CN111214625A

CN111214625A - Traditional Chinese medicine composition for treating Alzheimer's disease and preparation method thereof

Info

Publication number: CN111214625A
Application number: CN201811429822.6A
Authority: CN
Inventors: 贾英; 肖锋; 颜廷旭; 何博赛; 吴博; 杜易洋
Original assignee: Shenyang Pharmaceutical University
Current assignee: Shenyang Pharmaceutical University
Priority date: 2018-11-27
Filing date: 2018-11-27
Publication date: 2020-06-02

Abstract

the invention relates to a Chinese medicinal composition for treating Alzheimer's disease and process for preparation, wherein the composition is prepared from (by weight portions) Chinese medicinal materials including alpinia oxyphylla 120-200 parts, schisandra fruit 30-80 parts, the invention has the advantages of (1) dose-dependent reversion of unilateral intracerebroventricular injection of Abeta_1‑42induced brain damage in mice model for Alzheimer's disease by ameliorating A β_1‑42(2) the levels of IL-1 β and IL-6 in the hippocampus of a model mouse of the Alzheimer's disease induced by injecting lipopolysaccharide into a unilateral ventricle can be obviously reduced, and the level of IL-1 β in the cortex can be obviously improved, so that the learning and memory disorder is obviously improved.

Description

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

The technical field is as follows:

the invention relates to a traditional Chinese medicine composition for treating Alzheimer's disease, which is prepared from the following traditional Chinese medicine raw materials in parts by weight: 150 parts of fructus alpiniae oxyphyllae and 50 parts of schisandra chinensis.

Background art:

alzheimer's Disease (AD) is a central neurodegenerative disease characterized clinically by generalized dementia, progressive and irreversible, and is currently the leading cause of dementia. There are studies showing that the incidence is highest in the population over 65 years of age, and it is predicted that the population will increase 62% by 2030, i.e. there will be 10 billion patients with alzheimer's disease worldwide. The most important factor affecting AD is the increase in age, and in addition, AD is also affected by a number of factors, such as: family history, carrying related genes, cardiovascular diseases, education degree, social and cognitive activities, traumatic brain injury and the like. However, the pathogenesis of AD is not completely known, and it is well known that: amyloid formation and deposition, oxidative stress, inflammatory mechanisms, central cholinergic injury, and the like. With regard to treatment of AD, there are two major categories of drug therapy and non-drug therapy, but neither method can cure AD.

the amyloid plaque deposition in brain is one of two clinical pathological changes of AD, the main component of the amyloid plaque deposition is β -amyloid (A β), A β is obtained by decomposing Amyloid Precursor Protein (APP) through continuous action of β -and gamma-secretase, amyloid A β is easy to aggregate, a series of oligomers and soluble aggregates are formed, insoluble A β fibers are formed when the aggregates are further aggregated, and the insoluble A β fibers are the core of the amyloid deposition.

the cholinergic hypothesis was proposed based on the detection of a decrease in cholinergic system activity in the brain of AD patients, but AChEIs only ameliorate some symptoms and do not cure the disease.

Currently, clinical treatment of AD is mainly to maintain function, slow down or delay symptoms. FDA approved drugs for clinical treatment of AD fall into two classes, one class being achei, such as donepezil, rivastigmine, galantamine; another class is N-methyl-aspartate (NMDA) receptor antagonists, such as memantine. These neurotransmitter modulators are able to improve the cognitive abilities and the daily behavioral abilities of patients, and relieve the symptoms of mild and moderate AD patients, but are unable to fundamentally improve the clinical status of the disease or stop the progression of the disease, which may be attributed to the inability of single-target therapeutics to cope with the occurrence and development of AD disorders of complex etiology. The multi-target therapeutic drug is expected to become the key point of future anti-AD drug research.

The theory of traditional Chinese medicine considers that the kidney is the congenital foundation, stores essence in the kidney, and leads to the brain and marrow. Insufficient kidney essence can affect brain morphology and interfere with brain function. Therefore, kidney essence is abundant and has a relationship with maintaining cognitive function, memory function, motor function, and normal operation of the brain. The traditional Chinese medicine also has the characteristics of multiple components, multiple targets and multiple ways, and has become the focus of research and development of new anti-AD medicines in recent years by combining with rich clinical application experience under the guidance of the basic theory of the traditional Chinese medicine. And a plurality of traditional Chinese medicines and compatible varieties thereof supplement and replace AD treatment medicines, such as schisandra chinensis, fructus alpiniae oxyphyllae, polygala tenuifolia, ginkgo leaves, kudzuvine roots, ginseng, polygonum multiflorum, epimedium and the like. Active ingredients of traditional Chinese medicines studied for treating AD include flavones, alkaloids, triterpenoid saponins, lignans, polysaccharides, and the like.

Fructus alpiniae oxyphyllae has the effects of warming kidney, controlling nocturnal emission and reducing urination, and fructus schizandrae has the effects of tonifying kidney and calming heart, and both are traditional kidney-protecting traditional Chinese medicines. The traditional Chinese medicine prescription-Yizhi Wuwei Wan recorded in the book of twenty nine introductions (such as Yi Fang) from the Puji Fang (general remedy) comprises the following components: the eleven traditional Chinese medicines of the schisandra, the fructus alpiniae oxyphyllae, the cistanche deserticola, the ginseng and the like are mainly used for treating liver and kidney deficiency and essence and qi dissipation. The brain-strengthening pill and the brain-strengthening capsule of the patent formulation are recorded in 2015 edition (part of Chinese pharmacopoeia), both have the effects of tonifying kidney and strengthening brain, nourishing blood and tranquilization, can be used for treating mild cognitive impairment of the elderly, and are prepared from the Chinese magnoliavine fruit and the Chinese medicinal materials. Modern pharmacological research shows that the fructus alpiniae oxyphyllae and the schisandra chinensis have the effects of protecting a nervous system, improving learning and memory capacity, resisting inflammation and resisting oxidation and the like.

Fructus Alpinae Oxyphyllae is dried mature fruit of Alpinia oxyphylla Miq. Produced in Hainan, Guangdong, Guangxi and other places, and has rich resources. Growing in the shade or cultivating under the forest. Beginning with Bencao Shiyi (materia Medica): vomit stopping. For seminal emission deficiency and leakage, dribbling urination, qi-tonifying and tranquilization, deficiency tonifying, triple energizer benefiting, qi regulating, and urination at night, twenty-four pieces are smashed and decocted with salt for oral administration. The Chinese pharmacopoeia records that the medicine has pungent taste and warm nature and enters spleen and kidney meridians; can warm kidney, secure essence, reduce urination, warm spleen, check diarrhea and control saliva; can be used for treating renal deficiency, enuresis, frequent micturition, nocturnal emission, diarrhea due to spleen cold, abdominal psychroalgia, and excessive saliva.

Fructus Schisandrae chinensis is dried mature fruit of Schisandra chinensis (Turcz.) Baill. of Magnoliaceae, and is known as "Schisandra chinensis". It is produced in Heilongjiang, Jilin, Liaoning, inner Mongolia, Hebei, Shanxi, Ningxia and Gansu, etc. and has rich resource. Is grown in valley, side stream and hillside with elevation of 1200-1700 m. Also distributed in korea and japan. It is recorded in Shen nong Ben Cao Jing (Shen nong's herbal), Wu Wei Zi is sour and warm in taste. Mainly tonify qi, reverse qi, fatigue, emaciation, deficiency tonifying, yin strengthening and male essence benefiting. The Chinese pharmacopoeia records that the medicine has sour and sweet taste and warm nature, and enters lung, heart and kidney meridians; can astringe, benefit qi, promote the production of body fluid, tonify kidney, and calm heart; can be used for treating chronic cough, asthma, nocturnal emission, enuresis, frequent micturition, chronic diarrhea, spontaneous perspiration, night sweat, thirst due to body fluid consumption, internal heat, diabetes, palpitation, and insomnia.

Disclosure of Invention

The invention aims to provide a traditional Chinese medicine composition for treating AD, which consists of fructus alpiniae oxyphyllae and schisandra chinensis.

In a further embodiment of the invention, 120-200 parts of fructus alpiniae oxyphyllae and 30-80 parts of schisandra chinensis. Preferably, the two raw material medicaments comprise the following components in parts by weight: 150 parts of fructus alpiniae oxyphyllae and 50 parts of schisandra chinensis.

The components are proportioned according to the weight parts, and can be increased or reduced according to the corresponding proportion during production, but the proportion of the raw medicinal materials in the weight proportion among the components is unchanged.

Another purpose of the invention is to provide the traditional Chinese medicine composition for treating A beta menstruation_1-42And scopoletin induced two AD model mice.

injection of Abeta by unilateral ventricles_1-42And injecting scopolamine into the abdominal cavity, and respectively establishing an AD model.

the traditional Chinese medicine composition can improve the intracerebral oxidative stress level of the Abeta model mice and reverse the damage of the intracerebral cholinergic system of the scopolamine model mice.

In addition, the fructus alpiniae oxyphyllae belongs to a medicine-food homologous variety, and the schisandra chinensis can be added into health-care food, so that the safety of the fructus alpiniae oxyphyllae and the health-care food is high. The traditional Chinese medicine composition of the two medicines has simple components, easy preparation and definite drug effect, and can effectively play the role of improving the behavioural and learning and memory functions of AD patients.

Preferably, a method for preparing the Chinese medicinal composition comprises the following steps:

in the invention, the specific preparation method of the fructus alpiniae oxyphyllae and schisandra chinensis traditional Chinese medicine composition comprises the following steps:

(1) weighing the following raw materials in parts by weight: 120-200 parts of fructus alpiniae oxyphyllae and 30-80 parts of schisandra chinensis;

(2) pulverizing fructus Alpinae Oxyphyllae into coarse powder, reflux extracting with 50-90% ethanol, collecting extractive solution, filtering, recovering ethanol, and concentrating under reduced pressure to obtain fluid extract 1;

(3) diluting the fluid extract 1 with water, loading onto macroporous resin column, eluting with distilled water until the eluate is colorless, eluting with 20-70% ethanol, collecting eluate, recovering ethanol under reduced pressure, concentrating, and drying to obtain fructus Alpinae Oxyphyllae extract.

(4) Pulverizing fructus Schisandrae into coarse powder, reflux extracting with 35-85% ethanol, collecting extractive solution, filtering, recovering ethanol, and concentrating under reduced pressure to obtain fluid extract 2;

(5) diluting the fluid extract 2 with water, loading onto macroporous resin column, eluting with distilled water until the eluate is colorless, eluting with 20-60% ethanol, collecting eluate, recovering ethanol under reduced pressure, concentrating, and drying to obtain fructus Schisandrae extract.

(6) Mixing fructus Alpinae Oxyphyllae with fructus Schisandrae extract.

In the above preparation method, preferably, in step (2), fructus Alpinae Oxyphyllae is pulverized into coarse powder, and reflux-extracted with 60% ethanol solution; the relative density of the clear paste 1 is preferably 1.10 (measured at 60 ℃).

Diluting the clear paste 1 in the step (3) with water, adding macroporous resin, and eluting with 30% ethanol.

Pulverizing fructus Schisandrae into coarse powder, and reflux-extracting with 80% ethanol solution; the relative density of the clear paste 2 is preferably 1.10 (measured at 60 ℃).

And (5) after macroporous resin is added, eluting with 50% ethanol.

Drawings

FIG. 1 is a flow chart of an experiment in example 1 of the present invention.

FIG. 2 shows the results of the space exploration experiment in example 1 of the present invention.

Wherein, A is the time percentage of the target area; b: number of passes through stage (^*p<0.05,^**p<0.01 compared to control group;^#p<0.05, compared to model set).

FIG. 3 shows the levels of biochemical markers in hippocampus and cortex of a case model mouse in example 2 of the present invention.

Wherein A, B: superoxide dismutase (SOD) activity; c, D: malondialdehyde (MAD) level; e, F: reactive Oxygen Species (ROS) level: (^*p<0.05,^***p<0.001 compared to control group;^#p<0.05,^##p<0.01,^###p<0.001 compared to model group).

FIG. 4 is the result of observation of histopathology in example 3 of the present invention.

Wherein, A: a control group; b: a model group; c: donepezil group; and D, a high-dose group of the traditional Chinese medicine composition (magnification is 400 x).

FIG. 5 is a flowchart of an experiment in example 4 of the present invention.

FIG. 6 shows the results of the Y-maze in example 4 of the present invention.

Wherein, A: the number of arm feeding times; b spontaneous alternation rate (^**p<0.01 compared to control group;^#p<0.05,^##p<0.01 vs. model set).

FIG. 7 shows the levels of biochemical markers in hippocampus and cortex of a case model mouse in example 5 of the present invention.

Wherein, A, B: the level of Ach; c, D: AChE activity; e, F: ChAT; g, H: the M1 receptor; (p)<0.05,^**p<0.01,^***p<0.001 compared to control group; (ii) a^#p<0.05,^##p<0.01,^###p<0.001 compared to model group).

Detailed Description

The present invention may be further understood by the following examples, which are intended for the purpose of illustration only and are not intended to limit the scope of the invention in any way.

EXAMPLE 1 combination of Chinese herbs on A beta_1-42Behavioral experiments in induced AD model mice

1. Sample preparation method

Preparation of Chinese medicinal composition

(1) Weighing the following raw materials in parts by weight: 150g of fructus alpiniae oxyphyllae and 50g of schisandra chinensis;

(2) pulverizing fructus Alpinae Oxyphyllae into coarse powder, extracting with 50% ethanol under reflux for 2 times (2 hr each time), and mixing extractive solutions. Filtering the extractive solution, recovering ethanol, and concentrating under reduced pressure to relative density of 1.10 (measured at 60 deg.C) to obtain fluid extract 1;

(3) diluting the fluid extract 1 with 2 times of water, separating with HP20 macroporous resin column, eluting with distilled water until the eluate is colorless, eluting with 10 times of 35% alcohol, collecting eluate, recovering ethanol under reduced pressure, concentrating, and drying to obtain fructus Alpinae Oxyphyllae extract.

(4) Pulverizing fructus Schisandrae into coarse powder, extracting with 50% ethanol under reflux for 2 times (2 hr each time), and mixing extractive solutions. Filtering the extractive solution, recovering ethanol, and concentrating under reduced pressure to relative density of 1.10 (measured at 60 deg.C) to obtain fluid extract 2;

(5) diluting the fluid extract 2 with 2 times of water, loading onto AB-8 macroporous resin column, eluting with distilled water until the eluate is colorless, eluting with 10 times of 60% alcohol, collecting eluate, recovering ethanol under reduced pressure, concentrating, and drying to obtain fructus Schisandrae extract.

(6) Mixing fructus Alpinae Oxyphyllae and fructus Schisandrae extract, diluting with 0.5% CMC-Na solution to obtain Chinese medicinal composition containing extract 3, 6, and 12mg/mL, and storing at 4 deg.C.

Fructus Alpinae Oxyphyllae extract intragastric juice is prepared by diluting fructus Alpinae Oxyphyllae extract obtained in step (3) with 0.5% CMC-Na solution to 3, 6, 12mg/mL soaking paste, and storing at 4 deg.C.

Fructus Schisandrae extract gastric lavage solution is prepared by diluting fructus Schisandrae extract obtained in step (5) with 0.5% CMC-Na solution to content of 3, 6, 12mg/mL extract, and storing at 4 deg.C.

2. Animal grouping, model building and drug administration, experimental design and statistical analysis

Animal grouping 120 mice were randomly divided into 12 groups, and the specific grouping and administration were as follows:

(1) blank group (control);

(2) model group (vehicle) intracerebroventricular injection of Abeta_1-42Intragastric administration of 0.5% CMC-Na aqueous solution;

(3) positive drug (donepezil) intracerebroventricular injection of Abeta_1-42Gavage with donepezil (3 mg/kg);

(4) the fructus Alpinae Oxyphyllae extract can be administered to patients with low, medium, and high doses of gastric juice (A (L), A (M), A (H)): by intracerebroventricular injection of Abeta_1-42Gavage extract (300mg/kg,600mg/kg,1200 mg/kg);

(5) the low, medium and high dosage group (S (L), S (M), S (H)) of fructus Schisandrae extract perfused with gastric juice is prepared by injecting A β into ventricle_1-42Fructus Schisandrae extract for intragastric administration (300mg/kg,600mg/kg,1200 mg/kg);

(6) the Chinese medicinal composition has low, medium and high dosage groups (AS (L), AS (M), AS (H)) the brain ventricle is injected with Abeta_1-42The traditional Chinese medicine composition is used for intragastric administration (300mg/kg,600mg/kg and 1200 mg/kg);

model establishment and administration after one week of adaptive feeding of experimental mice, model creation is carried out, 4% chloral hydrate is adopted to anaesthetize mice in other groups except blank groups, the mice are fixed on a brain stereotaxic apparatus, and A β is injected into ventricles of brain on one side_1-42(1 mug/muL, 3 muL) and coordinates (AP, -0.5mm, ML, +/-1.1 mm, DV, -3.0mm), the injection is completed at a constant speed within 3min, the needle is kept for 3min to ensure that the needle is drawn slowly after full dispersion, the operation part is sprinkled with penicillin sodium powder to prevent infection, the scalp is sutured, and the penicillin sodium solution is injected intramuscularly for three consecutive days after the operation. Performing intragastric administration for administration of corresponding drugs from the 4 th day after the operation, continuously administering for 30 days, performing an ethological experiment from the 23 th day after administration, performing decapitation and sacrifice on the mice after the last day of the ethological experiment is finished, rapidly taking out brain tissues on an ice box, taking two random brains in each group for histopathological section preparation, and performing HE staining on the sections; separating hippocampal region and cortical region from the rest brain tissue, weighing, placing in 1.5mL EP tube, adding appropriate amount of precooled normal saline, ultrasonic crushing the tissue with cell crusher to obtain 10% tissue homogenate, centrifuging at 4 deg.C and 3500r/min for 10min with low temperature centrifuge, collecting supernatant, storing at-80 deg.C, and detecting various biochemical indexes. The specific experimental flow is shown in figure 1.

The Morris water maze consists of a round water pool and an automatic video recording and analyzing system. Adding water into a round water pool (diameter 80cm, height 30cm), dropping black ink to make water opaque, dividing the maze into 4 quadrants, and keeping water temperature at about 22 deg.C. There was another black circular platform (diameter 10cm, height 28cm) placed in the center of the fourth quadrant. During testing, the second quadrant is selected as a water entry point, the mouse is placed into water facing the pool wall, the time required for the animal to search and climb up the platform is recorded according to the water maze tracking system, namely the escape latency (escape latency), if the platform is not found within 90s, the animal is led to the platform and stays on the platform for 30s, and the escape latency is recorded as 90 s.

A platform visual experiment is carried out 2 days before the Morris water maze experiment, the platform is 1cm higher than the water surface, each mouse is trained for 1 time every day, and the incubation period of each group of mice is recorded. The Morris water maze experiment was started on day 3 for the localized voyage experiment, and each mouse was trained 2 times a day to record the latency of each group of mice. And the Morris water maze experiment is a space exploration experiment on the 8 th day, the platform is removed, the animal is placed in the water facing the pool wall in the second quadrant, and the swimming time of the animal in the target quadrant within 90s and the times of passing through the platform are recorded.

Statistical processing adopts the sps 19.0 software to process the difference of each component, data are expressed by mean +/-S.E.M., parameters among groups are statistically analyzed by One-way ANOVA and Tukey methods, and the statistical significance is achieved by taking p <0.05 as a significant difference.

3. Results of the experiment

(1) Platform visual experimental results the escape latencies for each group of mice to find the platform are shown in table 1, and on days 1 and 2 of the experiment, compared with the blank group, the escape latencies of other groups have no significant difference (p >0.05), indicating that each group of mice has no visual difference.

TABLE 1 escape latency (sec) for visible platform

(2) Results of localization voyage experiments the escape latency for each group of mice to find the platform is shown in table 2, and the latency for each group of mice from day 3 to day 6 of the experiment is not significantly different (p > 0.05). On the 7 th day of the experiment, the latency of the mice in the model group is obviously higher than that of the mice in the blank group (p is less than 0.05), and compared with the mice in the model group, the latency of the mice in the traditional Chinese medicine composition and the mice in the high-dose group is obviously reduced (p is less than 0.05).

The average daily swimming speed of each group of mice in the positioning navigation experiment is shown in table 3, and no significant difference (p >0.05) exists among the groups of mice, which indicates that the motor abilities of the groups of mice are basically consistent.

TABLE 2 hidden platform escape latency (sec)

^*p<0.05, compared to control group;^#p<0.05, comparison with model group

TABLE 3 positioning navigation experiment of hidden platform

(3) The residence time of each group of mice in the target quadrant of the space exploration experiment is shown in fig. 2(a), and compared with the control group, the residence time of the model group in the target quadrant is remarkably reduced (p < 0.05); the high dose administration of the Chinese medicinal composition resulted in a significant increase in the residence time of the mice in the target quadrant (p <0.05) compared to the model group.

The number of platform crossing times of each group of mice is shown in fig. 2(B), and compared with the control group, the number of platform crossing times of the model group is remarkably reduced (p < 0.01); compared with the model group, the number of times that the mice of the high-dose administration group of the traditional Chinese medicine composition pass through the platform is obviously increased (p is less than 0.05).

example 2 detection of Biochemical indicators of the Chinese medicinal composition on A β 1-42 induced AD model mice

1. Sample preparation method

Preparation of Chinese medicinal composition

(1) Weighing the following raw materials in parts by weight: 200g of fructus alpiniae oxyphyllae and 30g of schisandra chinensis;

(2) pulverizing fructus Alpinae Oxyphyllae into coarse powder, extracting with 65% ethanol under reflux for 2 times (2 hr each time), and mixing extractive solutions. Filtering the extractive solution, recovering ethanol, and concentrating under reduced pressure to relative density of 1.10 (measured at 60 deg.C) to obtain fluid extract 1;

(3) diluting the fluid extract 1 with 1 weight time of water, loading onto HP20 macroporous resin column, eluting with distilled water until the eluate is colorless, eluting with 10 times weight of 35% alcohol, collecting eluate, recovering ethanol under reduced pressure, concentrating, and drying to obtain fructus Alpinae Oxyphyllae extract.

(4) Pulverizing fructus Schisandrae into coarse powder, extracting with 70% ethanol under reflux for 2 times (2 hr each time), and mixing extractive solutions. Filtering the extractive solution, recovering ethanol, and concentrating under reduced pressure to relative density of 1.10 (measured at 60 deg.C) to obtain fluid extract 2;

(5) diluting the fluid extract 2 with 1 weight time of water, loading onto AB-8 macroporous resin column, eluting with distilled water until the eluate is colorless, eluting with 10 times of 40% alcohol, collecting eluate, recovering ethanol under reduced pressure, concentrating, and drying to obtain fructus Schisandrae extract.

(6) Mixing fructus Alpinae Oxyphyllae and fructus Schisandrae extract, diluting with 0.5% CMC-Na solution to obtain extract of 3, 6, 12mg/mL, and storing at 4 deg.C.

Preparation of Alpinia oxyphylla extract gastric lavage fluid

Preparation of gastric lavage solution containing Schisandra chinensis extract

2. Animal grouping, model building and administration, experimental design and statistical analysis, etc., as in example 1

The activity of superoxide dismutase (SOD) and the level of Malondialdehyde (MDA) are detected by adopting a test box, and the detection method is operated according to the actual condition and the specification of the test box. The level of Reactive Oxygen Species (ROS) is detected using a kit, and the procedure is performed according to kit instructions.

3. Results of the experiment

As shown in fig. 3: in terms of SOD activity, compared with a blank group, the SOD activity in both hippocampus and cortex of the model group mice is obviously reduced (p is less than 0.05); compared with the model group, the SOD activity in the mouse hippocampus of the positive medicine group, the intelligence-benefiting high-dose group and the traditional Chinese medicine composition high-dose group is obviously improved (p is less than 0.01, p is less than 0.05, and p is less than 0.001); SOD activity in mouse cortex of high-dose group, traditional Chinese medicine composition and high-dose group of fructus alpiniae oxyphyllae is obviously increased (p is less than 0.01). In terms of MDA level, compared with a blank group, the content of MDA in the hippocampus of the mice in the model group is remarkably increased (p is less than 0.001); compared with the model group, the MDA content in mice hippocampus of the low, medium and high dose groups and the high dose group of the traditional Chinese medicine composition of the schisandra chinensis is obviously reduced (p is less than 0.001); there was no significant difference in MDA content in the cortex of each group of mice (p > 0.05). In terms of ROS levels, ROS content was significantly increased in the hippocampus of model group mice compared to the blank group (p < 0.05); compared with the mouse in the model group, the content of ROS in the hippocampus of the mouse in the high-dose group of the traditional Chinese medicine composition is obviously reduced (p is less than 0.05); there was no significant difference in ROS content in the cortex of each group of mice (p > 0.05).

according to the existing experimental basis, ROS, SOD and MDA are used as biochemical indexes for detecting the oxidative stress level in the brain in the experiment, after detection, the SOD activity of a model group mouse is obviously reduced, the MDA and ROS levels are obviously increased, which is consistent with the report of the existing literature, after comparison of administration groups, the situation that the administration of the nootropic fructus schizandrae and the traditional Chinese medicine composition can improve the administration condition is observed, wherein the administration effect of the high-dose group of the traditional Chinese medicine composition (1200mg/kg) is most obvious.

histopathological observation of mice in blank group, model group, positive medicine group and high-dose group of traditional Chinese medicine composition shows that A β is injected into ventricles of brain on one side_1-42Can induce damage to neurons in hippocampus and cortex, and the high-dose group (1200mg/kg) of the traditional Chinese medicine composition can obviously improve the phenomenon.

example 3 Chinese medicinal composition for treatment of Abeta_1-42Histopathological observations of induced AD model mice

1. Preparation of Chinese medicinal composition

(1) Weighing the following raw materials in parts by weight: 120g of fructus alpiniae oxyphyllae and 60g of schisandra chinensis;

(2) pulverizing fructus Alpinae Oxyphyllae into coarse powder, extracting with 85% ethanol under reflux for 2 times (2 hr each time), and mixing extractive solutions. Filtering the extractive solution, recovering ethanol, and concentrating under reduced pressure to relative density of 1.10 (measured at 60 deg.C) to obtain fluid extract 1;

(3) diluting the fluid extract 1 with 1 weight time of water, loading onto HP20 macroporous resin column, eluting with distilled water until the eluate is colorless, eluting with 10 times weight of 40% alcohol, collecting eluate, recovering ethanol under reduced pressure, concentrating, and drying to obtain fructus Alpinae Oxyphyllae extract;

(4) pulverizing fructus Schisandrae into coarse powder, extracting with 85% ethanol under reflux for 2 times (2 hr each time), and mixing extractive solutions. Filtering the extractive solution, recovering ethanol, and concentrating under reduced pressure to relative density of 1.10 (measured at 60 deg.C) to obtain fluid extract 2;

(5) diluting the fluid extract 2 with 2 times of water, loading onto AB-8 macroporous resin column, eluting with distilled water until the eluate is colorless, eluting with 10 times of 30% alcohol, collecting eluate, recovering ethanol under reduced pressure, concentrating, and drying to obtain fructus Schisandrae extract.

Preparation of Alpinia oxyphylla extract gastric lavage fluid

Preparation of gastric lavage solution containing Schisandra chinensis extract

2. Animal grouping, model building and administration, experimental design and statistical analysis, hematoxylin-eosin staining (HE staining) as in example 1

Fixing whole brain of blank group, model group, positive drug group and high-dose group of Chinese medicinal composition in 4% formaldehyde solution, sequentially dehydrating with 70%, 80%, 90% and 95% absolute ethanol by volume percentage for 12h, embedding in paraffin, and slicing. After dewaxing, gradient dehydration is sequentially carried out on 95%, 90% and 80% ethanol, HE staining is carried out after paraffin section, hematoxylin solution staining is carried out for 3-5 min, differentiation is carried out on 1% hydrochloric acid ethanol, tap water returns to blue, then eosin solution staining is carried out for 30-60 s, washing is carried out, gradient ethanol dehydration is carried out, sealing is carried out after xylene permeabilization, and the cell morphology of the mouse hippocampus is observed by adopting a microscope.

3. Results of the experiment

As shown in fig. 4, nuclear compaction phenomenon was observed in hippocampus and cortex of model mice compared to the blank group; compared with the model group, the phenomenon in the hippocampus and cortex of the mice of the positive medicine group and the compatible high-dose group is reduced, and the cells of the hippocampus of the mice of the compatible high-dose group are orderly and tightly arranged.

Example 4 Effect of the Chinese medicinal composition on learning and memory function of scopolamine-induced AD model mouse

1. Preparation of Chinese medicinal composition

(1) Weighing the following raw materials in parts by weight: 170g of fructus alpiniae oxyphyllae and 40g of schisandra chinensis;

(2) pulverizing fructus Alpinae Oxyphyllae into coarse powder, extracting with 70% ethanol under reflux for 2 times (2 hr each time), and mixing extractive solutions. Filtering the extractive solution, recovering ethanol, and concentrating under reduced pressure to relative density of 1.10 (measured at 60 deg.C) to obtain fluid extract 1;

(3) diluting the fluid extract 1 with 2 times of water, separating with HP20 macroporous resin column, eluting with distilled water until the eluate is colorless, eluting with 10 times of 60% alcohol, collecting eluate, recovering ethanol under reduced pressure, concentrating, and drying to obtain fructus Alpinae Oxyphyllae extract.

(4) Pulverizing fructus Schisandrae into coarse powder, extracting with 60% ethanol under reflux for 2 times (2 hr each time), and mixing extractive solutions. Filtering the extractive solution, recovering ethanol, and concentrating under reduced pressure to relative density of 1.10 (measured at 60 deg.C) to obtain fluid extract 2;

(5) diluting the fluid extract 2 with 1 weight time of water, loading onto AB-8 macroporous resin column, eluting with distilled water until the eluate is colorless, eluting with 10 times of 50% alcohol, collecting eluate, recovering ethanol under reduced pressure, concentrating, and drying to obtain fructus Schisandrae extract.

2. Animal grouping, model building, drug administration and experimental design

40 mice were randomly divided into 4 groups, and the specific grouping and dosing were as follows:

(1) blank group (control);

(2) model group (vehicle): intragastric administration of 0.5% CMC-Na solution, intraperitoneal injection of scopolamine;

(3) positive drug (donepezil): gavage donepezil (3mg/kg), intraperitoneal injection of scopolamine;

(4) traditional Chinese medicine composition group (AS): the Chinese medicinal composition is administered at high dose by intragastric administration (1200mg/kg), and is administered by intraperitoneal injection with scopolamine;

administration was started 7 days after adaptive feeding of experimental mice. The administration is carried out continuously for 21 days, on the 22 th day, after the administration for 30min by intragastric administration, scopolamine (3mg/kg) is injected intraperitoneally, and the Y maze experiment is carried out after 30 min. After the last day of the behavioral experiment, the mice are killed by head breakage, brain tissues are rapidly taken out from an ice box, two random mice in each group are used for preparing histopathological sections, and HE staining is carried out on the sections; separating hippocampal region and cortical region from the rest brain tissue, weighing, placing in 1.5mL EP tube, adding appropriate amount of precooled normal saline, ultrasonic crushing the tissue with cell crusher to obtain 10% tissue homogenate, centrifuging at 4 deg.C and 3500r/min for 10min with low temperature centrifuge, collecting supernatant, storing at-80 deg.C, and detecting various biochemical indexes. The specific experimental procedure is shown in fig. 5.

The Y-maze consists of three arms with the same specification and an angle of 120 degrees, which are respectively named as A, B and C, and the material is an iron plate sprayed with black paint. The arm length of each arm is 40cm, the height is 12cm, the bottom width is 3cm, the top width is 10cm, and the intersection of the three arms is an equilateral triangle. The bottom of the Y maze was padded with fresh padding and each animal was replaced immediately after testing and sterilized with 75% ethanol to eliminate the odor from the previous animal interfering with the next animal. During the experiment, a certain wall of a mouse randomly facing away from the maze is placed in a central triangular area of the maze, then the mouse is freely explored in the maze for 8min, the total arm entering times (the whole body of the mouse enters the arm except the tail part, namely, the arm entering time) and the arm entering sequence of each mouse are recorded, and the spontaneous alternating reaction rate is calculated to be the correct alternating reaction times/(total times-2) multiplied by 100%.

3. Results of the experiment

As shown in fig. 6, there was no significant difference in total arm entry frequency for each group of mice. Compared with a blank group, the spontaneous reaction alternation rate of the mice in the model group is obviously reduced (p < 0.01); compared with the model group, the spontaneous reaction alternation rate of the mice of the positive medicine group and the traditional Chinese medicine composition group is obviously increased (p <0.05, p < 0.01).

Example 5 Effect of the Chinese medicinal composition on Biochemical indices of scopolamine-induced AD model mice

1. Preparation of Chinese medicinal composition

(1) Weighing the following raw materials in parts by weight: 150g of fructus alpiniae oxyphyllae and 70g of schisandra chinensis;

(2) pulverizing fructus Alpinae Oxyphyllae into coarse powder, extracting with 90% ethanol under reflux for 2 times (2 hr each time), and mixing extractive solutions. Filtering the extractive solution, recovering ethanol, and concentrating under reduced pressure to relative density of 1.10 (measured at 60 deg.C) to obtain fluid extract 1;

(3) diluting the fluid extract 1 with 1 weight time of water, loading onto HP20 macroporous resin column, eluting with distilled water until the eluate is colorless, eluting with 10 times weight of 20% alcohol, collecting eluate, recovering ethanol under reduced pressure, concentrating, and drying to obtain fructus Alpinae Oxyphyllae extract.

(4) Pulverizing fructus Schisandrae into coarse powder, extracting with 40% ethanol under reflux for 2 times (2 hr each time), and mixing extractive solutions. Filtering the extractive solution, recovering ethanol, and concentrating under reduced pressure to relative density of 1.10 (measured at 60 deg.C) to obtain fluid extract 2;

(5) diluting the fluid extract 2 with 1 weight time of water, loading onto AB-8 macroporous resin column, eluting with distilled water until the eluate is colorless, eluting with 10 times of 60% alcohol, collecting eluate, recovering ethanol under reduced pressure, concentrating, and drying to obtain fructus Schisandrae extract.

2. Animal grouping, model building, drug administration and experimental design, as in example 4

3. Biochemical index detection

The level of acetylcholine (Ach) and the activities of acetylcholinesterase (AChE) and acetylcholinesterase (ChAT) are detected by using the test kit, and the detection method is operated according to the actual conditions and the specification of the test kit. The level of muscarinic acetylcholine receptor subtype (M1) receptors is detected using a kit, according to the kit instructions.

4. Results of the experiment

The results of the experiment are shown in FIG. 7: in terms of acetylcholine (Ach), the levels of Ach in both hippocampus and cortex were significantly reduced in the model group mice compared to the blank group (p <0.01, p < 0.05); compared with the model group, the level of Ach in the cortex of the mice of the positive drug group and the compatible group is obviously increased (p is less than 0.05, and p is less than 0.01). In terms of acetylcholinesterase (AChE), AChE activity was significantly increased in hippocampus and cortex of model group mice compared to blank group (p <0.01, p < 0.05); compared with the model group, the activity of the AChE in the hippocampus and the cortex of the compatible group of mice is remarkably reduced (p <0.001 and p <0.01), while the activity of the AChE in the hippocampus of the positive drug mice is remarkably reduced (p < 0.001). There was no significant difference in choline acetyltransferase (ChAT) activity in both hippocampus and cortex of each group of mice (p > 0.05). In the aspect of muscarinic receptor 1(M1) receptor, levels of M1 receptor were significantly reduced in hippocampus and cortex of model mice compared to blank group (p < 0.001); compared with a model group, the traditional Chinese medicine composition group has the advantages that the M1 receptor level in the hippocampus and the cortex of a mouse is obviously increased (p is less than 0.01), and the M1 receptor level in the cortex of a mouse with a positive medicine group is obviously increased (p is less than 0.05).

Claims

1. The traditional Chinese medicine composition for treating the Alzheimer disease is characterized by being prepared from the following raw material medicines in parts by weight: 120-200 parts of fructus alpiniae oxyphyllae and 30-80 parts of schisandra chinensis.

2. A method for preparing the traditional Chinese medicine composition of claim 1, which comprises the following steps:

(6) Mixing fructus Alpinae Oxyphyllae with fructus Schisandrae extract.

3. A pharmaceutical composition comprising the Chinese medicinal composition of claim 1 and a pharmaceutically acceptable carrier.

4. The use of the Chinese medicinal composition of claim 1 in the preparation of a medicament for the treatment of alzheimer's disease.

5. Use of the pharmaceutical composition of claim 3 for the manufacture of a medicament for the treatment of alzheimer's disease.