CN108543037B

CN108543037B - Traditional Chinese medicine composition and preparation method and application thereof

Info

Publication number: CN108543037B
Application number: CN201810686652.3A
Authority: CN
Inventors: 张云龙; 刘妍
Original assignee: Guangzhou Medical University
Current assignee: Guangzhou Medical University
Priority date: 2018-06-28
Filing date: 2018-06-28
Publication date: 2021-02-26
Anticipated expiration: 2038-06-28
Also published as: CN108543037A

Abstract

The invention discloses a traditional Chinese medicine composition and a preparation method and application thereof. The traditional Chinese medicine composition is composed of coptis chinensis, phellodendron amurense, gardenia, salvia miltiorrhiza, turmeric and rhizoma acori graminei, can be used for remarkably improving learning and memory dysfunction of a 5 XFAD transgenic mouse, reducing expression of hippocampal Abeta 40/42, improving expression levels of pre-synaptic and post-synaptic proteins of the hippocampus of the 5 XFAD transgenic mouse, improving excitatory synaptic transmission dysfunction of the hippocampus of the 5 XFAD transgenic mouse, inhibiting apoptosis of neurons of the hippocampus of the 5 XFAD mouse and proliferation of microglial cells, inhibiting expression of serum and hippocampus inflammatory factors of the 5 XFAD mouse, and providing a reliable theoretical basis for preparation of medicaments for treating or assisting in treating Alzheimer's disease.

Description

Traditional Chinese medicine composition and preparation method and application thereof

Technical Field

The invention belongs to the field of medicines, and particularly relates to a traditional Chinese medicine composition as well as a preparation method and application thereof.

Background

Alzheimer's Disease (AD), senile dementia, is a progressive degenerative disease of the nervous system and also a major dementia disease. Epidemiological data from the "global Alzheimer's disease report" in 2016 showed that by 2016, about 4700 million people worldwide had AD, by 2030 it increased to 7470 million, and by 2050 it increased to over 1 hundred million 3150 million people^[1]. About 58% of these AD patients live in developing countries, and it is expected that more than 68% of new AD cases will occur in asia in 2050 developing countries and 49% of new AD cases in 2015^[1]. At present, the Chinese population is in the rapid aging development stage, the AD incidence rate is increased year by year, the number of AD patients is the first in the world at present, statistics shows that the number of the Chinese people suffering from AD is 569 ten thousand in 2010, the prevalence rate is 6.25 per thousand, and the number of the Chinese people suffering from AD is increased at the rate of 30 ten thousand per year^[2]. The clinical symptoms of AD are mainly manifested by progressive memory impairment, cognitive dysfunction, impairment of visuospatial ability, gradual loss of speech function, executive dysfunction, and personality and behavioral changes^[1,3-6]. At the same time, the treatment and care costs of AD are increasing year by year, and the total cost for treating senile dementia in 2016 is estimated to be $ 8180 billion worldwide, which is increased by $ 2140 billion compared with 2010^[1]. The treatment cost after the onset of AD is high, no specific treatment medicine and means exist at present, and heavy mental and economic burden is brought to families and society.

The etiology of AD is unknown, and it may be associated with gene instability and gene mutation, metabolic disorders, neuroinflammation, and slownessViral infection, craniocerebral trauma or environmental factors. It is now generally accepted that the most central pathological features of AD are the appearance of Amyloid Plaques (Amyloid Plaques) consisting of Amyloid beta (Abeta) outside the nerve cells in the brain, also known as Senile Plaques (SP), and intracellular Neurofibrillary Tangles (NFT)^[3-7]The lesion sites are mainly in the hippocampus and cortical areas. A β is released by the enzymatic degradation reaction of β Amyloid Precursor Protein (APP), and in particular a β 42 is widely and stably present in senile plaques and neurofibrillary tangles. Most researchers believe that a large amount of a β deposition is a direct cause of AD onset, and inflammatory responses are local secondary responses caused by a β deposition and are also important factors in degeneration of the central nervous system, and the main signs of these responses are activation of astrocytes and microglia^[8]. The inflammatory reaction is an important pathological mechanism of AD, and the obvious nonspecific immune inflammatory reaction exists in the brain of AD patients, and plays a role in the early and middle stages of plaque formation^[9]It is mainly expressed that inflammatory factors such as Interleukin-1 (Interleukin-1), Interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) are obviously up-regulated in parts such as hippocampus and cortex of AD brain tissue. In addition, A beta can directly activate microglia, and initiate inflammatory reaction^[10]And activated microglia can release peroxide to directly damage neurons, and can release cytokines to damage adjacent neurons or endothelial cells. These factors include IL-1 beta, TNF-alpha, NO, and various proteolytic enzymes, which cause or exacerbate degenerative changes in neurons^[11]。

For AD, there are many therapeutic drugs such as acetylcholinesterase inhibitors (e.g. donepezil), physostigmine, galantamine, huperzine a, etc., which can enhance the cognitive ability of AD patients, but their use is limited due to side effects of the central or peripheral system.

AD belongs to the category of 'coma, amnesia' and 'anorexia' in traditional Chinese medicine. The traditional Chinese medicine considers the basic disease of the diseaseThe mechanism is 'marrow-decreasing brain-disappearance and mental-dysfunction', namely senile viscera deficiency, kidney essence deficiency and brain marrow malnutrition, which cause mental-dysfunction and dementia. Wang Yongyan Si Shi thinks that the brain is the house of Qing Ling, most contraindicated for foul qi and turbid toxin, when the viscera are weak or invaded by exogenous pathogens, the viscera of the human body are dysfunctional, qi, blood and body fluids are abnormal in circulation, phlegm turbidity, blood stasis and the like in the body cannot be discharged in time, and the phlegm is vaporized and decomposed, and the heat is transformed into toxin, and the turbid toxin is generated internally. Once the interior excess and turbid toxin is formed, it is characterized by destroying the body and consuming the viscera, meridians and collaterals. Deficiency and failure of viscera can lead to deficiency and airflow stagnation, water and body fluid distribution and qi and blood circulation disorder, phlegm generation and stasis generation, mutual cohesion of phlegm and blood stasis, difficulty in moving of phlegm and blood, difficulty in transforming phlegm and blood stasis, toxic substance transformation caused by phlegm and blood stasis, body destruction, brain orifice obstruction and mental disorder, and dementia. Deficiency of viscera, deficiency of kidney essence, marrow depression and brain elimination, and failure of mental mechanism are the basic pathological manifestations of AD, but the main pathogenesis of AD is the generation of turbid toxin internally, phlegm reduction and stasis generation, and obstruction of brain orifices. The theory is the basic idea of the theory of 'toxic damage to brain collaterals' in modern traditional Chinese medicine, and the application of the traditional Chinese medicine for clearing heat and removing toxicity to prevent and treat AD also becomes a significant and new important way^[12]And the traditional Chinese medicine has rich resources, and the search for the medicine for treating AD in the traditional Chinese medicine has great social and economic benefits.

Currently, research and clinical studies show that: the coptis chinensis detoxification decoction has obvious effect on treating senile dementia with symptoms of heart-liver fire hyperactivity and toxic heat flourishing^[13-15]. Modern pharmacological research shows that the coptis chinensis detoxification decoction has the effects of resisting inflammation, reducing blood pressure and blood fat, improving gastrointestinal tract function, resisting oxidation and atherosclerosis, promoting learning and memory and the like^[16-20]. Research shows that the coptis chinensis detoxification soup can up-regulate the activity of superoxide dismutase (SOD) and reduce excessive Malondialdehyde (MDA) generated by lipid peroxidation, thereby playing a role in resisting lipid peroxidation^[21](ii) a The Coptidis rhizoma detoxicating decoction also has effects of inhibiting acetylcholinesterase (AchE) activity, increasing acetylcholinesterase (ChAT) activity, relieving acetylcholine hydrolysis rate, and improving learning and memory function of AD model^[22,23]. In the aspect of nerve immunity regulation, the Huanglianjiedu decoction can reduce cerebral componentsThe expression level of inflammatory factors IL-1 beta, IL-6 and the like in tissues is improved, the immune microenvironment is improved, the dysmnesia and the spatial direction discrimination disorder of an AD rat model and an APP/PS1 double-transgenic AD mouse model caused by Abeta ventricle injection are further obviously improved, the deposition of hippocampus and cortex Abeta plaques and the quantity of senile plaques and neurofibrillary tangles are reduced, and the neuroprotective effect is exerted^[24-26]. However, the formula is only suitable for the excessive toxic heat type Alzheimer disease, and for turbid toxin blood stasis type, the formula is lack of products for promoting blood circulation to remove blood stasis, eliminating phlegm and inducing resuscitation.

At present, the traditional Chinese medicine symptoms of AD are mainly divided into different symptoms of insufficient marrow sea, spleen and kidney deficiency, phlegm turbidity obstruction in orifices, blood stasis internal resistance and the like. The basic pathogenesis of the marrow-sea deficiency disease is kidney essence deficiency and marrow-sea insufficiency, and the marrow-sea insufficiency disease is mainly manifested by intelligence decline, obvious decline of memory and computational power, dizziness and tinnitus, relaxation and sleepiness, tooth dryness and hair scorching, waist soreness and bone softness, difficult walking, thin and light tongue, thin and white fur and deep and weak pulse, and the seven-fortune drink is used for treating kidney tonifying and marrow benefiting, essence replenishing and spirit nourishing; the basic pathogenesis of the spleen-kidney deficiency disease is dysfunction of the spleen in transport and deficiency of kidney essence, and the symptoms mainly include expression dull, silence and little speech, hypomnesis, shortness of breath and no speaking desire, muscle atrophy, poor appetite and anorexia, saliva overflow, soreness and weakness of waist and knees, or cold limbs, abdominal pain and press, diarrhea, pale tongue, swollen tongue, white fur, or red tongue, little or no fur, deep and weak pulse, and the medicine is prepared by reducing the number of pills, tonifying the kidney and strengthening the spleen, tonifying qi and producing sperm; phlegm turbidity obscures orifices, the basic pathogenesis is that spleen movement is not healthy, phlegm is generated and dampness is generated, phlegm dampness obscures orifices and is mainly manifested as dull expression, intelligence decline, poor appetite, epigastric and abdominal pain, fullness and discomfort, excessive mouth and saliva foam, pale tongue, white and greasy fur and slippery pulse, and the phlegm-removing decoction is used for treating the phlegm-removing decoction, spleen strengthening and turbidity-eliminating, phlegm-eliminating and orifice-opening; the basic pathogenesis of the internal blood stasis syndrome is blood stasis, obstruction and resuscitation are mainly changed into slow expression, difficult speech, amnesia, easy fright, scaly skin, dry mouth and no desire to drink, dim eyes, dark tongue or petechia petechiae and thready and unsmooth pulse, and the internal blood stasis syndrome is treated by the decoction for dredging the orifice, activating blood circulation, removing blood stasis, inducing resuscitation and restoring consciousness.

However, at present, clinically, turbid toxin is generated internally, phlegm is reduced, stasis is generated, and then brain orifices are shielded as a basic pathogenesis, and patients with turbid toxin stasis blood type Alzheimer's disease are in the majority, but related traditional Chinese medicine researches are not found in the disease.

Reference documents:

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14. Shaoweng, Huangbei, Panxiang, Zhangzhong, Chenguohua, Coptis chinensis detoxification decoction combined with huperzine A for treating senile dementia clinical observation, Hubei J.TCM 2012(09) 11-12.

15. Xulei, coptis decoction for detoxification, clinical and experimental research on treating senile dementia, Master, Hubei university of traditional Chinese medicine; 2015.

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18.Wang L,Zhu HY,He JZ,Yin X,Guo LH.Effect of Modified Huanglian Jiedu Decoction Purgation Combined Electroacupuncture in Intervening Gastrointestinal Dysfunction of Critically Ill Patients Undergoing Abdominal Surgery.Zhongguo Zhong Xi Yi Jie He Za Zhi,2015,35(8):966-970.

19.Li T,Han JY,Wang BB,Chen B,Li YM,Yu ZJ,Xue X,Zhang JP,Wang XB,Zeng H,Ma YL.Huanglian jiedu decoction regulated and controlled differentiation of monocytes,macrophages,and foam cells:an experimental study.Zhongguo Zhong Xi Yi Jie He Za Zhi,2014,34(9):1096-1102.

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21. dongxu, Duhui Lian, Korea Mega, Wangmei, Wangchun, Linli, Huanglian Jiedu Tang has effects on SOD activity, MDA content, I-kappa B and NF-kappa B protein expression of senile dementia rats, Chinese materia medica, 2012(08):1730-1732.

22. Research on antioxidation and acetylcholinesterase inhibition of Song Jianfang, Wang hong Jie, Senan, Bordetella and Huanglian Jiedu Tang, J.Chinese Experimental and prescriptions, 2010(05) 61-64.

23. Li bin, Shexiling, Penli Yan, Wangfei, Yangxin, Wuwenbin, Huanglian Jiedu Tang have influence on learning and memory ability and central cholinergic system of mice with acquired memory impairment.

24. Qixin, Chen Guo Hua, Mei rose, Wan 24354g, Wan Kaixin, Pan Song and Huang Lian Jie Tang has effects on the metabolism of free radicals and IL-6 and IL-1 beta contents of brain tissues of APP/PS1 double-transgenic Alzheimer's disease mice on stroke and neurological diseases, 2011(02):72-74.

25. Chen Guawa, Mei rose, Xin, Wan moon, Wan \24354, von pei, Huanglian Jiedu Tang has an interference effect on IL-6 and IL-1 beta levels and SP and NFTs amounts of brain tissues of APP/PS1 double-transgenic AD mice, and China journal of neuroimmunology and neurology, 2011(05): 352-.

26. The formula of the decoction is green, Zhanxianfu, solieria, sun plum, coptis chinensis detoxification decoction, the treatment effect of AD rats and the influence on the content of cytokines, China TCM journal, 2004(06), 86-89.

Disclosure of Invention

The invention aims to provide a traditional Chinese medicine composition and a preparation method and application thereof.

The inventor provides a traditional Chinese medicine composition taking coptis chinensis, phellodendron amurense, gardenia, salvia miltiorrhiza, turmeric and rhizoma acori graminei as active ingredients.

Further, the traditional Chinese medicine composition comprises the following substances in parts by weight: 10-20 parts of coptis chinensis, 5-10 parts of phellodendron, 10-20 parts of gardenia, 10-18 parts of salvia miltiorrhiza, 5-15 parts of turmeric and 5-15 parts of rhizoma acori graminei.

Further, the weight portions are as follows: 12-18 parts of coptis chinensis, 6-10 parts of phellodendron, 12-18 parts of gardenia, 13-17 parts of salvia miltiorrhiza, 8-12 parts of turmeric and 8-12 parts of rhizoma acori graminei.

Further: the Chinese medicinal composition comprises the following components in percentage by mass: (2-6): (1-3): (2-6).

Further: the Chinese medicinal composition comprises the following components in percentage by mass: 4: 2: 4.

further: the traditional Chinese medicine composition comprises the following components in parts by weight: 16 parts of coptis chinensis, 8 parts of golden cypress, 16 parts of gardenia, 15 parts of salvia miltiorrhiza, 10 parts of turmeric and 10 parts of rhizoma acori graminei.

The invention also provides a traditional Chinese medicine preparation, which is any one of powder, tablets, injection, capsules, decoction, granules, pills, paste and targeted preparations prepared from the composition.

The inventor also provides a preparation method of the decoction, which comprises the following steps: decocting Coptidis rhizoma, cortex Phellodendri, fructus Gardeniae, Saviae Miltiorrhizae radix, Curcuma rhizome and rhizoma Acori Graminei in water to obtain decoction.

Further, the traditional Chinese medicine composition is decocted for two times.

Further, adding 5-20 times of water for decoction for the first time.

Further, 8-15 times of water is added for the first time for decoction.

Further, 10 times of water was added for the first time to perform the decoction.

Further, the traditional Chinese medicine composition is soaked before decoction.

Further, the soaking time is 0.1-5 h.

Further, the soaking time is 0.5-3 h.

And further, adding 3-15 times of water for decoction for the second time.

Further, adding water with the volume 5-12 times of that of the mixture for the second time for decoction.

Further, adding 8 times of water for decoction for the second time

Furthermore, the first time and the second time of decoction are independent for 0.1-2 hours.

Furthermore, the first time and the second time of decoction are independent for 0.5-1.5 h.

Further, the first and second decocting time is 1h independently.

Further: each 2-4 g of the composition is prepared into 1mL of decoction.

Further: each 2.92g of the composition gave 1mL of decoction.

The inventor applies the decoction prepared above to 5 × FAD transgenic mice, and carries out related research, and the research result shows that: the decoction prepared by the traditional Chinese medicine composition has the functions of detoxifying and removing blood stasis, so that the decoction is called as detoxifying and removing blood stasis decoction, can obviously improve the learning and memory dysfunction of a 5 XFAD transgenic mouse, reduces the deposition of hippocampal Abeta, can obviously improve the expression levels of glutamic acid receptors, presynaptic proteins, postsynaptic proteins and the like of the hippocampal of the 5 XFAD transgenic mouse, and improves the transmission function of excitatory synapses of the hippocampal of the 5 XFAD transgenic mouse. Meanwhile, the detoxification stasis-removing decoction inhibits the proliferation of hippocampal microglia, the expression level of serum and hippocampal inflammatory factors, and further discovers that: the detoxifying and stasis removing soup also has obvious neuroprotective and immunoregulation effects.

The above experimental data show that: the traditional Chinese medicine composition has great potential in preparing medicines for treating or assisting in treating the Alzheimer disease.

Further, said alzheimer's disease is preferably alzheimer's disease of the turbid blood group.

The invention has the beneficial effects that:

1. the test proves that: the traditional Chinese medicine composition can obviously improve the learning and memory dysfunction of a 5 XFAD transgenic mouse, reduce the expression of hippocampal Abeta 40/42, improve the expression level of pre-synaptic and post-synaptic proteins of the hippocampi of the 5 XFAD transgenic mouse, improve the excitatory synaptic transmission dysfunction of the hippocampi of the 5 XFAD mouse, inhibit the apoptosis of the neurons of the hippocampi of the 5 XFAD mouse and the proliferation of microglia, and can up-regulate the expression of TREM2 of the hippocampi of the 5 XFAD mouse at the mRNA level, thereby providing a reliable theoretical basis for the preparation of medicaments for treating or assisting in treating Alzheimer disease.

2. The traditional Chinese medicine composition has obvious nerve protection and immunoregulation benefits, is composed of traditional Chinese medicines with definite pharmacology, is safe and reliable when being applied in practice, and has no obvious side effect.

Drawings

FIG. 1: (A) percent shuttle in T maze for different groups of mice; (B) percent shuttle in the Y maze for different groups of mice; (C) training mice of different groups for 5 days to reach the platform; (D) the time for the mice of different groups to reach the platform quadrant after training; (E) the residence time of the mice of different groups in the platform quadrant is determined; (F) passing through the platform for different groups of mice; (G) identifying a pattern diagram of an experimental training and testing phase for a new object; (H) for the training phase, each group of mice explores the percentage of time for the object A; (I) for the training phase, each group of mice explores the percentage of time for the B object; (J) for the testing phase, the percentage of time each group of mice explores for new objects;

FIG. 2: (A) LTP field potential slope percentage for Con, 5 × FAD, and JDHYT-L groups; (B) LTP field potential slope percentage for Con, 5 × FAD, and JDHYT-H groups; (C) field potential intensities of different groups of mouse LTP at different times after high-frequency stimulation;

FIG. 3: (A) protein expression Western blot patterns of different groups of mouse glutamate receptors NR2A, Abeta 40/42, Synapsin and PSD95 in hippocampus; (B) relative optical density values of the expression quantity of A beta 40/42 protein of mouse glutamic acid receptor NR2A of different groups; (C) relative optical density values of protein expression quantities of Synapsin and PSD95 of different groups of mice presynaptic proteins;

FIG. 4: (A) represents the current curve for AMPA receptor mediated mepscs; (B) graph (left) of cumulative frequency distribution of AMPA receptor-mediated mepscs frequencies and a plot of quantitative analysis of frequencies (right); (C) graphs of cumulative frequency distribution (left) and quantitative analysis of average amplitude (right) for APMA receptor-mediated mepscs amplitude;

FIG. 5: (A) morphological maps of hippocampal neurons observed for nissl staining of different groups of mice; (B) an Iba1 positive microglia in hippocampus expression profile (left) and an Iba1 positive cell quantification profile (right) for different groups of mice;

FIG. 6: (A) expression of IL-6 in sera of mice of different groups; (B) expression of IL-1 β in sera of mice of different groups; (C) expression of TNF-alpha in serum of mice of different groups; (D) expression of hippocampal TREM2mRNA for different groups of mice; (E) expression of IL-6mRNA from hippocampus of mice of different groups; (F) expression of IL-1 beta mRNA from hippocampus of mice of different groups; (G) expression of TNF-alpha mRNA from hippocampal in different groups of mice.

Detailed Description

The present invention will be described in further detail with reference to examples. It will also be understood that the following examples are included merely for purposes of further illustrating the invention and are not to be construed as limiting the scope of the invention, as the invention extends to insubstantial modifications and adaptations of the invention following in the light of the principles set forth herein. The specific process parameters and the like of the following examples are also only one example of suitable ranges, and the skilled person can make a selection within the suitable ranges through the description herein, and are not limited to the specific data of the following examples.

Example 1

A traditional Chinese medicine composition comprises the following traditional Chinese medicines in parts by mass:

16 parts of coptis chinensis, 8 parts of golden cypress, 16 parts of gardenia, 15 parts of salvia miltiorrhiza, 10 parts of turmeric and 10 parts of rhizoma acori graminei.

A preparation method of a decoction containing the traditional Chinese medicine composition comprises the following steps:

1) weighing 7 parts of the traditional Chinese medicine composition, and cutting into pieces by using scissors;

2) adding 10 times volume of double distilled water, soaking for 2h, decocting for 1h in a medicine tank, timing from slight boiling, and filtering the medicine liquid with 8 layers of gauze;

3) adding 8 times volume of single distilled water for the second time, decocting for 1 hr, and filtering with 8 layers of gauze;

4) pouring out the medicinal residue, squeezing with gauze, mixing filtrates, and cleaning medicinal canister and gauze with double distilled water for three times;

5) mixing the filtrate and the washing liquid again, subpackaging into 50mL centrifuge tubes, and centrifuging at 4000rpm for 5 min;

6) collecting the supernatant in a 2L round-bottom flask, concentrating by rotation at 55 deg.C to about 180mL (i.e. 2.92g crude drug/mL);

7) subpackaging into 5mL EP tubes, each tube is 3.5mL, labeling, storing at-80 deg.C, and thawing 1 tube per day.

The above-mentioned decoction is referred to in the present application as: decoction for removing toxic substance and removing blood stasis.

Example 2

15 parts of coptis chinensis, 10 parts of golden cypress, 15 parts of gardenia, 15 parts of salvia miltiorrhiza, 10 parts of turmeric and 10 parts of rhizoma acori graminei.

The preparation method of the decoction is as in the embodiment, and the formula of the traditional Chinese medicine is as in the embodiment 2.

1. Selecting a model:

there are many animal types of AD models including a β oligomer intracerebral injection model, APP/PS1 double transgenic mouse model, etc. 5 × FAD transgenic model mice carrying 5 familial mutations (5 × FAD) were selected for the study, wherein the mutations associated with the gene APP are K670N/M671L (Swedish), I716V (Florida), and V86717 25 (London), the mutations associated with the gene PS1 are M146 56286 and L V, the transgenic promoter is Thy1, and the homologous wild type is C57/B6 jlx. The 5 XFAD mouse is a relatively accepted AD model mouse, and can better simulate various AD pathological phenotypes: intracellular A beta 42 amyloid aggregation can be seen at 1.5 months, amyloid deposition begins to appear at 2 months, memory impairment appears at about 4 months, and various AD pathological characteristics such as synaptic structure, functional impairment, neuroinflammation and pyramidal neuron loss appear, and the pathological phenotype of 5 x FAD mice appears earlier in various AD model mice. In the present application, 4-month-old 5 × FAD mice were selected for the experiments.

2. The administration scheme is as follows:

the equivalent dose of the mouse is calculated by referring to the administration dose of the human body and 75g of crude drug per 70 kg/day, a conversion coefficient 9 is determined by the standard of traditional Chinese medicine pharmacology experimental methodology, and the equivalent dose of the mouse is 9.64g of crude drug per kg/day after the conversion coefficient 9 is multiplied.

The decoction prepared by the traditional Chinese medicine formula in example 1 is selected for testing.

According to the pathological characteristics of 5 × FAD transgenic AD mice, the administration scheme adopted by the application is as follows:

(1) the mice are continuously administered for 1 month and 1 time per day by intragastric administration from 4 months of age, the daily administration dose of the mice is 9.64g/kg (crude drug amount/mouse body weight) according to the conversion ratio of the drug dose of the human to the mouse, the high dose group is detoxication and stasis-removing soup (namely: 19.28g/kg, recorded as JDHYT-H) with 2 times of the adult equivalent dose, the low dose group is detoxication and stasis-removing soup (namely: 4.82g/kg, recorded as JDHYT-L) with 1/2 times of the adult equivalent dose, and the mice are administered for 1 month and 1 time per day by intragastric administration according to the standard that the administration volume is 0.1mL/10 g;

(2) the 5 XFAD transgenic mice of the same age are used as a control group, and the stomach-perfusing solvent double distilled water is perfused by 0.1mL/10g of the body weight of the mice, and the soup for removing the toxin and the blood stasis without the perfusion is recorded as: 5 × FAD;

(3) the age-matched wild type C57BL/6J mouse is used as a normal reference group mouse, and the stomach-perfusing solvent double-distilled water is used for perfusing 0.1mL/10g mouse body weight, and the soup for removing toxin and removing blood stasis without perfusing stomach is recorded as: con;

the experimental study is carried out in multiple batches, the study on the behavior of the mice is firstly carried out after the drug treatment, and then the study on the biochemistry, the molecular biology, the morphology and the electrophysiology is respectively carried out.

3. The experimental method comprises the following steps:

(1) study of behaviours

A: morris water maze experiment: the Morris water maze consists of a large circular water pool, an underwater platform and an image acquisition and processing system. During the experiment, the pool is divided into four quadrants (quadrant E, S, W, N) on average, one fixed point in each quadrant is a water inlet point, the mouse is placed into the pool from the water inlet point, and the camera system records the movement track of the mouse and the time for finding the underwater platform. If the mouse still cannot find the platform within 60s, the experimenter directs it to the platform and stays there for 10 s. Each mouse enters water from four water entry points every day, and how to successfully find the platform is learned and continuously trained for 5 days. Training to 5 days, removing the platform, putting a mouse on the opposite side of the platform facing the pool wall, and recording the movement time and the track of the mouse in each quadrant within 60s by the camera system, wherein the movement time and the track comprise the latency from entering water to reaching the platform, the swimming distance, the swimming speed, the residence time in the platform quadrant, the crossing times and the like.

B: t maze experiment: the T-maze consists of 3 identical arms, 3 arms being T-shaped. The mouse is placed in the center of the T maze, and the camera system records the times of entering each arm and the staying time of the mouse within 5 min. The percent shuttle within 5min was statistically analyzed for each mouse.

C: y maze experiment: the Y maze consists of 3 identical arms, and the included angle of each arm is 120 degrees. The mice were placed in the center of the Y maze and the camera system recorded the number of times the mice entered each arm and the residence time within 5min and counted the shuttle percentage for each mouse.

D: new object identification experiment: the first day of the experiment, the mice were familiar with the surrounding environment in an open field box; on the next day, the mouse is placed in an open field box to freely search and adapt to two same objects, and the camera system records the respective identification time of the two objects within 5 min; on the third day, after the mouse freely explores two identical objects for 1 hour, one of the objects is changed into another new object with different color and shape, and the camera system records the identification time of the animal to the new object and the old object within 5min from the time of placing the animal in the open field box. According to the formula: time to identify new object/(time to identify new object + time to identify old object) the search preference (explicit preference) is calculated and the result is expressed in percentage (%).

(2) Western blot experiment

Extracting protein sample of hippocampal tissue, determining protein concentration by using BCA kit, adding sample buffer solution according to a certain proportion, and carrying out boiling water bath at 100 ℃ for 5 min; transferring the protein to a polyvinylidene fluoride (PVDF) membrane after electrophoresis, rinsing the PVDF membrane for 5min by using a TBST buffer solution at room temperature, blocking the PVDF membrane for 2h by using a 5% BSA blocking solution, and rinsing the PVDF membrane for 3 times and 5min each time; incubating at 4 ℃ for one night, and rinsing the PVDF membrane for 3 times and 5min each time by using TBST buffer solution; the PVDF membrane and the secondary antibody are incubated for 1h at room temperature, and rinsed by TBST buffer solution. And (3) mixing the chemiluminescence reagent A solution and the chemiluminescence reagent B solution in equal amount, incubating the PVDF membrane for 1min, exposing on an exposure instrument, analyzing the intensity of the strip by using Image J Image analysis software, performing statistical analysis processing on data by using SPSS16.0, and determining that the p is less than 0.05 and has statistical difference.

(3) qRT-PCR experiment

Extracting total RNA of hippocampal tissue with Trizol reagent, adding corresponding volume of 5 XgDNA Eraser Buffer, gDNA Eraser, RNase Free dH₂O at 42 ℃ for 2min to remove genomic DNA, 5 XPrimeScript Buffer 2, PrimeScript RT Enzyme Mix 1, RT Primer Mix, RNase Free dH were added to the reaction mixture for removing genomic DNA₂O, finishing reverse transcription reaction at 37 ℃ for 15min and 85 ℃ for 5sec, adding SYBR Premix Ex Taq and upstream and downstream primers into a DNA template to perform fluorescent quantitative PCR, wherein the PCR reaction condition is 95 ℃ and 5 s; 30s at 55 ℃; 72 ℃ for 30 s; 40 cycles. Comparing the target gene with internal reference to obtain relative CT value, performing data statistical analysis by using SPSS16.0, and p<0.05 is statistically different.

(4) Immunohistochemical staining

Perfusion and fixation: after mice were anesthetized by isoflurane inhalation, the hearts were exposed and transfixed via the left ventricle to the ascending aorta using pre-cooled normal saline and 4% paraformaldehyde for perfusion. The animals had convulsions of the muscles of their limbs, and the hardening of their limbs indicated successful perfusion. Fixing mouse brain in 4% paraformaldehyde at 4 deg.C for about 6 hr, sequentially transferring into 20% and 30% sucrose solution, gradient dehydrating in 4 deg.C refrigerator, and embedding tissue with OCT embedding medium;

(II) slicing: continuously coronal slicing by a constant-temperature freezing slicer, sticking on an anti-drop sheet, baking for 3.5h in a constant-temperature oven at 65 ℃, and storing in a refrigerator at-80 ℃ for later use;

(III) antigen retrieval: placing the slices in a 0.01M citrate buffer solution staining jar, carrying out water bath at 98 ℃ for 10-15 min, naturally cooling to room temperature, washing with PBS for 3 times, and washing for 5min each time;

(IV) membrane rupture: breaking membrane with 0.3% Triton for 30min, washing with PBS for 3 times, 5min each time;

(V) sealing: blocking with 5% BSA for 10 min;

(VI) incubating the primary antibody: dripping primary antibody, standing overnight in a wet box at 4 deg.C, taking out from refrigerator, rewarming at room temperature for 45min, and washing with PBS for 5min for 3 times;

(VII) incubation of secondary antibody: incubating at 37 deg.C for 60min, washing with PBS for 3 times, each time for 5 min;

(VIII) color development: dripping the mixture into slices, detecting the reaction time under a mirror at room temperature, and flushing the slices with running water for 10 min;

(IX) counterstaining: counterstaining with hematoxylin for 2min, soaking in tap water, washing for 10min, separating with hydrochloric acid and ethanol for 1 time, washing with tap water, and washing for 10 min;

(X) gradient alcohol dehydration: 70% ethanol for 3min, 80% ethanol for 3min, 90% ethanol for 3min, 95% ethanol I for 3min, 95% ethanol II for 3min, 100% ethanol I for 3min, 100% ethanol II for 5min, xylene I for 5min, and xylene II for 5 min;

(XI) Gum seal piece, microscopic examination.

(5) Enzyme-linked immunosorbent assay (ELISA)

Taking serum or preparing hippocampal tissue homogenate, adding a sample to be detected into a coated pore plate, then incubating the sample with a biotin-labeled antibody for 30 min-1 h, washing, adding avidin-labeled horse radish peroxidase, washing again, removing unbound enzyme conjugate, finally adding a substrate, reacting with the enzyme conjugate to generate color, wherein the shade of the color is in a proportional relation with the concentration in the sample, and detecting the absorbance value by using an enzyme-labeling instrument.

(6) Electrophysiological patch clamp experiment

After anesthesia, the mouse is cut off and the brain is taken out quickly, the brain tissue is adhered to the objective table of the microtome quickly, and the artificial cerebrospinal fluid (95% O) filled with ice water at the temperature of 0-4 ℃ is placed in the brain tissue₂/5％CO₂Saturated) was cut out into 400 μm thick hippocampal brain slices using a vibrating microtome;

transferring brain slice to 32 deg.C incubation tank for incubation for 30min, standing at room temperature for further incubation, wherein the incubation tank contains 95% O₂/5％CO₂Saturated artificial cerebrospinal fluid;

observing action potential emitting frequency and threshold of hippocampal neurons of different groups of mice by adopting a whole-cell current clamp technology so as to know excitability change conditions of the hippocampal neurons of the mice;

recording excitatory and inhibitory synaptic transmission conditions of hippocampus of different groups of mice in a whole-cell voltage clamp mode, wherein the excitatory and inhibitory synaptic transmission conditions of hippocampus neurons are recorded by AMPA micro excitatory postsynaptic currents (mEPSC), and the presynaptic and postsynaptic excitatory synaptic transmission conditions are known; the field-excitation post-synthetic potential (fEPSP) and input-output (input-output) curves of the hippocampal tissue section are recorded outside the cells respectively, so that the synaptic plasticity conditions of LTP and the like of Schafer fiber-CA 1 pyramidal cell layer channels in a hippocampal CA3 area are known, and the neuronal excitability and the synaptic transmission function of different groups of mice are comprehensively analyzed.

(7) Statistical analysis

Experimental data were subjected to t-test or One-Way analysis of variance (One Way ANOVA) using SPSS16.0 statistical software, and One Way ANOVA was subjected to analysis of group-to-group variation using post-hoc Dunnett's. All measurements are expressed as mean. + -. standard error (x. + -. S.E.), and p<0.05 is a significant difference in the number of molecules,^*p<0.05，^**p<0.01，^*indicating the difference between each group and the normal reference group;^#p<0.05，^##p<0.01，^#shows the difference between the detoxification stasis-removing decoction treatment group and the 5 XFAD transgenic mouse group.

4. And (4) analyzing results:

(1) improving learning and memory dysfunction of 5 XFAD mice

As can be seen from fig. 1(a) and 1 (B): the percent shuttle between arms was significantly higher in both JDHYT-H and JDHYT-L groups than in the 5 XFAD group in the T and Y maze;

as is clear from FIGS. 1(C) to (F): in the Morris water maze, the time to reach the plateau was gradually decreased for each group of mice as the number of training days increased, but the JDHYT-H and JDHYT-L groups reached the plateau significantly shorter than the 5 x FAD mice during the 5-day training period, and the JDHYT-H group was comparable to the normal reference group (fig. 1 (C)); on day 6, the platform was removed, the time to reach the platform was recorded for different groups of mice, and the results showed: the JDHYT-H and JDHYT-L groups reduced the time for 5 × FAD mice to reach the plateau, and the time for the JDHYT-H group was also shorter than the normal reference group (fig. 1(D)), while increasing the dwell time of the 5 × FAD mice in the target quadrant and the number of passes through the target quadrant (fig. 1(E) - (F));

from FIGS. 1(G) to (J): the JDHYT-H group and the JDHYT-L group can obviously improve the recognition function of the 5 XFAD mice on new objects;

the above shows that the detoxifying and stasis removing decoction with low and high dose can obviously improve the learning and memory functions of the 5 XFAD mice.

From FIGS. 2(A) to (C), it can be seen that: the long-term potentiation (LTP) of Schafer fiber-CA 1 pyramidal cell layer pathway in CA3 region of hippocampus of 5 XFAD mice is improved by low and high doses of the detoxification stasis-removing decoction, the LTP is a persistent potentiation phenomenon occurring in signal transmission of two neurons, and can stimulate the two neurons synchronously, because memory is considered to be encoded by change of synaptic strength, the LTP is generally regarded as one of main molecular mechanisms forming the basis of learning and memory, and thus, the low and high doses of the detoxification stasis-removing decoction are further proved to obviously improve learning and memory dysfunction of the 5 XFAD mice from electrophysiological results.

(2) Improvement of 5 XFAD mouse hippocampal synaptic dysfunction

Through Western blot and electrophysiological experiment, the expressions of low and high dose detoxication and stasis-removing decoction on postsynaptic proteins, glutamate receptors and Abeta 40/42 of hippocampal of 5 XFAD mice and the influence of the postsynaptic proteins on excitatory synaptic transmission are analyzed.

After 5 XFAD mice were given different doses of detoxification stasis-removing soup (i.e., JDHYT-H group and JDHYT-L group) for 1 month, hippocampal tissues were harvested, total protein was extracted, and Western blot was used to detect the expression levels of glutamate receptors, Abeta 40/42, proteins before and after synapses, etc., and the results are shown in FIG. 3:

as is clear from fig. 3(a) to (C): compared with the 5 XFAD group, the JDHYT-H group and the JDHYT-L group significantly increased the expression of the glutamate receptor NR2A, which is much higher than that of the normal reference group, and thus the detoxification and stasis-removing decoction can up-regulate the expression of the NMDA receptor and improve excitatory synaptic transmission; meanwhile, the detoxifying and stasis-removing decoction also obviously reduces the expression of Abeta 40/42 of the hippocampus, and the detoxifying and stasis-removing decoction is prompted to obviously reduce the deposition of Abeta 40/42, a pathological marker of AD, in the hippocampus, and improve the expression levels of presynaptic and postsynaptic proteins and the expression levels of presynaptic Synapsin and postsynaptic PSD-95 of a 5 XFAD mouse.

The microphthalmic postsynaptic current (mEPSC) mediated by AMPA receptor of pyramidal neurons in CA1 region of hippocampus was recorded in gap-free mode at-65 mV potential clamped by whole cell patch clamp, and 20 μ M Bicuculline (GABA receptor blocker) and 1 μ M TTX (tetrodotoxin, sodium channel blocker) were added to the perfusate during recording, as shown in fig. 4:

as can be seen from fig. 4 (a): the JDHYT-H group and the JDHYT-L group can obviously improve the frequency and amplitude of mEPSC mediated by AMPA receptors;

as can be seen from fig. 4(B) and 4 (C): by quantifying the graph in fig. 4(a), it can be seen that the frequency and amplitude of AMPA receptor-mediated mepscs in the 5 × FAD mice are both significantly reduced compared to the normal reference group, while the JDHYT-H group and the JDHYT-L group are both significantly increased compared to the 5 × FAD mice, and after the treatment of the detoxification stasis-removing decoction, the frequency and amplitude of AMPA receptor-mediated mepscs are both significantly increased;

excitatory postsynaptic potential (EPSC) is the change in membrane potential of depolarizing nature produced in postsynaptic neurons by the action of excitatory synapses, which may reflect the release of presynaptic transmitters and their effect on postsynaptic receptors, and thus the work of excitatory synaptic transmission, as demonstrated by the electrophysiological results described above: the hippocampus of 5 × FAD mice is accompanied by the down-regulation of excitatory synaptic transmission function mediated by glutamate receptor APMA receptor, while the detoxification stasis-removing decoction improves the excitatory synaptic transmission disorder mediated by 5 × FAD mouse APMA receptor, consistent with the above-mentioned results of molecular biological experiments, and thus, it is known from the consistent results of molecular biological experiments and electrophysiological experiments that: the detoxification and stasis-removing decoction can relieve the synaptic dysfunction of hippocampus of 5 XFAD mice.

(3) Improvement of neuroinflammatory response in 5 xFAD mouse brain

5 XFAD mice were treated with detoxifying and stasis removing decoction (JDHYT-H and JDHYT-L) at different doses for 1 month, and then the material was obtained. Nissl staining was used to detect the morphology of hippocampal neurons, and pictures were taken under a microscope, with the results shown in FIG. 5 (A);

5 XFAD mice were given different doses of detoxification stasis-resolving decoction (i.e., JDHYT-H group and JDHYT-L group) for 1 month, and were drawn, Iba1 labeled microglia, and immunohistochemical assay was performed to detect hippocampal microglia expression, the results are shown in FIG. 5 (B);

5 XFAD mice are given different doses of detoxification stasis-removing soup (namely JDHYT-H group and JDHYT-L group) for 1 month, blood is taken, ELISA experiment is carried out to detect the expression of IL-6, IL-1 beta and TNF-alpha in the blood serum of the mice of different groups, and the results are shown in FIGS. 6(A) - (C);

after 5 XFAD mice are given different doses of detoxification stasis-removing soup (namely, JDHYT-H group and JDHYT-L group) for 1 month, hippocampal tissues are taken, total RNA is extracted, qRT-PCR experiment is carried out to detect the expression of hippocampal TREM2, IL-6, IL-1 beta and TNF-alpha of different groups of mice, and the result is shown in figures 6(D) to (G):

as can be seen from fig. 5 (a): vacuolar degeneration, apoptosis and deletion of neurons are accompanied in the hippocampal tissues of 5 xFAD mice, and the detoxification stasis-removing decoction improves the state and activity of the neurons of the hippocampus of the 5 xFAD mice, which shows that the detoxification stasis-removing decoction has a protective effect on the neurons of the hippocampus of the 5 xFAD mice;

as can be seen from fig. 5 (B): the detoxification and stasis-removing decoction inhibits the proliferation of 5 XFAD mouse hippocampal microglia, which shows that the detoxification and stasis-removing decoction can inhibit microglial cell-mediated neuroinflammation reaction to a certain extent;

as is clear from fig. 6(a) to (G): in the serum and hippocampal tissues of 5 × FAD mice, there was concomitant upregulation of inflammatory factor (IL-6, IL-1 β and TNF- α) expression, while the detoxification stasis-resolving decoction decreased the expression of IL-6, IL-1 β and TNF- α at protein and mRNA levels in the serum and hippocampal tissues of 5 × FAD mice, particularly with high dose effect, while the low and high dose detoxification stasis-resolving decoction was also found to upregulate the expression of 5 × FAD mouse hippocampal TREM2 at mRNA levels (see FIG. 6(D)), in combination with the previous Western blot results, presumably: the detoxifying and stasis removing decoction can play a role in resisting AD by regulating the immunoregulation.

Claims

1. A traditional Chinese medicine composition for treating or assisting in treating Alzheimer disease is characterized in that: the composition consists of coptis chinensis, phellodendron, gardenia, salvia miltiorrhiza, turmeric and rhizoma acori graminei, and the composition comprises the following substances in parts by mass: 12-18 parts of coptis chinensis, 6-10 parts of phellodendron, 12-18 parts of gardenia, 13-17 parts of salvia miltiorrhiza, 8-12 parts of turmeric and 8-12 parts of rhizoma acori graminei.

2. The composition of claim 1, wherein: the mass part ratio of the coptis chinensis to the golden cypress to the gardenia is 2: 1: 2.

3. a traditional Chinese medicine preparation is characterized in that: the preparation is any one dosage form of powder, tablets, injections, capsules, decoctions, granules, pills, paste and targeted preparations prepared from the composition of any one of claims 1-2.

4. A preparation method of a decoction is characterized in that: the composition of any one of claims 1 to 2, wherein the composition comprises Coptidis rhizoma, Phellodendri cortex, Gardeniae fructus, Saviae Miltiorrhizae radix, Curcuma rhizome and rhizoma Acori Graminei, and is prepared by decocting with water to obtain decoction.

5. The method of claim 4, wherein: 1mL of decoction is obtained per 2-4 g of the composition.

6. The method of claim 4, wherein: the composition is decocted for two times, wherein 5-20 times of water is added for the first time for decoction, and 3-15 times of water is added for decoction for the second time.

7. Use of a composition according to any one of claims 1 to 2 in the manufacture of a medicament for the treatment or co-treatment of alzheimer's disease.

8. Use according to claim 7, characterized in that: the Alzheimer disease is Alzheimer disease of turbid virus blood stasis type.