CN113750180A

CN113750180A - Application of traditional Chinese medicine compound kidney-tonifying brain-benefiting preparation in preparation of medicine for preventing and treating Alzheimer disease

Info

Publication number: CN113750180A
Application number: CN202111107789.7A
Authority: CN
Inventors: 匡海学; 王秋红; 刘娟; 王知斌; 刘祥成
Original assignee: Individual
Current assignee: Individual
Priority date: 2021-09-22
Filing date: 2021-09-22
Publication date: 2021-12-07

Abstract

The invention relates to application of a traditional Chinese medicine compound kidney tonifying and brain benefiting preparation in preparation of a medicine for preventing and treating Alzheimer disease, belonging to the technical field of medicines. In order to solve the problems of great side effect and complicated medication method of the existing western medicines for preventing and treating the Alzheimer disease, the invention provides the application of the traditional Chinese medicine compound kidney-tonifying brain-benefiting preparation in the aspect of preparing the medicines for preventing and treating the Alzheimer disease. Pharmacological experiments prove that the traditional Chinese medicine compound kidney-tonifying brain-benefiting preparation has the effects of improving pathological damage of hippocampus, inhibiting inflammatory cytokines and removing oxygen free radicals, can effectively overcome spatial cognitive disorder of model animals, improve behavioral abnormality of the model animals, and improve learning and memory capacity, thereby effectively controlling the state of an illness. The traditional Chinese medicine compound kidney tonifying and brain benefiting preparation is applied to clinical practice of preventing and treating the Alzheimer disease, has good application basis and prospect, and is beneficial to providing a new research direction for preventing and treating the traditional Chinese medicine of the Alzheimer disease.

Description

Application of traditional Chinese medicine compound kidney-tonifying brain-benefiting preparation in preparation of medicine for preventing and treating Alzheimer disease

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to an application of a traditional Chinese medicine compound kidney-tonifying and brain-benefiting preparation in preparation of a medicine for preventing and treating Alzheimer disease.

Background

Alzheimer's disease, also known as senile dementia, is a degenerative disease of the central nervous system, has an insidious onset and mainly shows persistent and irreversible intelligent decline, and the course of the disease is chronic and progressive.

One of the typical symptoms of alzheimer's disease is memory impairment, but alzheimer's disease is different from general insomnia and amnesia. The common insomnia and amnesia is the loss of memory trace in the brain, which is characterized in that things sensed before cannot be recalled, the memory is temporary, and the memory can be recovered under proper conditions. And the self-knowledge of the ordinary insomnia and amnesia patients is generally kept intact, namely, the patients realize the abnormal mental activities and actively seek medical help for the suffering, and the symptoms of the insomnia and amnesia can be improved by psychology adjustment or other relaxation modes. The self-learning ability of the patients suffering from the Alzheimer disease is generally deficient, the patients cannot realize the pathological manifestations of the patients, and the patients have the characteristic of permanent forgetfulness and can not remember all the events or experiences in a period of time. This is because memory impairment in alzheimer's disease often underlies brain organic lesions.

The hippocampus is an important tissue of the central nervous system and mainly participates in higher physiological functions such as learning, memory, emotion and the like. Damage to hippocampal neurons can affect hippocampal function, eventually developing learning impairment, memory dysfunction, depression, temporal lobe epilepsy, and the like, and is one of the pathological features manifested by alzheimer's disease. By inhibiting hippocampal injury and improving hippocampal neural plasticity, the effect of relieving Alzheimer disease symptoms can be achieved to a certain extent.

Neuroinflammation also plays an important role in neurodegenerative diseases. Neuroinflammation is an inflammatory reaction occurring in the brain and may be caused by a variety of factors, such as brain trauma, beta-amyloid deposition, disorders of energy metabolism, and even aging. There are studies showing that the release of pro-inflammatory factors in large quantities damages neurons and causes loss of synapses and neuronal death, associated with the occurrence of a patient's condition, which plays an important role in assessing the condition of alzheimer's disease.

Researches find that the reduction of the oxidation resistance and the accumulation of free radicals in the body of the patient with the Alzheimer disease are important causes of the neurocytotoxicity, and further the learning and memory functions are influenced. SOD and GSH-PX are important antioxidant enzymes in vivo, and have activity reduced in learning and memory disorder patients, and free radicals are generated in large amount to cause brain tissue function damage.

At present, the progress of the Alzheimer disease cannot be reversed or prevented, but intervention treatment aiming at the etiology is carried out on the basis of supporting and symptomatic treatment strategies in the early stage, so that the decline of the daily life quality of a patient can be delayed. At present, clinical Alzheimer disease drugs mainly comprise two main classes of cholinesterase inhibitors and N-methyl-D-aspartate receptor antagonists. In addition, antipsychotics, antidepressants may also be used in the treatment of symptoms associated with alzheimer's disease. However, the elderly are more sensitive to adverse reactions of drugs, so that the risks of adverse events such as cardiovascular accidents and pulmonary infection are increased, and consciousness turbidity, falling, respiratory depression, drug dependence and the like can be caused. Before the western medicines are taken, risks, benefits and cautious use need to be balanced, part of the western medicines are taken from low dosage and slowly added, the dosage needs to be gradually reduced or stopped after symptoms are improved, the administration method is complicated, and many cautions need to be taken. Therefore, researchers are engaged in searching for a prescription capable of preventing and treating the Alzheimer disease from a traditional Chinese medicine prescription with mild drug effect, small side effect and convenient taking.

The invention with the application number of 200910252002.9 discloses a traditional Chinese medicine composition with the functions of tonifying kidney and strengthening brain, which is a prescription consisting of American ginseng, cordyceps sinensis, pseudo-ginseng powder, salvia miltiorrhiza, ligusticum chuanxiong hort, rhizoma sparganii, curcuma zedoary, charred triplet, glossy privet fruit, radix bupleuri, eclipta alba, radix ophiopogonis and schisandra chinensis and has the effects of regulating human endocrine, enhancing human immunity, improving microcirculation, tonifying kidney, promoting blood circulation and preventing and treating neurasthenia. However, the existing traditional Chinese medicine formula for tonifying kidney and strengthening brain mainly aims at common memory impairment, mainly improves microcirculation and nourishes brain cells, and has no curative effect on dysmnesia of patients suffering from Alzheimer's disease caused by pathological injury, inflammatory cytokines or oxygen free radicals.

Disclosure of Invention

The kidney-tonifying and brain-nourishing preparation has the effects of tonifying kidney, raising essence, tonifying qi and nourishing blood, is used for treating palpitation, shortness of breath, insomnia, amnesia, spermatorrhea, night sweat, soreness of waist and legs, tinnitus and deafness caused by deficiency of kidney essence and deficiency of both qi and blood, and is clinically used for treating nephropathy as a main treatment direction. In order to solve the problems of great side effect and complicated medication method of the existing western medicines for preventing and treating the Alzheimer disease, the invention provides a new application of a traditional Chinese medicine compound kidney-tonifying brain-benefiting pill in the aspect of preparing medicines for preventing and treating the Alzheimer disease.

The technical scheme of the invention is as follows:

application of a compound Chinese medicinal preparation for invigorating kidney and nourishing brain in preparing medicine for preventing and treating Alzheimer disease is provided.

Furthermore, the prevention and treatment of the Alzheimer disease can inhibit the release of inflammatory cytokines in the brain of the patient with the Alzheimer disease, clear oxygen free radicals, improve pathological damage of the hippocampus of the patient and improve learning and memory ability.

Further, the traditional Chinese medicine compound kidney tonifying and brain benefiting preparation comprises the following components in parts by weight: 85-125 parts of prepared rehmannia root, 30-60 parts of ligusticum wallichii, 35-85 parts of polygala tenuifolia, 35-85 parts of poria cocos, 35-85 parts of angelica sinensis, 6-18 parts of pilose antler, 35-85 parts of red ginseng, 35-85 parts of bran-fried Chinese yam, 30-60 parts of fructus psoraleae, 30-60 parts of achyranthes bidentata, 30-60 parts of wolfberry fruit, 30-60 parts of radix scrophulariae, 35-85 parts of radix ophiopogonis, 30-60 parts of schisandra chinensis, 35-85 parts of fried spina date seed and 10-20 parts of cinnabar.

Furthermore, the preparation for tonifying the kidney and benefiting the brain in the medicament for preventing and treating the Alzheimer disease is the only active ingredient.

Furthermore, the medicament for preventing and treating the Alzheimer disease also comprises pharmaceutically acceptable auxiliary materials and/or carriers.

Furthermore, the dosage form of the medicine for preventing and treating Alzheimer disease is granules, tablets, capsules, pills, dripping pills or oral liquid preparations.

Further, the traditional Chinese medicine compound kidney-tonifying and brain-nourishing preparation is a kidney-tonifying and brain-nourishing pill, wherein the weight ratio of cooked rehmannia root, ligusticum wallichii, polygala tenuifolia, poria cocos, angelica sinensis, pilose antler, red ginseng, bran-fried Chinese yam, salt fructus psoraleae, achyranthes root, wolfberry fruit, figwort root, radix ophiopogonis, schisandra chinensis, fried spina date seed and cinnabar in the kidney-tonifying and brain-nourishing pill is 194: 70: 91: 91: 91: 14.4: 94: 91: 70: 70: 72: 70: 91: 70: 91: 24.

further, the preparation method of the kidney tonifying and brain benefiting pill comprises the following steps: refining Cinnabaris with water to obtain fine powder; decocting the angelica, the radix ophiopogonis, the radix scrophulariae, the polygala tenuifolia and the achyranthes bidentata twice in water for 2 hours each time, filtering, combining the filtrates, standing for more than 8 hours, filtering, and concentrating the filtrate to obtain clear paste with the relative density of 1.16-1.20 at the temperature of 50 ℃; pulverizing radix rehmanniae Preparata, rhizoma Ligustici Chuanxiong, Poria, cornu Cervi Pantotrichum, Ginseng radix Rubri, rhizoma Dioscoreae parched with bran, fructus Psoraleae preparata, fructus Lycii, fructus Schisandrae chinensis and semen Ziziphi Spinosae preparata into fine powder; grinding the obtained fine powder and Cinnabaris superfine powder, sieving, mixing, adding the above fluid extract, mixing, making into concentrated watered pill, drying, polishing, and making into 1000 g.

Further, the content detection requirements of the kidney tonifying and brain benefiting pill are as follows: the content of fructus Psoraleae containing salt per 1g of the pill is not less than 0.47mg based on the total amount of psoralen and isopsoralen; the content of fructus Schisandrae in 1g of the pill is not less than 0.27mg, calculated as schizandrol A.

Furthermore, the medicine for preventing and treating the Alzheimer disease is administrated in an oral mode, 2g of a kidney-tonifying and brain-benefiting preparation is contained in the medicine for oral administration every time, the medicine is orally taken twice every day, and one treatment course is 90 days.

The invention has the beneficial effects that:

pharmacological experiments prove that after 60 days of administration, the escape latency of mice of each administration group is obviously shortened, pathological injuries of a hippocampal CA1 area and a CA3 area are obviously improved, and the synthesis and release levels of inflammatory mediators (IL-1 beta, IL-6, COX-2 and TNF-alpha) in serum are obviously reduced, the cascade reaction of the inflammatory mediators is blocked, and excessive inflammatory reaction is favorably controlled, so that the condition of an illness is effectively controlled, and the death rate of the illness is reduced. Simultaneously, the activity of oxygen free radical scavenging enzymes such as superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) is increased, the scavenging capability of the organism to oxygen free radicals is improved, and the damage of the organism caused by free radical attack is reduced. Therefore, the traditional Chinese medicine compound kidney-tonifying brain-benefiting preparation can improve the pathological morphological damage of brain tissue hippocampus by adjusting inflammatory factors and oxidative stress related factors, so as to improve the behavioral abnormality of an Alzheimer disease model mouse, overcome the spatial cognitive disorder of a model animal and improve the learning and memory ability of the model animal. The traditional Chinese medicine compound kidney-tonifying brain-benefiting preparation has the effects of improving pathological damage of hippocampus, inhibiting inflammatory cytokines and removing oxygen radicals, is applied to clinical practice of preventing and treating Alzheimer disease, has good application basis and prospect, and is beneficial to providing a new research direction for preventing and treating the Alzheimer disease by traditional Chinese medicines.

Drawings

FIG. 1 is a graph comparing escape latencies of groups of mice in the Morris water maze positioning navigation experiment of example 2;

FIG. 2 is a comparative graph of representative movement traces of groups of mice in the Morris water maze space exploration experiment of example 3;

FIG. 3 is a graph comparing the number of times each group of mice correctly crossed the platform region in the Morris water maze space exploration experiment in example 3;

FIG. 4 is a graph comparing the cumulative time of each group of mice entering the platform quadrant in the Morris water maze space exploration experiment in example 3;

FIG. 5 is a graph showing the comparison of the expression levels of the inflammatory factor IL-6 in the serum of each group of mice in the ELISA experiment of example 4;

FIG. 6 is a graph comparing the expression levels of the inflammatory factor COX-2 in the serum of mice of each group in the ELISA experiment of example 4;

FIG. 7 is a graph comparing the expression levels of the inflammatory factor IL-1 β in the serum of mice of each group in the ELISA experiment of example 4;

FIG. 8 is a graph comparing the expression levels of the inflammatory factor TNF- α in serum of mice of each group in the ELISA experiment of example 4;

FIG. 9 is a graph comparing the expression levels of SOD in serum of mice of each group in the ELISA experiment of example 4;

FIG. 10 is a graph comparing GSH-Px expression levels in sera of mice of each group in ELISA experiment of example 4;

FIG. 11 is a graph comparing the results of HE staining in CA1 region of hippocampus of mice in each group in example 5;

FIG. 12 is a graph comparing the results of HE staining in CA3 region of hippocampus of mice in each group in example 5;

FIG. 13 is a graph comparing the results of histopathological scoring of hippocampus of mice in each group in example 5.

Detailed Description

The technical solutions of the present invention are further described below with reference to the following examples, but the present invention is not limited thereto, and any modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention. The process equipment or apparatus not specifically mentioned in the following examples are conventional in the art, and if not specifically mentioned, the raw materials and the like used in the examples of the present invention are commercially available; unless otherwise specified, the technical means used in the examples of the present invention are conventional means well known to those skilled in the art.

Example 1

This example establishes alzheimer's disease model mice and methods for grouping and administering them.

(one) experimental animals: 72 SPF grade ICR mice, male and female halves. The weight of the product is 22-25g, which is purchased from Liaoning Biotechnology GmbH, NO. SCXK 2020- - -0001.

(II) drugs and reagents

The kidney-tonifying and brain-benefiting preparation used in the embodiment is a kidney-tonifying and brain-benefiting pill produced by the limited pharmaceutical industry of peony, Jiangtiantai, and the approved literature is a Chinese medicine standard character Z20025764; product lot numbers 190703, 200501, 200902, 2009-03, 201201 and 210302.

The comparative western medicine used in this example was donepezil hydrochloride tablets, 10 mg/tablet, lot number 1911040, manufactured by wei-defense pharmaceutical products, inc.

Aβ_1-42Limited by Beijing Boaosen biotechnologyFrom Inc., cat # bs-0107p, lot # BJ 02263853.

The sodium penicillin for injection is produced by Harbin pharmaceutical group pharmaceutical manufacturing company, product batch No. 20110628-2.

Mouse interleukin 6(IL-6), mouse tumor necrosis factor alpha (TNF-alpha), mouse cyclooxygenase 2(COX-2), mouse interleukin 1 beta (IL-1 beta), mouse glutathione peroxidase (GSH-PX) and mouse superoxide dismutase (SOD) ELISA kits are purchased from Jiangsu enzyme immunity industry Co., Ltd and have the product numbers of MM-0163M2, MM-0132M2, MM-0356M2, MM-0040M2, MM-0758M2 and MM-0389M 2.

(III) Experimental apparatus

DW-200 type brain stereotaxic instrument (Chengdutai Union science and technology Co.);

tai union TM-Vision behavioural experimental system (WMT-100Morris water maze video analysis system (white rat version));

a cryogenic refrigerator (hel DW-86L 626);

microsyringe (Shanghai Tingn microsyringe, 10 μ L standard);

medical silk non-absorbable suture (Yangzhou Jinhuan medical instrument and instruments factory, specification and model 3-0, production lot 20200103);

absorbent cotton balls (Yanggu Jingyanggang sanitary materials factory);

a common hemostatic forceps (Zhang Jiagang city Jinfeng deer knife and scissor factory, 12.5cm straight specification, Susu food and drug supervision instrument No. 2007, 1010051);

a microplate reader (Ledu Life sciences GmbH, model: RT-6100);

an electric heating constant temperature incubator (Wuhan-Hengsu clean scientific instruments Co., Ltd.: BPS-50 CL);

plate washing machine (Beijing celestial stone model: ZMX-988B);

a micro high-speed centrifuge (Hunan instrument centrifuge instrument, Inc. model number: TG16W in the development area of the Changsha high and new technology industry);

high speed tissue milling instruments (Wuhan Severe Biotech Co., Ltd.: KZ-II);

upright white light photographing microscope (Nikon (Japan) model Eclipse Ci-L); scan browsing software 3dhistech (hungary) model caseviewer 2.4.

(IV) Experimental groups

After 72 ICR mice, male and female, were bred adaptively for one week, and then were randomly divided into a sham-operated group (Control), an Alzheimer disease model group (AD model), a Donepezil hydrochloride group (Donepezil), a kidney-tonifying and brain-benefiting pill low dose group (BSYNW-L), a kidney-tonifying and brain-benefiting pill medium dose group (BSYNW-M), and a kidney-tonifying and brain-benefiting pill high dose group (BSYN-W-H) by body mass and sex, and 12 mice were each group.

(V) Alzheimer disease model construction

After the model group, the donepezil hydrochloride group, the kidney tonifying and brain benefiting pill low dose group, the kidney tonifying and brain benefiting pill medium dose group and the kidney tonifying and brain benefiting pill high dose group are anesthetized with urethane 350mg/kg, the mice are fixed, and the hair of the head is cut off, and the head skin is disinfected by iodophor and alcohol. The skin of the mouse head was cut about 2cm, the position of the glottis was found, the glottis was used as the origin, the glottis was moved rightward by 1mm, the glottis was moved backward by 0.5mm and the depth was 3mm, and 3. mu.l of aged Abeta was slowly injected by a microsyringe within 10min_1-42(5. mu.g/. mu.l) and dispersed for 5 min. And (5) smearing a proper amount of penicillin sodium powder on the wounds after disinfection, and suturing the wounds. In case of feeding in mouse cage, penicillin (1 time/d) was given 5 days before operation to avoid infection while paying attention to postoperative warming. Sham groups injected with equal volumes of sterile saline.

(VI) administration of drugs

The dosage of the kidney-tonifying brain-benefiting pill in the high, middle and low groups is 1.44g/kg, 0.72g/kg and 0.36g/kg respectively, and the dosage of the donepezil hydrochloride group (1.5mg/kg) is administrated by intragastric administration once a day. 6 equal batches of kidney-tonifying and brain-benefiting pills are taken, ground into superfine powder and fully and uniformly mixed, and the donepezil hydrochloride is also ground into powder for later use. The administration is continued for 60 days, and the mice in the sham operation group and the model group are perfused with distilled water with the same volume.

Example 2

In the embodiment, the influence of the kidney tonifying and brain benefiting preparation on the learning and memory capacity of the mouse is investigated through a Morris water maze positioning navigation experiment.

The specific method of the Morris water maze positioning navigation experiment comprises the following steps:

the Morris water maze body was a white circular pool of water 120cm in diameter and 50cm in height, which was filled with water and skimmed milk powder during the test, and divided into four equal area quadrants. A platform 25cm in height and 10cm in diameter was placed in the very center of the target quadrant, submerged 1cm below the water surface. During the experiment, the mice are placed in the opposite angle quadrant of the target quadrant where the platform is located in a back-to-back mode, the total time is 60s, and all groups of mice are trained sequentially. If the mouse finds the platform within 60s, recording the time as the escape latency of the mouse, allowing the mouse to stay on the platform for 10s, wiping the mouse and putting the mouse back into the cage, and if the mouse does not find the target platform within 60s, guiding the mouse to reach the platform, wherein the escape latency of the mouse is recorded as 60 s.

Example 1 groups of mice established after 60 days of treatment 6 mice (3 female and 3 male) per group were randomly selected for 6 days of training and their swimming paths were recorded using image tracking software. And analyzing and searching the latency of the escape platform by using statistical analysis software.

FIG. 1 is a graph comparing escape latencies of groups of mice in the Morris water maze positioning navigation experiment of the present embodiment; as shown in the comparison results in FIG. 1, the escape latency of each group of mice decreased to different degrees as the number of training days increased. The experimental data show that the escape latency of mice in the alzheimer model group from the third day is statistically significant compared to other groups of mice (P <0.05 or P < 0.01). The kidney tonifying and brain benefiting pills can effectively improve the decline of the spatial memory capacity of the senile dementia animal model and show dose dependence. Wherein, compared with the Control group, the AD model group mice have obviously prolonged escape latency time (P <0.01), the BSYNW-H group mice have obviously reduced escape latency compared with the AD model group (P <0.01), and the BSYNW-L group mice and the BSYNW-M group mice have different degrees of reduction of escape latency (P <0.01 or P < 0.05).

Example 3

In the embodiment, the influence of the kidney tonifying and brain benefiting preparation on the learning and memory capacity of the mouse is investigated through a Morris water maze space exploration experiment.

The specific method of the Morris water maze space exploration experiment comprises the following steps:

example 2 after the positioning navigation detection is finished for 24h, that is, the seventh day, the transparent platform is removed, the mouse back platform is placed at the edge of the diagonal quadrant of the target quadrant, the mouse freely moves for 60s, and the number of times that each mouse passes through the platform and the accumulated time that each mouse enters the target quadrant where the platform is located are recorded in the period. The ambient environment should be kept quiet during all experiments, and the positions of other reference objects cannot be freely moved.

Fig. 2 is a comparison graph of representative movement trajectories of each group of mice in the Morris water maze space exploration experiment, fig. 3 is a comparison graph of the number of times that each group of mice correctly passes through the platform area in the Morris water maze space exploration experiment, and fig. 4 is a comparison graph of the cumulative time that each group of mice enters the platform quadrant in the Morris water maze space exploration experiment.

The Morris water maze space exploration experiment result shows that compared with the Control group, the accumulated residence time of the AD model group mouse in the target quadrant is obviously shortened, the times of accurately passing through the position of the platform are less, and the obvious difference is achieved (P is less than 0.01). Compared with the AD model group, the cumulative residence time of the mice in the BSYNW-H group and the mice in the Donepzil group in the target quadrant is obviously prolonged (P <0.01, P <0.05), and the times of accurately crossing the position of the original platform are obviously increased (P < 0.01). The pill for tonifying the kidney and benefiting the brain has good curative effect on improving the spatial learning and memory capacity of the model mouse with the Alzheimer disease.

Example 4

In the embodiment, the influence of the kidney-tonifying and brain-benefiting preparation on the levels of inflammatory factors and oxidative stress related factors in the serum of the mouse is examined through an enzyme-linked immunosorbent assay.

The specific method of the enzyme-linked immunosorbent assay comprises the following steps:

example 3 at the end of the behavioural test, the mouse eyes were bled and sacrificed by decapitation, and after centrifugation at 135000r/min at 4 ℃ for 20min, serum was separated, leaving the sample to be tested, and the remaining samples were stored at-80 ℃ for future use. The procedures were performed exactly as described in the kit instructions.

FIGS. 5-8 are graphs comparing the expression levels of inflammatory factors IL-6, COX-2, IL-1 beta and TNF-alpha in the sera of mice of each group in the ELISA experiment of this example; FIG. 9 and FIG. 10 are the comparison of the expression levels of SOD and GSH-Px in the serum of each group of mice in the ELISA experiment of this example.

As shown in FIGS. 5 to 10, the serum levels of IL-1. beta., TNF-. alpha., COX-2, and IL-6 were increased, and the levels of SOD and GSH-PX were decreased in the AD model group mice, which were significantly different from those in the control group (P < 0.05). Compared with the AD model group, the serum contents of IL-1 beta, TNF-alpha, COX-2 and IL-6 in mice in the BSYNW-H group are all reduced, while the contents of SOD and GSH-PX are increased, and the difference has statistical significance (P is less than 0.05). Compared with the AD model group, the serum contents of IL-1 beta, TNF-alpha, COX-2 and IL-6 in mice in the BSYNW-M group and the BSYNW-L group are reduced with different degrees, and the contents of SOD and GSH-PX are increased with different degrees and are in dose dependence. The serum IL-6 level of mice in the BSYNW-M group is also obviously different from that in the AD model group (P is less than 0.05), which shows that the kidney-tonifying and brain-benefiting pill can inhibit the release of inflammatory factors in the serum of mice in an Alzheimer disease model and increase the release of related antioxidant factors, and the action mechanism of the medicament is possibly related to anti-inflammatory and antioxidant effects.

Example 5

In this example, pathological morphological changes of the mouse hippocampal CA1 region and CA3 region were detected by HE staining.

The HE dyeing method comprises the following steps:

after the mice are killed by decapitation, the whole brain tissue is quickly taken out under the low-temperature aseptic condition, washed by ice-cold isotonic saline, divided into left and right half brains from the midline of the brain, fixed by 4 percent paraformaldehyde solution for 48 hours, washed by tap water for 24 hours, dehydrated and transparent, and then paraffin-embedded HE staining is carried out. The remaining brains were stored at-80 ℃ for further analysis.

The slices were soaked in alum for 5min, rinsed with tap water, and then eosin-stained for 1 min. The slices were dehydrated in an ascending alcohol solution, made transparent with xylene, and then sealed.

Pathological changes in the hippocampal CA1 region and CA3 region were examined by Leica DM3000LED light microscope in Germany and analyzed by Motic MED6.0 digital medical image analysis system. Then, microscopic examination and photographing are carried out under a microscope, and the pathological morphology scoring is carried out according to the pathological scoring standard. The scoring criteria are shown in table 1 below.

TABLE 1 Hippocampus CA1 region, CA3 region histopathology scoring criteria

Statistical analysis: the experimental data are expressed as (x + -s), the hippocampal histomorphometry HE staining score is tested by nonparametric analysis, the Morris water maze escape latency is analyzed by variance of repeated measurement data, the rest of the intergroup data are compared by one-factor variance analysis, and the results are statistically analyzed and plotted by GraphPad Prism 5. P <0.05 is considered statistically significant.

FIG. 11 is a graph comparing the results of HE staining in CA1 region of hippocampus of each group of mice in this example; FIG. 12 is a graph comparing the results of HE staining in CA3 region of hippocampus of each group of mice in this example; FIG. 13 is a graph comparing the histopathological scoring results of the hippocampus of the mice of each group of this example.

The significant cytopathological changes of neurons in CA1 region and CA3 region of hippocampus of AD model mice, including disordered pyramidal cell arrangement, thinning, reduced cell volume, enlarged cell gap and irregular shape, can be observed by comparing the pictures shown in FIGS. 11-13. The arrangement of nerve cells is disordered and the number of the nerve cells is obviously reduced, the cell consolidation and vacuolation are extremely obviously seen, and compared with a Control group, the pathological morphology score of the nerve cells is obviously increased; compared with the AD model group, the kidney-tonifying and brain-nourishing pill has the advantages that the number of hippocampal nerve cells of mice in each administration group is obviously increased, the arrangement disorder state is improved, the phenomena of cell consolidation and vacuolation are obviously relieved, and the pathological morphology score is obviously reduced. Among them, the cells in the CA1 region and CA3 region of the hippocampus of mice in the BSYNW-H group and the Donepzil group were slightly cytopathic compared to those in the AD model group, but the cells were not present in the normal mice. Observed under a light lens, and injected with Abeta_1-42Then, compared with the Control group, the number of neurons in the CA1 area and CA3 area of the hippocampus of the AD model group mice is obviously reduced, which indicates that the kidney-tonifying and brain-benefiting pillHas good effect on improving pathological morphological damage of the hippocampus of an AD model mouse.

A series of experiments in examples 1-5 prove that the compound traditional Chinese medicine kidney-tonifying and brain-benefiting preparation can treat learning and memory ability of mice of an Alzheimer disease model, improve pathological morphological injury of hippocampus, inhibit synthesis and release of inflammatory mediators, block cascade reaction of inflammatory mediators, and facilitate control of excessive inflammatory reaction, thereby effectively controlling the state of an illness and reducing the death rate. In the aspect of antagonizing oxygen free radicals, the Chinese herbal compound kidney-tonifying brain-benefiting preparation improves the scavenging capacity of organisms to oxygen free radicals and lightens the damage of the organisms caused by free radical attack by increasing the activities of oxygen free radical scavenging enzymes such as superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX).

Claims

1. Application of a compound Chinese medicinal preparation for invigorating kidney and nourishing brain in preparing medicine for preventing and treating Alzheimer disease is provided.

2. The application of the compound traditional Chinese medicine kidney-tonifying and brain-benefiting preparation in preparing a medicine for preventing and treating alzheimer disease according to claim 1, wherein the prevention and treatment of alzheimer disease is to inhibit the release of inflammatory cytokines in the brain of alzheimer disease patients, eliminate oxygen radicals, improve pathological damage of hippocampus of the alzheimer disease patients, and improve learning and memory abilities.

3. The application of the traditional Chinese medicine compound kidney-tonifying and brain-benefiting preparation in preparing the medicine for preventing and treating the alzheimer disease according to claim 1 or 2 is characterized in that the traditional Chinese medicine compound kidney-tonifying and brain-benefiting preparation contains the following components in parts by weight: 85-125 parts of prepared rehmannia root, 30-60 parts of ligusticum wallichii, 35-85 parts of polygala tenuifolia, 35-85 parts of poria cocos, 35-85 parts of angelica sinensis, 6-18 parts of pilose antler, 35-85 parts of red ginseng, 35-85 parts of bran-fried Chinese yam, 30-60 parts of fructus psoraleae, 30-60 parts of achyranthes bidentata, 30-60 parts of wolfberry fruit, 30-60 parts of radix scrophulariae, 35-85 parts of radix ophiopogonis, 30-60 parts of schisandra chinensis, 35-85 parts of fried spina date seed and 10-20 parts of cinnabar.

4. The application of the compound traditional Chinese medicine kidney-tonifying and brain-benefiting preparation in preparing a medicine for preventing and treating alzheimer disease according to claim 3, wherein the kidney-tonifying and brain-benefiting preparation in the medicine for preventing and treating alzheimer disease is the only active ingredient.

5. The application of the compound traditional Chinese medicine kidney tonifying and brain strengthening preparation in preparing a medicine for preventing and treating alzheimer's disease according to claim 4, wherein the medicine for preventing and treating alzheimer's disease further comprises pharmaceutically acceptable auxiliary materials and/or carriers.

6. The application of the compound traditional Chinese medicine kidney tonifying and brain strengthening preparation in preparing a medicine for preventing and treating alzheimer's disease according to claim 5, wherein the dosage form of the medicine for preventing and treating alzheimer's disease is granules, tablets, capsules, pills, dripping pills or oral liquid preparations.

7. The application of the traditional Chinese medicine compound kidney-tonifying and brain-benefiting preparation in preparing a medicine for preventing and treating alzheimer disease as claimed in claim 6, wherein the traditional Chinese medicine compound kidney-tonifying and brain-benefiting preparation is a kidney-tonifying and brain-benefiting pill, and the weight ratio of cooked rehmannia root, ligusticum wallichii, polygala tenuifolia, poria cocos, angelica sinensis, pilose antler, red ginseng, bran-fried Chinese yam, fructus psoraleae, achyranthes bidentata, wolfberry fruit, radix scrophulariae, radix ophiopogonis, schisandra chinensis, fried spina date seed and cinnabar in the kidney-tonifying and brain-benefiting pill is 194: 70: 91: 91: 91: 14.4: 94: 91: 70: 70: 72: 70: 91: 70: 91: 24.

8. the application of the traditional Chinese medicine compound kidney-tonifying and brain-benefiting preparation in preparing a medicine for preventing and treating alzheimer disease according to claim 7 is characterized in that the preparation method of the kidney-tonifying and brain-benefiting pill comprises the following steps: refining Cinnabaris with water to obtain fine powder; decocting the angelica, the radix ophiopogonis, the radix scrophulariae, the polygala tenuifolia and the achyranthes bidentata twice in water for 2 hours each time, filtering, combining the filtrates, standing for more than 8 hours, filtering, and concentrating the filtrate to obtain clear paste with the relative density of 1.16-1.20 at the temperature of 50 ℃; pulverizing radix rehmanniae Preparata, rhizoma Ligustici Chuanxiong, Poria, cornu Cervi Pantotrichum, Ginseng radix Rubri, rhizoma Dioscoreae parched with bran, fructus Psoraleae preparata, fructus Lycii, fructus Schisandrae chinensis and semen Ziziphi Spinosae preparata into fine powder; grinding the obtained fine powder and Cinnabaris superfine powder, sieving, mixing, adding the above fluid extract, mixing, making into concentrated watered pill, drying, polishing, and making into 1000 g.

9. The application of the traditional Chinese medicine compound kidney-tonifying and brain-benefiting preparation in preparing a medicine for preventing and treating alzheimer disease according to claim 8 is characterized in that the content detection requirement of the kidney-tonifying and brain-benefiting pill is as follows: the content of fructus Psoraleae containing salt per 1g of the pill is not less than 0.47mg based on the total amount of psoralen and isopsoralen; the content of fructus Schisandrae in 1g of the pill is not less than 0.27mg, calculated as schizandrol A.

10. The application of the traditional Chinese medicine compound kidney-tonifying and brain-benefiting preparation in preparing a medicine for preventing and treating alzheimer disease according to claim 9 is characterized in that the medicine for preventing and treating alzheimer disease is orally administered, 2g of the kidney-tonifying and brain-benefiting preparation is contained in the medicine for oral administration every time, the medicine is orally administered twice every day, and one treatment course is 90 days.