the transcriptional level expression of BDNF factor in the brain homogenate of mice in each group in Example 3;

[0036] Figure 12 It is the comparison diagram of the expression levels of Aβ at the transcriptional level and protein level in the brain homogenate of mice in each group in Example 3. Among them, A is the detection of the transcriptional level of Aβ in the brains of mice in each group by RT-qPCR, with β-Actin as the internal reference, and 2^ -ΔΔCt is used to calculate the change in the transcriptional level of the gene after drug administration treatment, *P<0.05, **P<0.01; B is the detection of the Aβ protein level in the brains of mice in each group by WB, with β-Actin as the internal reference, and the molecular weight is 42kD; C is the Aβ oligomer level in the brains of mice in each group, and ImageJ is used to perform gray scale analysis on the bands, *P<0.05;

[0037] Figure 13 It is the comparison diagram of the MDA content in the brain homogenate of mice in each group in Example 3, with ddH 2 O as the Control, *P<0.05;

[0038] Figure 14 It is the comparison diagram of the GSH content in the brain homogenate of mice in each group in Example 3, with ddH 2 O as the Control, ***P<0.001;

[0039] Figure 15 It is the comparison diagram of the transcriptional level expression of antioxidant stress-related factors in the brain homogenate of mice in each group in Example 3. Among them, A is the transcriptional level of HO-1, *P<0.05, ****P<0.0001; B is the transcriptional level of CAT, **P<0.01, ***P<0.001. Detailed implementation manners

[0040] In order to better understand the above technical solutions, the exemplary embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although the exemplary embodiments of the present invention are shown in the drawings, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided to enable a more clear and thorough understanding of the present invention and to be able to convey the scope of the present invention completely to those skilled in the art.

[0041] The decoction, dry extract or freeze-dried powder prepared based on the formula of the traditional Chinese medicine composition for treating Alzheimer's dementia in the embodiments of the present invention is abbreviated as "A17-3".

[0042] Preparation of Dry Extract of Traditional Chinese Medicine Composition for Treating Alzheimer's Dementia in Example 1

[0043] A traditional Chinese medicine composition for treating Alzheimer's dementia, comprising raw materials by weight:

[0044] Astragalus membranaceus 30g, Ganoderma lucidum 12g, processed Polygonum multiflorum 12g, Cistanche deserticola 10g, Dendrobium officinale 10g, Lycium barbarum 8g, Poria cocos 7g, Ginseng 6g, Cimicifuga foetida 5g, Pseudostellaria heterophylla 5g.

[0045] It is prepared by the following method, including the following steps:

[0046] S1. Weigh the raw materials according to the parts by weight, mix them, add water 5 times the total weight of the raw materials, soak for 30 minutes, then add water to 10 times the total weight of the raw materials, decoct for 45 minutes to obtain the first decoction liquid and the medicinal residues, and then add water 10 times the total weight of the raw materials to the medicinal residues, decoct for 45 minutes to obtain the second decoction liquid;

[0047] S2. Combine the first decoction liquid and the second decoction liquid, filter, concentrate the filtrate under reduced pressure to a thick paste, and dry to obtain the dry extract of the traditional Chinese medicine composition for treating Alzheimer's dementia.

[0048] Add pharmaceutically acceptable excipients to the above dry extract, and tablets, capsules, granules, pills or oral liquid preparations can be prepared according to conventional methods.

[0049] Preparation of Freeze-dried Powder of Traditional Chinese Medicine Composition for Treating Alzheimer's Dementia in Example 2

[0050] The main difference between this example and Example 1 is that the form of the traditional Chinese medicine composition for treating Alzheimer's dementia obtained is different, and the rest remains unchanged.

[0051] The specific preparation method includes the following steps:

[0052] S1. Weigh the raw materials according to the parts by weight, mix them, add water 5 times the total weight of the raw materials, soak for 30 minutes, then add water to 10 times the total weight of the raw materials, decoct for 45 minutes to obtain the first decoction liquid and the medicinal residues, and then add water 10 times the total weight of the raw materials to the medicinal residues, decoct for 45 minutes to obtain the second decoction liquid;

[0053] S2. Combine the first decoction liquid and the second decoction liquid, and perform freeze-drying treatment including pre-cooling, vacuum pumping, primary sublimation, and analytical drying to obtain the freeze-dried powder of the traditional Chinese medicine composition for treating Alzheimer's dementia.

[0054] Application of Traditional Chinese Medicine Composition in Treating Alzheimer's Dementia in Example 3

[0055] 1. Materials

[0056] 1.1 Experimental animals

[0057] Transgenic AD Caenorhabditis elegans models CL4176, CL2355, CL2122 and wild-type Caenorhabditis elegans N2 were used; transgenic AD mouse model APP / PS1 and its wild-type littermate negative control were used to observe the pharmacological effects of the traditional Chinese medicine composition of the present invention on the classical AD model and determine its effect in treating Alzheimer's dementia.

[0058] 1.2 Experimental drugs

[0059] The traditional Chinese medicine composition prepared in Example 1 or 2 was used as the test drug (the daily dosage for adults was equivalent to 105 g of crude drug), and donepezil hydrochloride (brand name: Aricept, batch number H20050978) was used as the positive control drug (specification: 5 mg per capsule, and the clinical daily dosage was 1 capsule).

[0060] Dosage of the traditional Chinese medicine composition: The dry extract of the traditional Chinese medicine composition in Example 1 was administered by gavage at a dose of 0.1 ml / 10 g of mouse body weight. The high-concentration (10X, diluted with water by 10 times the daily human dosage) and low-concentration (5X, diluted with water by 5 times the daily human dosage) dosing doses were specifically calculated according to the mouse body weight.

[0061] The freeze-dried powder of the traditional Chinese medicine composition in Example 2 was used for subsequent C. elegans medication.

[0062] Dosage of the positive control drug: Calculated based on the daily dosage of 5 mg for an adult (60 kg), and expanded 10 times to obtain the final positive dosing dose for mice, which was calculated to be 0.833 g / kg. Donepezil hydrochloride was taken and dissolved in water to form an 83.33 mg / mL solution. The dosing volume was 0.1 ml / 10 g of body weight, and the test dose for mice was specifically calculated according to the body weight.

[0063] 1.2 Grouping and dosing of mice

[0064] The transgenic APP / PS1 mice were randomly divided into a model group, a positive control group (donepezil hydrochloride group), a high-concentration traditional Chinese medicine composition group (A17-3(10X) group), and a low-concentration traditional Chinese medicine composition group (A17-3(5X) group), with 7 - 9 mice in each group. Gavage administration started at 6 months of age. Each group of mice was gavaged once a day at a volume of 0.1 ml / 10 g for three consecutive months. The wild-type control group and the model group were given an equal volume of solvent water, and each intervention group was given the corresponding drug. After the dosing was completed, specimens were collected for analysis. The specific grouping and dosing of mice are shown in Table 1.

[0065] Table 1

[0066]

[0067] 2. Experimental methods

[0068] 2.1 Determination methods for each index of the nematode model

[0069] Synchronization treatment of nematodes:

[0070] First, rinse and collect the nematodes on the culture medium with M9 buffer, centrifuge at 1200 rpm for 2 min to remove OP50 in the collected liquid. Add 1 mL of lysis solution and vortex for 4 min. Immediately after lysis, add 5 - 7 mL of M9 buffer, centrifuge at 4000 rpm for 3 min, and aspirate the supernatant. Resuspend the eggs with S-complete, place the synchronized eggs at the culture temperature corresponding to the strain, and observe the hatching of the eggs under a microscope 24 h later.

[0071] (1) Paralysis experiment to determine the paralysis rate and median paralysis time of CL4176 nematodes

[0072] Take an EP tube, add 10 μL of Escherichia coli bacterial solution, 10 μL of the drug solution, and 30 - 40 synchronized CL4176 nematodes, and make up to 100 μL with S-complete; after mixing, transfer to an NGM medium without OP50, culture at 16 °C for 36 h, then transfer to 23 °C and continue to culture for 24 h; record and analyze the paralysis situation under a microscope.

[0073] (2) Chemotaxis experiment to determine the chemotaxis of CL2355 and CL2122 nematodes

[0074] Place the synchronized CL2355 transgenic nematodes and their control strain CL2122 in an NGM medium containing the test drug and OP50 for culture; culture the nematodes at 16 °C for 36 h and then transfer to 23 °C for 36 h; collect the nematodes, wash them with M9 buffer, centrifuge at 1200 rpm for 2 min, discard the supernatant, and wash three times; transfer the collected nematodes to the center of a solid NGM plate without bacteria coating. Drop 10 μL of the attractant (1 M sodium acetate: 1 M sodium azide = 1:1 v / v) and 10 μL of the blank solution (sterile water: 1 M sodium azide = 1:1 v / v) on both sides of the plate respectively. After placing it upside down at room temperature for 2 h, record the number of nematodes on the side of the attractant, the side of the blank control, and in the center of the plate, count the nematodes in each quadrant respectively, and calculate the chemotaxis index (CI).

[0075] (3) Paraquat-induced oxidative stress experiment to determine the antioxidant stress ability of N2 nematodes

[0076] Dilute the synchronized worm solution to 120 worms / mL with S-com; after mixing evenly, aliquot it into transparent 96-well plates (110 μL / well), seal the plates with high-transparency sealing film after aliquoting, and incubate at 20 °C for 45 h. Subsequently, add 25 μL of 1.08 mM FUdR solution to each well to inhibit nematode egg laying and hatching of the laid eggs in the plates. After sealing, shake and mix evenly for 1 min, and continue to incubate for 24 h; then add 15 μL of the test drug, seal and shake to mix evenly, and continue to incubate; collect the nematodes after the adult D5 of the drug treatment, wash away OP50 and the drug with M9 containing 1% Tween-20, and then spread the collected nematodes in the drug-treated group and the control group on 35 mm sterile NGM medium for standby; pick the collected nematodes into 96-well plates containing 90 μL of M9 at 15 worms / well, and the number of nematodes in each treatment group is not less than 60. Add 50 mM paraquat solution prepared with 30 μL of M9 to the 96-well plates, shake and mix evenly for 1 min, and place them in a 20 °C incubator; set the time when paraquat is added as the 0th hour, and then record the number of dead nematodes in the plates every 24 h until all the nematodes in the plates die. Finally, analyze and draw the corresponding paraquat tolerance survival curve.

[0077] 2.2 Determination methods for each index of the mouse model

[0078] (1) The novel object recognition test was used to measure the exploratory ability and learning and memory ability of mice in each group

[0079] For the experiment, a soundproof box with a size of 60 cm × 60 cm was selected, and marks were made at the diagonal positions at the bottom. Before the experiment started, transfer the mice to the behavioral laboratory 1 - 2 days in advance to let the animals adapt to the experimental environment. On the first day of the training period, place two exactly the same objects at the set positions in the test box, and then put the experimental animals into the test box from a certain fixed corner. Each animal was set with a 10-min free exploration time. On the second day of the test period, replace one of the objects with a novel object, place the animal at the same position as in the training period, and the test time is 10 min. Record the time for the animal to explore the novel and old objects. According to PI = a / (a + b), calculate the PI value, where a is the time to explore the novel object and b is the time to explore the old object.

[0080] (2) The Morris water maze test was used to measure the spatial learning and memory ability of mice in each group

[0081] The water maze apparatus was a 100 cm × 100 cm pool, with a platform set in one of the quadrants. The water level was 1 cm above the platform to explore the ability of animals to learn to find the hidden platform. All experimental animals were transferred to the experimental environment one day in advance to adapt for one night. From the 1st day to the 5th day was the acquisition training. The animals were placed into the water with their heads facing the pool wall from any quadrant, and a platform was set in one of the quadrants. Observe the time it takes for the animals to reach the platform. If the mice cannot find the platform within 60 s, they were guided to the platform and allowed to stay on the platform for 20 s. Each mouse was trained in four quadrants every day. The 6th day was the test period. After removing the platform, the animals were placed into the water with their heads facing the pool wall from the diagonal quadrant of the platform, and relevant indicators such as the latency to reach the target area, the number of times reaching the platform, and the residence time in the target quadrant were recorded.

[0082] Mouse sampling and brain homogenate preparation: On the second day after the mouse behavior experiment ended, the mice were anesthetized (with 5% chloral hydrate), the chest cavity was quickly opened, and perfusion was carried out through the heart with 0.86% normal saline. When the color of the blood gradually changed from dark red to transparent, the mice were decapitated, the cranial cavity was opened, and the brain tissue was taken on ice. The left and right brains were separated. The left brain was first fixed with 4% formaldehyde and placed in a 4℃ refrigerator. After 24 h, it was dehydrated with 20% sucrose, and then after another 24 h, it was dehydrated with 30% sucrose. After dehydration, the brain was embedded with embedding agent for later use; the right brain was accurately weighed, 0.86% normal saline was added at a ratio of 1:9, and it was ground thoroughly with a grinding pestle for 4 min, centrifuged at 3500 rpm for 10 min, and the supernatant was taken as 10% brain homogenate, which was aliquoted into 0.6 mL EP tubes, labeled, and stored in a -80℃ refrigerator for later use.

[0083] (3) HE staining was used to determine the pathological changes in the brains of mice in each group

[0084] After the mice were sacrificed and the brain tissue was taken, it was fixed with paraformaldehyde, dehydrated and cleared, infiltrated with wax, embedded, sectioned with paraffin, dewaxed, stained with 0.5% hematoxylin, differentiated with hydrochloric acid alcohol, stained with eosin, dehydrated, cleared, and mounted. The results were observed under a microscope.

[0085] (4) Immunohistochemistry was used to determine the expression of Aβ in the brains of mice in each group

[0086] Take the paraffin-embedded brain tissue and section it at a thickness of 5 μm. Dewax it and perform antigen repair for 15 min. Wash the slides with PBS, and incubate them with 3% hydrogen peroxide and 3% fetal bovine serum at room temperature in the dark for 25 min and 30 min respectively. Wash the sections, add the primary antibody Aβ (1:200), and incubate overnight at 4℃. Add the secondary antibody (1:200) and incubate at room temperature for 1 h. Wash the sections, add DAB chromogenic solution, dehydrate with gradient ethanol and xylene, and mount. Observe the sections under an upright optical microscope and take pictures.

[0087] (5) WB and RT-qPCR were used to detect the expression of Aβ in the brains of mice in each group

[0088] Extract RNA from the previously prepared mouse brain homogenate, then perform reverse transcription to obtain cDNA, and use the cDNA for RT-qPCR. Extract proteins from the brain homogenate, prepare a three-layer Tricine-SDS-PAGE gel and then perform gel electrophoresis. Since the molecular weight of the Aβ protein is relatively small, a semi-dry transfer membrane apparatus is used for membrane transfer at a constant voltage of 12 V for 17 min. Take out the transferred membrane and place it in a blocking box, pour in the rapid blocking solution, and slowly shake it on an oscillator for 15 - 20 min. After blocking, quickly wash it with 1×TBST, once every 10 min, for three times. Incubate with the primary antibody (dilution ratio 1:1000) overnight in a 4°C refrigerator. Recover the primary antibody, quickly wash it with 1×TBST, once every 10 min, for three times. Incubate with the secondary antibody (dilution ratio 1:2000) at room temperature for 1 h. Recover the secondary antibody, quickly wash it with 1×TBST, once every 10 min, for three times. Drop the developing solution on the surface of the membrane and develop it in a developer after 1 min.

[0089] (6) Detect the contents of GSH and MDA in the brain homogenates of mice in each group

[0090] Take the previously prepared mouse brain homogenate and operate according to the kit instructions to detect the contents of GSH and MDA in the brain homogenates of mice in each group.

[0091] (7) Detect the neuroprotective factor BDNF, oxidative stress-related factors CAT, and HO-1 in the brains of mice in each group

[0092] Extract RNA from the previously prepared mouse brain homogenate, then perform reverse transcription to obtain cDNA, and use the cDNA for RT-qPCR to detect each factor.

[0093] All the above experimental data were analyzed using Graphpad Prism 8.0 software, and the experimental data were expressed as X±SD. The experimental results were evaluated for inter-group differences using the oneway-anova test method. When P<0.05, it indicates that the difference is statistically significant.

[0094] 3. Experimental results

[0095] 3.1 C. elegans model analysis

[0096] The present invention uses a C. elegans model to preliminarily evaluate the efficacy of the traditional Chinese medicine composition of the present invention by examining its paralysis rate, chemotaxis, antioxidant stress ability, etc.

[0097] From Figure 1 and Table 2, it can be seen that compared with the median paralysis time (PT of the control group 50) Compared with [comparison object not provided], A17-3 at 4 different concentrations could all delay the paralysis rate of CL4176 nematodes, and the effects of A17-3 at concentrations of 0.1 mg / mL, 0.5 mg / mL, and 1 mg / mL in delaying paralysis were significant. After administration, the percentage of PT 50 elongation were 11.81%, 26.58%, and 27.71% respectively. This result preliminarily proves that the traditional Chinese medicine composition of the present invention has the effect of delaying the paralysis of CL4176 nematodes caused by Aβ.

[0098] Table 2

[0099]

[0100] Note: Compared with the control group, * represents P < 0.05, *** represents P < 0.001, **** represents P < 0.0001. Using S-complete as the Control, the experiment was repeated in three batches, and the number of worms in each group in each batch was not less than 40.

[0101] From Figure 2 and Table 3, it can be seen that the control strain CL2122 without the Aβ gene transfer had normal chemotactic behavior, manifested as most of the nematodes in the detection plate being sensitive to the chemical attractant and tending to move towards the side of the attractant, with a CI index of 0.2721; while the CL2355 nematodes showed obvious chemotactic behavior defects, with similar numbers of individuals moving to the side of the attractant and the blank control side, and a CI index of -0.0628, showing a significant difference between the two. After treatment with 0.1 mg / mL and 0.5 mg / mL of A17-3, the chemotactic behavior of CL2355 was significantly improved, restoring its sensitivity to chemical odors to a certain extent, manifested as more nematodes crawling towards the side of the attractant, and the CI values increased to 0.0867 and 0.1448 respectively, both showing a significant difference from the CL2355 control group. This result preliminarily proves that the traditional Chinese medicine composition of the present invention has a protective effect on neuron damage caused by β-amyloid protein.

[0102] Table 3

[0103]

[0104] Note: Compared with the CL2355 control group, * represents P < 0.05, ** represents P < 0.01. Using sterile water as the Control, the total number of nematodes on the attractant side and the control side within 1 h was counted, and the CI value was calculated.

[0105] From Figure 3It can be seen that, compared with the control group, both 0.1 mg / mL and 0.5 mg / mL of A17-3 can prolong the average death time of N2 nematodes, and the oxidative stress tolerance of nematodes at the concentration of 0.5 mg / mL is extremely significantly improved (P < 0.001), and its average death time is prolonged by about 19.39% compared with the control group. This result preliminarily proves that the traditional Chinese medicine composition of the present invention has a certain antioxidant stress effect. Since the pathogenesis of AD is closely related to the oxidative stress inside the body, it is speculated that there is a certain connection between the antioxidant effect and the anti-AD effect of the traditional Chinese medicine composition of the present invention.

[0106] 3.2 Mouse model analysis

[0107] The present invention uses a mouse model to further investigate the improvement effect of the traditional Chinese medicine composition of the present invention on Alzheimer's disease and the corresponding mechanism analysis.

[0108] As Figure 4 shown, compared with wild-type mice, the exploration time of the APP / PS1 model group for new objects showed a significant decrease, and its PI index was significantly reduced, showing significant learning and memory ability defects. Compared with the model group mice, all three drug administration groups showed an increase in the PI index. Among them, the increases in the positive donepezil hydrochloride group and the A17-3 (10X) group were both significantly different, P < 0.01 and P < 0.05 respectively. This result shows that the traditional Chinese medicine composition of the present invention can improve the exploration ability and learning and memory ability of mice for new objects.

[0109] The effect of the traditional Chinese medicine composition of the present invention on the spatial learning and memory ability of AD mouse models is as Figures 5 - 8 shown. Figure 5 The results show that, compared with the model group, the wild-type control group, the positive donepezil hydrochloride group, and the A17-3 drug administration treatment group did not have a significant impact on the movement speed and total movement distance in the pool, indicating that there was no obvious difference in movement ability among the groups, and the subsequent behavioral index results were available. Figure 6 A The results show that in the 5-day place navigation experiment, as the training days increased, the latency of each group of mice to find the platform gradually shortened compared with the previous day; on the 5th day of training, the escape latency of the test mice was measured. Figure 6 B The results show that, compared with the wild-type control group, the escape latency of the model group mice was significantly increased, indicating that there was an obvious learning disorder in AD model mice. Compared with the model group, the escape latencies of the positive drug group, the high-concentration A17-3 group, and the low-concentration A17-3 group all showed a significant decrease. Figure 7 It is a typical swimming path diagram of each group of mice recorded in the spatial exploration experiment (test period). Figure 8For the residence time of mice in the quadrant where the platform is located during the space exploration experiment (testing period), it can be seen that the residence time of the model group is significantly lower than that of wild-type mice, showing symptoms of dementia; compared with the model group, after treatment with positive donepezil, the residence time of mice can be increased (P < 0.0001), about 12 s longer; compared with the model group, each concentration group of A17-3 showed an increase in residence time, and there were significant differences. Among them, the low-concentration group was about 8.3 s longer (P < 0.001), and the high-concentration group was about 10.6 s longer (P < 0.0001). The above results comprehensively show that the traditional Chinese medicine composition of the present invention has the effect of improving the spatial learning and memory ability of APP / PS1 mice in the AD transgenic model.

[0110] The effect of the traditional Chinese medicine composition of the present invention on the pathological changes of the brain tissue of the AD mouse model is as Figure 9 shown. The cells in the CA3 region of the hippocampus of wild-type control mice are arranged neatly and densely, with regular morphology; the cells in the CA3 region of the hippocampus of model group mice are abnormal, arranged disorderly, sparsely and irregularly, with large cell gaps and lighter coloring, showing significant differences compared with the wild-type control group; compared with the model group, the cells in the CA3 region of the hippocampus of each administration group mice are arranged tightly, the tissue is intact, and the number of pyramidal cells increases significantly, showing significant differences. This result shows that the traditional Chinese medicine composition of the present invention can improve the pathological morphology of the brain tissue of the AD mouse model and protect the brain tissue structure.

[0111] The effect of the traditional Chinese medicine composition of the present invention on the expression of Aβ in the brain tissue of the AD mouse model is as Figure 10 shown. Compared with the wild-type control group, the expression of Aβ in the cerebral cortex and hippocampus of model group mice is significantly increased; while the expression level of Aβ in the administration group is significantly lower than that in the model group. This result shows that the traditional Chinese medicine composition of the present invention can reduce the production of amyloid protein in the brain of APP / PS1 mice by reducing the transcriptional level of Aβ, thereby alleviating the neurotoxic damage caused by Aβ expression.

[0112] The effect of the traditional Chinese medicine composition of the present invention on the expression of BDNF in the brain homogenate of the AD mouse model is as Figure 11 shown. Compared with the wild-type control group, the expression of BDNF mRNA in model group mice is significantly decreased; while compared with the model group, the expression of BDNF mRNA can be increased in the donepezil hydrochloride group and high- and low-concentration A17-3 groups, and generally shows a dose-dependent effect, but there are no significant differences. This result proves that the traditional Chinese medicine composition of the present invention may play an anti-AD role by regulating the neuroprotective factor BDNF.

[0113] The effect of the traditional Chinese medicine composition of the present invention on the expression of Aβ in the brain homogenate of the AD mouse model is as Figure 12 shown. Figure 12The results of A indicate that after administration of different concentrations of A17-3, the mRNA level of Aβ in the brains of mice can be significantly reduced, indicating that it can affect the transcriptional expression of Aβ in the brains of mice. Figure 12 The results of B and C show that after administration of different concentrations of A17-3, the contents of Aβ oligomers and monomers in the brains of mice can be significantly reduced; a large number of studies have shown that the neurotoxicity of Aβ oligomers is significantly stronger than that of Aβ monomers. Compared with the model group, the contents of Aβ oligomers in the low-concentration and high-concentration A17-3 groups were reduced by 45.22% and 48.89% respectively. The above results further prove that the traditional Chinese medicine composition of the present invention can reduce the production of amyloid protein in the brains of APP / PS1 mice by reducing the transcriptional level of Aβ and alleviate the toxic damage caused by Aβ expression.

[0114] From Figure 13 it can be seen that the content of MDA in the brains of mice in the model group was significantly higher than that in the wild-type control group, indicating that the brains of mice in the model group showed significant damage after being attacked by free radicals compared with wild-type mice; while the content of MDA in the brains of mice in the positive drug donepezil hydrochloride group and the high- and low-concentration A17-3 groups decreased significantly compared with the model group. Combined with Figure 14 , the content of GSH in the brains of mice in the model group was lower than that in wild-type mice, indicating that the ability of the model group of mice to scavenge free radicals in the brain was lower than that of the wild-type group; compared with the model group, the positive donepezil hydrochloride group could significantly increase the content of GSH in the brains of mice, and each concentration group of A17-3 could increase the content of GSH in the brains of mice, but there was no significant difference. Combining the results of the two oxidative stress-related indexes, it is preliminarily shown that the traditional Chinese medicine composition of the present invention can reduce the oxidative stress damage in the brains of APP / PS1 mice and improve its antioxidant ability to achieve the effect of anti-AD dementia.

[0115] Furthermore, the effect of the traditional Chinese medicine composition of the present invention on the transcriptional expression levels of oxidative stress-related factors in the brain homogenate of an AD mouse model was investigated. From Figure 15 it can be seen that compared with the wild-type control group, the expressions of CAT and HO-1 mRNA in the model group of mice were significantly reduced, indicating that the antioxidant stress ability of the AD mouse model was significantly down-regulated. Compared with the model group, the positive drug donepezil hydrochloride group and each concentration group of A17-3 could significantly increase the expressions of CAT and HO-1 mRNA, improve the antioxidant ability of the AD mouse model, and after treatment with different concentrations of A17-3, CAT and HO-1 generally showed a dose-dependent up-regulation of transcriptional levels, and were significantly up-regulated under high-concentration treatment. This result indicates that the traditional Chinese medicine composition of the present invention can activate the antioxidant pathway.

[0116] In summary, the components in the traditional Chinese medicine composition of the present invention are mutually compatible, achieving the effect of effectively improving the symptoms of the Alzheimer's disease mouse model. At the same time, combined with the results of various experiments, it is proved that the anti-AD pharmacological action mechanism of the traditional Chinese medicine composition of the present invention is mainly related to up-regulating the neuroprotective factor BDNF, inhibiting the deposition of Aβ amyloid protein in the brain tissue, and enhancing the antioxidant stress ability of the body.

[0117] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, rather than limiting them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A Chinese medicine composition for treating Alzheimer's dementia, characterized in that: Including raw materials by weight: 15-45 parts of Astragalus, 6-18 parts of Ganoderma lucidum, 6-18 parts of processed Polygonum multiflorum, 5-15 parts of Cistanche deserticola, 5-15 parts of Dendrobium, 4-12 parts of Lycium barbarum, 4-12 parts of Poria, 3-9 parts of ginseng, 2.5-7.5 parts of Cimicifuga heracleifolia, and 2.5-7.5 parts of Pseudostellaria pseudoginseng.

2. The Chinese medicine composition for treating Alzheimer's dementia according to claim 1, characterized in that: Including raw materials by weight: 30 parts of Astragalus, 12 parts of Ganoderma Lucidum, 12 parts of processed Polygonum Multiflorum, 10 parts of Cistanche, 10 parts of Dendrobium, 8 parts of Lycium barbarum, 7 parts of Poria, 6 parts of Ginseng, 5 parts of Cimicifuga, and 5 parts of Pseudostellariae Radix.

3. A method for preparing a Chinese medicine composition for treating Alzheimer's dementia as claimed in claim 1 or 2, characterized in that: The following steps are involved: S1, weigh the raw materials by weight, mix them, add 3-6 times the total weight of the raw materials in water, soak for 30-90 minutes, then add water to 10 times the total weight of the raw materials, decoct for 45 minutes, obtain a primary decoction and medicinal residues, then add 10 times the total weight of the raw materials to the medicinal residues. Water, decoction for 45 minutes, obtain a secondary decoction; S2. Combining the first decoction and the second decoction to obtain a water decoction of the traditional Chinese medicine composition for treating Alzheimer's dementia.

4. The preparation method according to claim 3, characterized in that: After step S2, the method further includes filtering the combined decoction, concentrating the filtrate under reduced pressure to a thick paste, and drying to obtain a dry paste of the traditional Chinese medicine composition for treating Alzheimer's dementia.

5. The preparation method according to claim 3, characterized in that: After step S2, the combined decoction is freeze-dried to obtain freeze-dried powder of the traditional Chinese medicine composition for treating Alzheimer's dementia.

6. The preparation method according to claim 5, characterized in that: The freeze-drying process comprises the following steps: precooling, vacuuming, primary sublimation, and analytical drying.

7. Use of the Chinese medicine composition as claimed in claim 1 or 2 in the preparation of a medicament for treating Alzheimer's dementia.

8. The use according to claim 7, characterized in that Pharmaceutically acceptable excipients are added to the traditional Chinese medicine composition to prepare an oral preparation, and the dosage form of the oral preparation includes tablets, capsules, granules, pills or oral liquid preparations.

Images