CN112022941A

CN112022941A - Chinese medicinal composition freeze-dried powder and preparation method and application thereof

Info

Publication number: CN112022941A
Application number: CN202011103439.9A
Authority: CN
Inventors: 王凯; 张伯礼; 张玉莲; 崔远武; 马妍; 林翠茹; 张琳琳; 刘爽; 李强; 韩文文; 孙伟明; 徐家淳; 周震; 倪道艳; 李霖; 于臻; 宋宛珊
Original assignee: SECOND AFFILIATED HOSPITAL OF TIANJIN UNIVERSITY OF TCM
Current assignee: SECOND AFFILIATED HOSPITAL OF TIANJIN UNIVERSITY OF TCM
Priority date: 2020-10-15
Filing date: 2020-10-15
Publication date: 2020-12-04

Abstract

The invention provides a traditional Chinese medicine composition freeze-dried powder as well as a preparation method and application thereof, wherein the traditional Chinese medicine composition freeze-dried powder comprises the following raw materials: herba Epimedii, fructus Ligustri Lucidi, radix Polygoni Multiflori Preparata, Cistanchis herba, rhizoma Acori Graminei and rhizoma Ligustici Chuanxiong; the epimedium, the glossy privet fruit, the prepared fleece-flower root and the cistanche in the Chinese medicinal composition freeze-dried powder are designed aiming at the pathogenesis of AD kidney deficiency, the glossy privet fruit and the prepared fleece-flower root are used for enhancing the function of nourishing kidney yin, the epimedium and the cistanche are used for strengthening the force of warming kidney yang, and the four medicaments are used for seeking yang in yin and seeking yin in yang; the grassleaf sweelflag rhizome and the Szechuan lovage rhizome are respectively designed for the pathogenesis of AD phlegm and stasis and turbid toxin, and are used for eliminating phlegm, inducing resuscitation, promoting blood circulation and promoting qi circulation so that body fluid can be distributed and qi and blood can be passed; the six medicines are precise and appropriate in compatibility, and the medicines complement each other, so that the traditional Chinese medicine composition has the effects of nourishing the origin, promoting the brain, inducing resuscitation, tonifying the kidney, resolving turbidity, and treating both principal and secondary aspects of diseases, can effectively treat and even delay the progress of AD diseases, and plays the roles of improving the learning and memory capacity, relieving the neuron injury and reducing the generation of AD pathological products.

Description

Chinese medicinal composition freeze-dried powder and preparation method and application thereof

Technical Field

The invention belongs to the field of materials, and relates to a traditional Chinese medicine composition freeze-dried powder, and a preparation method and application thereof.

Background

At present, the prevalence rate of senile dementia in people over 65 years old is 10%, and 20% in people over 80 years old. This disease has been identified as an important topic in geriatric medicine and geriatric psychiatry. The pathological changes in senile dementia are atrophy of cerebral cortex, decrease of cortical nerves, large amount of neurofibrillary tangles and senile plaques in cerebral cortex and hippocampus. Clinically, the disease is characterized by the overall decline of high-level nerve functions such as cognition, thinking, emotion, personality, behavior and language in a clear state of consciousness, and typical symptoms are decline of learning and memory ability, loss of speech, language disorder and the like. Due to the characteristics of complex etiology and heterogeneous etiology of senile dementia, no effective treatment method is available in western medicine. Alzheimer's Disease (AD), also called senile dementia, is a degenerative disease of the central nervous system, with an insidious onset and a chronic progressive course, and is one of the most common types of senile dementia. AD is manifested as neuropsychiatric symptoms such as progressive cognitive dysfunction, personality changes and behavioral disturbances, which severely affect social, occupational and life functions. The etiology and pathogenesis of AD are not clarified, and the characteristic pathogenesis is changed into extracellular senile plaques formed by deposition of beta amyloid protein, nerve intracellular neurofibrillary tangles formed by hyperphosphorylation of tau protein, neuron loss with glia cell proliferation and the like. The current therapeutic drugs in the market mainly comprise cholinesterase inhibitors (ChEIs), glutamate receptor antagonists (NMDAR antagonists), drugs for inhibiting the formation and deposition of Abeta, calcium ion antagonists, free radical scavenging agents, anti-oxidation drugs, estrogen and the like, and the drugs mainly comprise chemical drugs, but the chemical drugs have the drug effect limitation and certain toxic and side effects, so that the clinical application of the drugs is limited to a certain extent. Therefore, scholars at home and abroad turn the attention to Chinese herbal medicines.

At present, a lot of medicines are used for treating senile dementia, for example, CN101129608A discloses a capsule for treating Alzheimer disease, which contains ten traditional Chinese medicines such as ginseng, angelica, fleece-flower root, pseudo-ginseng, medlar and the like, and the dosage form is capsule; CN102028910B discloses a Chinese medicinal preparation for treating senile dementia and a preparation method thereof, wherein the Chinese medicinal preparation comprises eleven Chinese medicaments such as liquorice, stiff silkworm, rhubarb, safflower, mint, cicada slough, turmeric, coptis root and the like, and is viscous liquid obtained by concentrating medicinal juice; CN101637590B discloses a traditional Chinese medicine for treating senile dementia, which comprises fifteen traditional Chinese medicines such as poria with hostwood, rhizoma acori graminei, polygala tenuifolia, centipede, fructus alpiniae oxyphyllae, peach kernel, rheum officinale, natrii sulfas exsiccatus, cassia twig, dragon bone, dogwood and the like, and the main dosage form of the traditional Chinese medicine is honeyed pills. However, the therapeutic effect of the above-mentioned drugs is yet to be further improved.

Therefore, it is necessary to provide a Chinese medicinal composition lyophilized powder with good therapeutic effect, little side effect, good stability and convenient administration.

Disclosure of Invention

The invention aims to provide a traditional Chinese medicine composition freeze-dried powder and a preparation method and application thereof, the raw materials of the traditional Chinese medicine composition freeze-dried powder comprise epimedium, glossy privet fruit, prepared fleece-flower root, desertliving cistanche, grassleaf sweelflag rhizome and Szechuan lovage rhizome, wherein the epimedium, the glossy privet fruit, the prepared fleece-flower root and the desertliving cistanche are designed aiming at the pathogenesis of AD kidney deficiency, the glossy privet fruit and the prepared fleece-flower root are used for enhancing the function of nourishing kidney yin, the epimedium and the desertliving cistanche are used for strengthening the power of warming kidney yang, and the four medicines are used for; the grassleaf sweelflag rhizome and the Szechuan lovage rhizome are respectively designed for the pathogenesis of AD phlegm and stasis and turbid toxin, and are used for eliminating phlegm, inducing resuscitation, promoting blood circulation and promoting qi circulation so that body fluid can be distributed and qi and blood can be passed; the six medicines are precise and appropriate in compatibility, and the medicines complement each other, so that the traditional Chinese medicine composition has the effects of nourishing the origin, promoting the brain, inducing resuscitation, tonifying the kidney, resolving turbidity, and treating both principal and secondary aspects of diseases, can effectively treat and even delay the progress of AD diseases, and plays the roles of improving the learning and memory capacity, relieving the neuron injury and reducing the generation of AD pathological products.

In order to achieve the purpose, the invention adopts the following technical scheme:

one of the purposes of the invention is to provide a traditional Chinese medicine composition freeze-dried powder, which comprises the following raw materials:

herba Epimedii, fructus Ligustri Lucidi, radix Polygoni Multiflori Preparata, Cistanchis herba, rhizoma Acori Graminei and rhizoma Ligustici Chuanxiong.

The first four kidney-tonifying traditional Chinese medicines in the traditional Chinese medicine composition freeze-dried powder are designed aiming at AD kidney deficiency, and reflect a treatment method of strengthening body resistance and tonifying kidney to balance yin and yang, replenish essence and benefit marrow. Wherein the glossy privet fruit is sweet and bitter, has cool nature, enters liver and kidney meridians at the same time, can nourish yin of liver and kidney, has the effects of blackening beard and improving eyesight, is a product for refreshing and tonifying although tonifying but not greasy, and is recorded in Shen nong Ben Cao Jing to' calm five internal organs, nourish spirit and eliminate diseases for a hundred days. It is good for health after long-term administration. Herba Epimedii is warm in nature, pungent and sweet in flavor, enters liver and kidney meridians, and has effects of warming kidney, tonifying yang, strengthening tendons and bones, and dispelling pathogenic wind and dampness; the book Ben Cao Bei Yao (materia Medica) carries the functions of nourishing life gate and benefiting essence and qi, Li Shi Zhen is clear and suitable for people with deficiency of true yang, and the book Ri Hua Zi Ben Cao (Rihua Zi materia) directly says that it can be used for treating senile dizziness and middle-aged amnesia. Cistanchis herba is warm in nature, sweet and salty in taste, enters kidney and large intestine meridians, and has effects of invigorating kidney yang, replenishing essence and blood, and moistening intestine dryness. The said herbal medicine is that "Bu Yin, Yue Yin, Yan Nian, Bu Zhu Yang". The Yun Rong Shen from the said herbal medicine is that "Yan Mingmen, Zi Shen Qi, and Bu Jing Xue … … are the mild tonics", because it is warm but not dry, tonics are not severe, and the effect of the medicine is mild. Radix Polygoni Multiflori Preparata is sweet and astringent, has slightly warm nature, enters liver and kidney channels, and has effects of invigorating essence and blood, reinforcing kidney and blackening beard. "Kaibao Ben Cao" (a compendium of materia Medica) says that it can tonify blood and qi, soothe moustache, … … take long-time bones and muscles, benefit essence and marrow, prolong life, tonify liver and kidney, astringe essence and qi, and is a "tonification good medicine" and "Gong is above rehmannia root and cochinchnese asparagus root" in Ben Cao gang mu (compendium of materia Medica). In the four medicines, the glossy privet fruit is matched with the radix polygoni multiflori preparata to nourish kidney yin, seek yang in yin, the epimedium and the cistanche are combined to warm the kidney and assist yang, seek yin in yang, and the four medicines achieve the effect of regulating yin and yang and mutual use.

The last two traditional Chinese medicines in the traditional Chinese medicine composition freeze-dried powder are designed aiming at AD phlegm stasis and turbidity as a standard, show the functions of eliminating pathogenic factors and dissolving turbidity, and are used for reducing phlegm, inducing resuscitation, promoting blood circulation and promoting qi circulation. Rhizoma acori graminei is pungent and bitter in taste and warm in nature, enters heart and stomach meridians, can induce resuscitation and refresh mind, calm mind and benefit intelligence, and can treat amnesia (newly compiled materia Medica). The book Jing also records that the people can take … … for a long time without forgetting and puzzling and can prolong life. Chuan Xiong is pungent in flavor and warm in nature, enters liver, gallbladder and pericardium channels, and has the actions of activating blood and moving qi, so it is called qi-flowing medicine in blood in Ben Cao gang mu, Zhang Yuan Lun, which is good at ascending head and eyes, so it is specially indicated for diseases of head and brain. The two medicines are matched to mutually strengthen the power of mutually reducing phlegm and removing blood stasis, so that phlegm and dampness are transformed, blood stasis is dispersed, turbid pathogenic factors are eliminated, qi and blood circulation is recovered, the two medicines are used together with the first four traditional Chinese medicines for tonifying the kidney, all the medicines are carried to directly reach the disease site, and simultaneously, the dynamic and static combination enables the tonifying to be not stagnant and the nourishing to be not greasy. The whole formula is comprehensively viewed, the traditional Chinese medicine can treat both symptoms and root causes, tonify yin and yang, tonify middle-jiao and purge, mainly tonify kidney, dissolve turbidity to assist tonifying, give consideration to both phases, and achieve the effect of tonifying kidney and dissolving turbidity together by slowing down the picture, and aims to focus on the pathogenesis of AD deficiency and marked excess.

The epimedium, the glossy privet fruit, the prepared fleece-flower root and the cistanche in the Chinese medicinal composition freeze-dried powder are designed aiming at the pathogenesis of AD kidney deficiency, the glossy privet fruit and the prepared fleece-flower root have the function of nourishing kidney yin, the epimedium and the cistanche have the function of warming kidney yang, and the four medicaments are combined to solve the problem of solving yang in yin and solving yin in yang; the grassleaf sweelflag rhizome and the Szechuan lovage rhizome are respectively designed for the pathogenesis of AD phlegm and stasis and turbid toxin, and are used for eliminating phlegm, inducing resuscitation, promoting blood circulation and promoting qi circulation so that body fluid can be distributed and qi and blood can be passed; the six medicines are precise and appropriate in compatibility, and the medicines complement each other, so that the traditional Chinese medicine composition has the effects of nourishing the origin, promoting the brain, inducing resuscitation, tonifying the kidney, resolving turbidity, and treating both principal and secondary aspects of diseases, can effectively treat and even delay the progress of AD diseases, and plays the roles of improving the learning and memory capacity, relieving the neuron injury and reducing the generation of AD pathological products.

Compared with traditional Chinese medicine composition freeze-dried powder and traditional Chinese medicine composition decoction, the invention discovers that the freeze-dried powder has better action trend in reducing the escape latency time of 7-month-old SAMP8 mice in the experiment of improving the learning and memory abilities of AD model mice; based on the current situation that AD can not be cured, under the guidance of the idea of 'preventive treatment of disease' in traditional Chinese medicine, SAMP8 mice and primary neurons of SD fetal mice of 3 months old are selected as research objects aiming at the early stage of AD, and the effect mechanism of the traditional Chinese medicine composition freeze-dried powder is found to be the reduction of A beta expression, the reduction of neuron loss and synaptic function reduction caused by cytotoxicity, the further protection of neurons and the improvement of learning and memory capacity decline, so that a part of experimental basis is provided for the expansion of the effect range of the traditional Chinese medicine composition in the prevention and treatment of AD and the clinical popularization, and the traditional Chinese medicine composition freeze-dried powder has high social application value.

The invention adopts modern biological technology, combines the similarity of pathogenesis evolution process of dementia kidney deficiency phlegm-generating stasis accumulation into turbid toxin and AD early pathogenesis, and finds that the Chinese medicinal composition freeze-dried powder has the effects of reducing APP in neurons from being hydrolyzed into A beta monomers and inhibiting A beta aggregation from generating neurotoxicity in the process of regulating A beta self-aggregation toxicity effect.

Preferably, the adding amount of the epimedium is 5-15 g, the adding amount of the glossy privet fruit is 5-15 g, the adding amount of the prepared fleece-flower root is 4-8 g, the adding amount of the cistanche is 5-15 g, the adding amount of the grassleaf sweelflag rhizome is 4-8 g, and the adding amount of the szechuan lovage rhizome is 4-8 g.

More preferably, the adding amount of the glossy privet fruit is 10 g, the adding amount of the prepared fleece-flower root is 6 g, the adding amount of the cistanche is 10 g, the adding amount of the grassleaf sweelflag rhizome is 6 g and the adding amount of the szechuan lovage rhizome is 6 g based on the adding amount of the epimedium herb being 10 g.

The second purpose of the invention is to provide a preparation method of the traditional Chinese medicine composition freeze-dried powder, which comprises the following steps:

extracting the mixture of herba Epimedii, fructus Ligustri Lucidi, radix Polygoni Multiflori Preparata, Cistanchis herba, rhizoma Acori Graminei and rhizoma Ligustici Chuanxiong with water, collecting extractive solution, concentrating to obtain concentrated solution, and freeze drying to obtain Chinese medicinal composition lyophilized powder.

In the invention, the preparation method of the Chinese medicinal composition freeze-dried powder is simple, and the raw materials are easily obtained and easy to realize.

In the present invention, the amount of water added is 8 to 15 g based on 1 g of the mixture added.

In the invention, the extraction time is 2-3 h, and the extraction mode is reflux extraction.

In the invention, the extracting solution collecting process comprises the following steps: separating the extracted primary filtrate and the filter residue, extracting the filter residue with water under reflux for 1-2 h, filtering to obtain secondary filtrate, and mixing the primary filtrate and the secondary filtrate to obtain the extract.

In the invention, the concentration mode is decompression concentration at 50-70 ℃.

In the invention, the freeze drying comprises eight steps: firstly, drying for 2.5 h at the temperature of minus 10 ℃; secondly, drying for 2.5 hours at the temperature of minus 5 ℃; thirdly, drying for 3 hours at the temperature of 0 ℃; fourthly, drying for 5 hours at the temperature of 5 ℃; fifthly, drying for 5 hours at 10 ℃; sixthly, drying for 8 hours at 15 ℃; seventhly, drying for 5 hours at 20 ℃; eighth step, drying at 25 ℃ for 41 h.

The preparation method also comprises the step of crushing the dried substance obtained after freeze drying.

The invention also aims to provide application of the traditional Chinese medicine composition freeze-dried powder in preparation of the Alzheimer disease medicine.

Compared with the prior art, the invention has the following beneficial effects:

(1) the raw materials of the traditional Chinese medicine composition freeze-dried powder comprise herba epimedii, glossy privet fruit, prepared polygonum multiflorum, cistanche, rhizoma acori graminei and ligusticum wallichii, the six medicines are compatible and are exquisite, the medicines complement each other, the effects of nourishing essence, promoting the brain resuscitation, tonifying the kidney, dissolving turbidity and treating both principal and secondary aspects of diseases are achieved, the AD disease process can be effectively treated and even delayed, the effects of improving the learning and memory capacity, relieving the neuron injury and reducing the generation of AD pathological products are achieved.

(2) Animal experiments show that the Chinese medicinal composition freeze-dried powder has a better action trend in reducing the escape latency time of an AD model mouse; based on the current situation that AD can not be cured, the action mechanism of the traditional Chinese medicine composition freeze-dried powder is to reduce A beta expression, reduce neuron deficiency and synapse function reduction caused by cytotoxicity, further protect neurons, improve learning and memory capacity reduction, provide partial experimental basis for expanding the action range of the traditional Chinese medicine composition freeze-dried powder in preventing and treating AD and clinical popularization, and have higher social application value.

(3) The traditional Chinese medicine composition freeze-dried powder has the effects of reducing the hydrolysis of APP in neurons into A beta monomers and inhibiting A beta aggregation to generate neurotoxicity in the regulation of A beta self-aggregation toxicity effect.

Detailed Description

The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

Example 1

The embodiment provides a Chinese medicinal composition freeze-dried powder, which comprises 10 g of epimedium, 10 g of glossy privet fruit, 6 g of prepared fleece-flower root, 10 g of desertliving cistanche, 6 g of grassleaf sweelflag rhizome and 6 g of Szechuan lovage rhizome.

The preparation method of the Chinese medicinal composition freeze-dried powder in the embodiment comprises the following steps:

extracting the mixture of herba Epimedii, fructus Ligustri Lucidi, radix Polygoni Multiflori Preparata, Cistanchis herba, rhizoma Acori Graminei and rhizoma Ligustici Chuanxiong with 8 times of water for 2 h, separating filtrate and residue, extracting the residue with 6 times of water under reflux for 1h, filtering, mixing the two filtrates to obtain extractive solution, concentrating the extractive solution at 60 deg.C, freeze drying (see Table 1), and pulverizing to obtain Chinese medicinal composition lyophilized powder. Sealing and storing in a shady and dry place.

TABLE 1

Examples	Temperature/. degree.C	Time per hour
			Example 1	-10	2.5
Example 2	-5	2.5
			Example 3	0	3
Example 4	5	5
			Example 5	10	5
Example 6	15	8
			Example 7	20	5
Example 8	25	41

The Chinese medicinal composition freeze-dried powder is tested as follows:

I. in vivo experiments:

30 male 7-month-old SAMP8 mice were randomly divided into 3 groups, a model (SAMP 8) group, a Chinese medicinal composition decoction group, and a Chinese medicinal composition lyophilized powder group, each group containing 10 mice. As a control group (SAMR 1), 10 male 7-month-old SAMR1 mice were selected. Each group of mice was intervened by gavage 1 time a day for 4 weeks. The Chinese medicinal composition decoction and Chinese medicinal composition lyophilized powder are administered respectively, and the dosage is calculated by referring to pharmacological experiment methodology, and is 6.24 g.kg^-1·d^-1(ii) a The SAMP8 and SAMR1 groups were dosed with equal amounts of distilled water. The Morris water maze is adopted to detect the change of escape latency time and the frequency of passing through a platform of each group of mice, and the influence of the Chinese medicinal composition decoction and the Chinese medicinal composition freeze-dried powder on the learning and memory ability of the AD model mice is evaluated.

II. In vivo experiments:

40 male 3-month-old mice, SAMP8, were randomly divided into 2 groups, a model (SAMP 8) group, and a Chinese medicinal composition lyophilized powder group, each of which was 20 mice. As a control group (SAMR 1), 20 male 3-month-old SAMR1 mice were selected. Each group of mice was intervened by gavage 1 time a day for 12 weeks. The Chinese medicinal composition lyophilized powder is administered to the Chinese medicinal composition lyophilized powder group, and the dosage is calculated by referring to pharmacological experiment methodology, and is 6.24 g.kg^-1·d^-1(ii) a SAMP8 and SAMR1Groups were given equal amounts of distilled water.

(1) The Morris water maze is adopted to detect the changes of search modes, escape latency time, platform crossing times, initial angles and original platform quadrant path ratios of each group of mice, and the influence of the freeze-dried powder of the traditional Chinese medicine composition on the early learning and memory capacity of the SAMP8 mice is evaluated.

(2) The arrangement state, the number and the microstructure of the neurons in the CA1 region of the hippocampus of each group of mice and the change of the synaptic transmission function of the hippocampus are observed by adopting Nie's staining and a transmission electron microscope and in vivo neuro-electrophysiological detection.

(3) And observing the expression level change of different forms of Abeta in the hippocampal region of each group of mice by adopting glycine silver staining, Western blot and ELISA detection.

(4) By using Western blot and RT-PCR detection, the change of relative expression amounts of key proteins (BACE 1, SOLL 1, VPS35, QPCT and PKA) and genes (BACE 1 and SOLL 1) in the self-aggregation toxicity effect of A beta in the hippocampal region of each group of mice is observed.

III, in vitro experiment:

separating and culturing primary neurons of SD fetal rat, performing purity identification by staining MAP2 with immunofluorescence method, randomly dividing 60 healthy male SD rat into Chinese medicinal composition lyophilized powder group and control group, calculating dosage with reference to pharmacological experiment methodology, and respectively adding Chinese medicinal composition lyophilized powder with crude drug amount of 4.32 g.kg^-1·d^-1And adding equal amount of distilled water, continuously performing intragastric administration for 3 days, 1 time per day, and collecting two groups of sera 1h after the last intragastric administration.

(1) Through CCK-8 and CCK-8, LDH and Abeta 40 ELISA detection kits, the influence of different concentrations of the traditional Chinese medicine composition freeze-dried powder containing drug serum intervention on primary neurons at different times and different concentrations of BACE1 inhibitor (AZD 3839) is observed, and the optimal administration dose and action time of subsequent experiments are screened.

(2) Randomly dividing the inoculated primary neurons into 5 groups including a blank control group, a serum control group, a traditional Chinese medicine composition freeze-dried powder drug-containing serum group, a BACE1 inhibitor group and a traditional Chinese medicine composition freeze-dried powder drug-containing serum + BACE1 inhibitor group, observing and comparing the activity of the neuron cells, the fluorescence intensity of Abeta and the protein expression condition by adopting CCK-8 and Abeta 40 immunofluorescence staining and Western blot detection, and preliminarily determining the generation effect of the traditional Chinese medicine composition freeze-dried powder on Abeta of the neurons in the self-aggregation toxic effect.

And (3) testing results:

in vivo experiments:

(1) experimental results for 7-month-old mice:

morris water maze: compared with the SAMR1 group, the SAMP8 group mice escaped the latency time and crossed the platform significantly less (P < 0.05); compared with SAMP8 mice, the Chinese medicinal composition decoction can remarkably reduce escape latency time and remarkably increase the number of times of crossing a platform (P < 0.05), wherein the Chinese medicinal composition lyophilized powder has better action trend in reducing the escape latency time of the mice, and the fact that the Chinese medicinal composition lyophilized powder can effectively improve AD learning memory capacity decline is prompted.

(2) Experimental results for 3-month-old mice:

(ii) Morris water maze: compared with SAMR1 group mice, SAMP8 group mice have significantly increased escape latency time and initial angle radian, and significantly reduced platform crossing times and target quadrant path occupation ratio (P < 0.05); compared with SAMP8 mice, the Chinese medicinal composition lyophilized powder can significantly reduce the escape latency time and initial angle radian, significantly increase the times of crossing the platform and the platform quadrant path ratio (P < 0.05), and effectively improve the platform search strategy and space exploration mode, wherein the initial angle in the escape latency and space exploration experiments in the positioning navigation experiment has no statistical difference from that in the SAMR1 mice, and the Chinese medicinal composition lyophilized powder can better improve the early learning and memory capacity decline of AD.

The results of neuron Nie staining and transmission electron microscopy show that: the neurons in the CA1 region of the hippocampus of mice in the SAMR1 group are arranged orderly and clearly, the organelles such as mitochondria, rough endoplasmic reticulum and the like in cytoplasm are rich, the structure is more and more intact, and the morphology of the nucleus is regular; the neuron arrangement of the CA1 region of the hippocampus of mice in the SAMP8 group is poor, the outline is fuzzy, the damage of a mitochondrial crest membrane in cytoplasm is heavy, the phenomenon of degranulation of a rough endoplasmic reticulum is obvious, the nucleus is deformed, the chromatin is uneven, the neuron count is obviously reduced compared with that of the SAMR1 group (P is less than 0.05), the synapse number is also reduced, a presynaptic membrane and a postsynaptic membrane are discontinuous, the synaptic cleft is obviously widened, the outline of a synaptic vesicle is fuzzy and reduced, and the compact region on the inner side of the postsynaptic membrane is obviously reduced. The general morphology and microstructure of neurons in CA1 area of mice in the traditional Chinese medicine composition freeze-dried powder group are improved to a certain extent, the number of the neurons is remarkably increased (P is less than 0.05) compared with SAMP8 group, the neurons have no statistical difference (P is more than 0.05) compared with SAMR1 group mice, the number of synapses is large, structures of presynaptic membranes and postsynaptic membranes are clear and complete, synaptic gaps are slightly widened, the outline of synaptic vesicles is good and slightly reduced, and the dense area of the postsynaptic membranes is partially reduced. The electrophysiological detection results of the hippocampus in the body and the hippocampus also show that: after TBS and LFS stimulation, the relative change amplitude of the amplitude and the slope of LTP and DEP fEPSP recorded in the hippocampus of mice in the SAMP8 group is obviously reduced (P < 0.05) compared with that of the SAMR1 group, while the relative change amplitude of the amplitude and the slope of the fEPSP of the LTP and DEP in the traditional Chinese medicine composition freeze-dried powder group is obviously increased (P < 0.05) compared with that of the SAMP8 group, and the statistical difference between the LTP and the SAMR1 group of mice is not generated (P > 0.05), which indicates that the traditional Chinese medicine composition freeze-dried powder can effectively protect the neuron morphological structure in the hippocampal region of the SAMP8 mice, reduce the neuron loss, improve the synaptic transmission function and further improve the learning and memory.

Expression level of hippocampal Α β: although no typical senile plaque deposition is found by silver glycine staining, more silver particles which are abnormally accumulated are found in the CA1 area of the hippocampus of mice in the SAMP8 group compared with those in the SAMR1 group, and the abnormal deposition particles of the traditional Chinese medicine composition are reduced after the traditional Chinese medicine composition is lyophilized. Western blot and ELISA results show that compared with SAMR1 group, the expression of A beta oligomer, A beta 40 and A beta 42 of SAMP8 group are remarkably increased (P < 0.05), and the content of the traditional Chinese medicine composition freeze-dried powder group is remarkably reduced (P < 0.05) compared with SAMP8 group, wherein the expression of A beta 42 has no statistical difference (P > 0.05) compared with SAMR1 group, which suggests that the traditional Chinese medicine composition freeze-dried powder can effectively reduce different forms of A beta in hippocampus of SAMP8 mice, so as to reduce the neurotoxicity of the mice and protect neurons.

And fourthly, displaying the results of Western blot and RT-PCR: compared with SAMR1 group mice, SAMP8 group mice have significantly increased expression of hippocampal BACE1, QPCT and PKA proteins and BACE 1mRNA (P < 0.05), and have reduced expression of major molecules involved in APP reverse transport, namely SOLL 1 and VPS35 proteins and SOLL 1mRNA, but the difference has not reached statistical significance (P > 0.05); compared with SAMP8 group, the traditional Chinese medicine composition lyophilized powder group has significantly reduced expression of BACE1, QPCT, PKA protein and BACE 1mRNA (P < 0.05), and the expression of BACE 1mRNA, protein and PKA protein has no statistical difference (P > 0.05) compared with SAMR1 group, while the expression of SORL1, VPS35 protein and SORL1mRNA is increased, but the difference does not reach the statistical significance (P > 0.05), which suggests that the traditional Chinese medicine composition lyophilized powder can reduce the neurotoxicity generated by A beta generation and aggregation by regulating A beta self-aggregation toxic effector chain reaction, and play a role of neuroprotection.

In vitro experiments:

(1) the effect of the traditional Chinese medicine composition freeze-dried powder and the BACE1 inhibitor on primary neurons is as follows: CCK-8 results show that compared with a control group, the cell viability of primary neurons can be remarkably improved by the drug-containing serum of the Chinese medicinal composition freeze-dried powder (P is less than 0.05), wherein the effect of 10% of the drug-containing serum in 48 hours of intervention is the best (P is less than 0.05); CCK-8, LDH and Abeta 40 ELISA results show that the higher the concentration of a BACE1 inhibitor, the higher the cell viability of primary neurons, and the lower the secretion amounts of LDH and Abeta 40, when the concentration reaches 1.08 mu mol/L, compared with a control group, the three reach statistical difference (P is less than 0.05), and at the moment, the cell viability is the highest, the cell damage is the smallest, and the effect of inhibiting the expression of Abeta is the most obvious.

(2) CCK-8 and Abeta 40 immunofluorescence and Western blot results show that: compared with two control groups, the traditional Chinese medicine composition freeze-dried powder contains the serum and the BACE1 inhibitor, both the cell activity (P < 0.05) of primary neurons can be remarkably promoted, the fluorescence expression intensity and the protein expression quantity (P < 0.05) of Abeta 40 can be remarkably reduced, the difference between the two is not statistically significant (P > 0.05), the combination of the two medicines has the tendency of improving the cell activity, weakening the fluorescence expression of Abeta 40 (P > 0.05) and remarkably reducing the protein expression quantity of Abeta 40 (P < 0.05), and the traditional Chinese medicine composition freeze-dried powder is preliminarily prompted to reduce the expression of Abeta in the neurons, so that the effect of protecting the neurons is exerted.

And (4) conclusion:

(1) therapeutic effect against AD animal models: the Chinese medicinal composition can effectively improve the learning and memory abilities of 7-month-old SAMP8 mice by water decoction and freeze-dried powder, and the freeze-dried powder has a better action trend in reducing the escape latency time of the mice.

(2) Pre-intervention in AD animal models: the freeze-dried powder of the traditional Chinese medicine composition can effectively improve the early learning and memory capacity decline of 3-month-old SAMP8 mice and delay the progress of AD diseases, and the effect is related to protecting the morphological structure of neurons, relieving the neuron deficiency and improving the synaptic transmission function. ② the possible mechanism of the freeze-dried powder of the traditional Chinese medicine composition for reducing the defect of the function of hippocampal neurons and synapses of SAMP8 mice lies in that the A beta self-aggregation toxicity effect is regulated, namely the generation of the A beta is reduced mainly by down-regulating the expression of BACE1, and the neurotoxicity generated by the self-aggregation of the A beta is inhibited, thereby playing the role of neuroprotection and delaying the pathological process of AD. ③ the Chinese medicinal composition freeze-dried powder is also proved to be capable of effectively reducing the generation of A beta in neurons in vitro, improving the cell activity and playing a role in protecting nerves, and has certain effect on preventing and treating AD similar to BACE1 inhibitor.

The applicant declares that the above description is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be understood by those skilled in the art that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are within the scope and disclosure of the present invention.

Claims

1. The traditional Chinese medicine composition freeze-dried powder is characterized by comprising the following raw materials:

2. The Chinese medicinal composition freeze-dried powder according to claim 1, wherein the addition amount of the epimedium herb is 5-15 g, the addition amount of the glossy privet fruit is 5-15 g, the addition amount of the prepared fleece-flower root is 4-8 g, the addition amount of the cistanche is 5-15 g, the addition amount of the grassleaf sweelflag rhizome is 4-8 g, and the addition amount of the szechuan lovage rhizome is 4-8 g.

3. The Chinese medicinal composition freeze-dried powder according to claim 2, wherein the addition amount of the glossy privet fruit is 10 g, the addition amount of the prepared fleece-flower root is 6 g, the addition amount of the cistanche is 10 g, the addition amount of the grassleaf sweelflag rhizome is 6 g, and the addition amount of the szechuan lovage rhizome is 6 g, calculated on the basis of the addition amount of the epimedium herb being 10 g.

4. The preparation method of the Chinese medicinal composition freeze-dried powder according to any one of claims 1 to 3, which is characterized by comprising the following steps:

5. The method according to claim 4, wherein the amount of water added is 8 to 15 g based on 1 g of the mixture.

6. The preparation method according to claim 4, wherein the extraction time is 2-3 h, and the extraction mode is reflux extraction.

7. The method according to claim 4, wherein the extraction liquid collection process comprises: separating the extracted primary filtrate and the filter residue, extracting the filter residue with water under reflux for 1-2 h, filtering to obtain secondary filtrate, and mixing the primary filtrate and the secondary filtrate to obtain the extract.

8. The method according to claim 4, wherein the concentration is carried out at 50-70 ℃ under reduced pressure, and the concentration of the concentrated solution is 0.5-1 g/mL.

9. The method of claim 4, wherein the freeze-drying comprises eight steps: firstly, drying for 2.5 h at the temperature of minus 10 ℃; secondly, drying for 2.5 hours at the temperature of minus 5 ℃; thirdly, drying for 3 hours at the temperature of 0 ℃; fourthly, drying for 5 hours at the temperature of 5 ℃; fifthly, drying for 5 hours at 10 ℃; sixthly, drying for 8 hours at 15 ℃; seventhly, drying for 5 hours at 20 ℃; eighthly, drying at 25 ℃ for 41 h;

10. The use of a lyophilized powder of a Chinese medicinal composition according to any one of claims 1-3 in the preparation of a medicament for treating alzheimer's disease.