CN114949013B - Application of yellow water branch alcohol extract in preparation of medicines for treating or protecting and regulating Alzheimer disease - Google Patents
Application of yellow water branch alcohol extract in preparation of medicines for treating or protecting and regulating Alzheimer disease Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
Abstract
The application discloses an application of a yellow water branch alcohol extract in preparing a medicament for treating or protecting and regulating Alzheimer disease. The application adopts methanol or ethanol to extract the yellow water branch and removes the solvent to obtain the extract; the SCOP intraperitoneal injection is adopted to build a learning and memory disorder mouse model, donepezil hydrochloride is used as a positive medicine, the yellow water branch alcohol extract is adopted to conduct protection research on the learning and memory disorder of the mouse, the anti-Alzheimer disease effect of the yellow water branch alcohol extract is discovered, the mechanism of the yellow water branch alcohol extract is initially discussed, and finally the yellow water branch alcohol extract is found to have better protection effect on scopolamine-induced learning and memory disorder mice, so that the yellow water branch alcohol extract can be used for preparing medicines for treating or protecting and regulating Alzheimer disease.
Description
Technical Field
The application belongs to the technical field of application of traditional Chinese medicines, and particularly relates to application of a yellow water branch alcohol extract in preparation of a medicine for treating or protecting and regulating Alzheimer disease.
Background
Alzheimer's Disease (AD), which is also known as Alzheimer's disease, senile dementia, and cerebral degeneration, is a persistent neurological disorder, and is the most common cause in the mental failure, and the symptoms are manifested as gradually serious cognitive disorders (memory disorders, learning disorders, attention disorders, spatial cognitive functions, and disorders of problem solving ability), and patients gradually cannot adapt to society. The traditional Chinese medicine for treating Alzheimer's disease has the advantages of high curative effect, low toxic and side effects, low cost and the like, and is paid attention to gradually.
The pathological features of AD are mainly: the affected brain areas develop extensive oxidative stress, cholinergic dysfunction, amyloid plaques, neuronal fiber tangles, neuronal loss, etc., especially in the hippocampus and prefrontal cortex, which are closely related to learning and memory. Abnormal accumulation of beta amyloid aβ formed by hydrolysis and cleavage of the beta amyloid precursor protein APP leads to neurodegeneration, causes neuroinflammation, causes impaired neuronal function, and causes cognitive dysfunction; a large amount of aβ deposits also cause Tau hyperphosphorylation, affecting tubulin group binding, causing neurofibrillary tangles, which in turn affect the normal operation of cells in brain tissue, ultimately leading to AD onset. When suffering from AD, cholinergic neurons in the brain decrease, acetylcholinesterase (AChE) activity increases and acetylcholinesterase activity decreases, resulting in decreased ACh synthesis, storage and release, which in turn causes dysfunction in learning and memory. Meanwhile, both Abeta and ROS can cause the overactivation of the small limb plasma cells and astrocytes, promote the release of pro-inflammatory factors IL-1 beta, IL-6 and TNF-alpha, and thus trigger neuroinflammation. The pro-inflammatory factors can further activate microglia and astrocytes, accelerate Abeta aggregation and form malignant circulation, so that nerve injury is caused, and the disease course of AD is affected more seriously.
Scopolamine (SCOP) injected into abdominal cavity can block the combination of central acetylcholine and its receptor through blood brain barrier, so that cholinergic system dysfunction and oxidative stress are increased to reduce learning and memory ability. The method has the advantages of simple and convenient operation and economy, is widely applied to the research field of AD learning and memory impairment. Donepezil is a second generation specific reversible central AChE inhibitor with little effect on peripheral AChE. By inhibiting AChE activity, the decomposition of synaptic cleft ACh is slowed down, thereby increasing ACh content and improving cognitive function in AD patients. At present, the compound is also widely applied to the research of resisting Alzheimer's disease as a positive drug.
The yellow water branch (Tiarella polyphenla D. Don) is a plant of the genus yellow water branch of the family Saxifragaceae, has the effects of promoting blood circulation, removing blood stasis, relieving swelling and pain, and has the main chemical components of flavonoids, triterpenoid saponins, volatile oil, etc., and has the effects of promoting blood circulation, removing blood stasis, relieving swelling and pain, resisting tumor, resisting complement, resisting asthma, protecting liver, etc. There are some documents concerning the extraction of yellow water branches, for example, document "research on drug effect substance of folk medicine yellow water branches" (Zhang Xiaodan, university of middle and south, 2008-06-30) discloses that 9 compounds are separated from 85% ethanol extract of yellow water branches by means of repeated chromatography such as silica gel column chromatography, polyamide column chromatography, TLC and the like and recrystallization technique, and chemical structures of the compounds in 7 are identified by spectroscopic methods such as NMR, IR and the like and physical and chemical methods, and the chemical structures of the compounds are as follows in sequence: stigmasterol, fatty acid heptadecanoic acid, ethyl gallate, quercetin, protocatechuic acid, myricetin, and gallic acid. Compounds 8 and 9 are being identified, wherein compounds stigmasterol, fatty acid heptadecanoic acid, ethyl gallate, protocatechuic acid and gallic acid are first isolated from such plants. However, the document does not disclose the use of the extract of the yellow water branch. SiHP, seiRO, keunYJ, etal.AnticomplementactivityofoleanoicacidmonodesmosidesandbisdesmosidesisolatedfromTiarellapolyphylla A.J. Arch PharmRes 1999,22 (4): 428-431. 7 oleanolic acid saponins have been reported to be isolated from methanol extract of yellow water branch and have good anticomplement effect. However, the document does not disclose that the extract of the yellow water branch can be used for preparing a medicament for treating or protecting and regulating Alzheimer disease, and the extraction modes are different, and the extracted substances are not necessarily the same.
Patent application CN110229204a discloses a method for extracting plants and application of the extract, which comprises pulverizing penthorum chinense pursh (penthorum chinense pursh) stems, extracting with 70% ethanol by ultrasonic, sequentially extracting with petroleum ether and ethyl acetate, concentrating under reduced pressure, freeze-drying the extract to obtain Huang Caojing polyphenol fragments PSE, separating by high-speed countercurrent chromatography (hscc) to obtain A, B fraction, further purifying the fraction a by preparative HPLC, manually collecting effluent according to chromatographic peaks, and concentrating under reduced pressure to obtain monomer compound PGHG. PSE and PGHG can obviously inhibit the reduction of the ATP level of SH-SY5Y cells induced by 6-hydroxydopamine, and improve the cell activity; wherein PGHG can regulate the expression of apoptosis proteins and inflammatory proteins by activating Nrf2/HO-1 antioxidant pathway; the neuroprotection has a remarkable effect in preventing and treating Parkinson's disease. However, penthorum chinense pursh (penthorum chinense pursh) is a plant of penthorum genus of Saxifragaceae family, has the effects of promoting diuresis, removing dampness, removing blood stasis and relieving pain, and is used for jaundice, edema, traumatic injury and swelling and pain. Although the yellow water branch and the penthorum chinense pursh belong to the saxifragaceae plant, the yellow water branch and the penthorum chinense pursh belong to different genera in the same family, so the yellow water branch and the penthorum chinense pursh are different traditional Chinese medicinal materials.
Patent application CN201810578059.7 discloses a quercetin modified nano sulfur, a preparation method and application thereof in anti-Alzheimer disease drugs, wherein the method utilizes the micro-size and high surface energy effect of the nano sulfur to modify the quercetin on the nano sulfur, thereby not only utilizing the effect of the quercetin in reducing the endoplasmic reticulum stress in cells, but also increasing the water solubility of the quercetin, and further obtaining the quercetin modified nano sulfur with high biocompatibility and AD treatment effect. The quercetin modified nano sulfur is further embedded into a microbubble shell formed by alpha-butyl cyanoacrylate by adopting a one-pot method, so that a pharmaceutical preparation containing the quercetin modified nano sulfur is obtained, microbubbles can be destroyed under the action of ultrasound, a sound hole effect is generated, the blood brain barrier is temporarily opened, and the quercetin modified nano sulfur can penetrate through the blood brain barrier and enter the brain to play a role in treatment, so that the pharmaceutical preparation has high brain targeting. However, the method discloses that nano sulfur containing quercetin modification can be applied to anti-Alzheimer's disease drugs, but not quercetin can be applied to anti-Alzheimer's disease drugs, and the yellow water branch alcohol extract contains substances such as stigmasterol, protocatechuic acid and the like instead of only quercetin.
Patent application CN200710177085.0 discloses two crystal forms of quercetin, a preparation method thereof, a pharmaceutical composition and application thereof, and provides that the quercetin can improve the muscle trembling effect of an animal model of Parkinson's disease through neuroprotection and monoamine oxidase inhibition, and meanwhile, the quercetin can be applied to treatment in nervous system diseases, cardiovascular and cerebrovascular system diseases, immune system diseases, anti-inflammatory and other system diseases. However, the method does not disclose that quercetin can be used for preparing a medicament for treating or protecting and regulating Alzheimer disease.
Therefore, no report on related literature exists in the prior art of using the yellow water branch alcohol extract for preparing the medicines for treating or protecting and regulating Alzheimer disease.
Disclosure of Invention
The application aims to solve the technical problems and provides application of the yellow water branch alcohol extract in preparing a medicament for treating or protecting and regulating Alzheimer disease. The application adopts methanol or ethanol to extract the yellow water branch and removes the solvent to obtain the extract; according to the application, a learning and memory disorder mouse model is established by SCOP intraperitoneal injection, donepezil hydrochloride is used as a positive medicine, a yellow water branch alcohol extract is used for carrying out a protection effect study on the learning and memory disorder of the mouse, the anti-Alzheimer disease effect of the yellow water branch alcohol extract is discovered, the mechanism of the yellow water branch alcohol extract is initially discussed, and finally, the yellow water branch alcohol extract is found to have a better protection effect on scopolamine-induced learning and memory disorder mice, so that the yellow water branch alcohol extract can be used for preparing medicines for treating or protecting and regulating Alzheimer disease diseases.
In order to achieve the above purpose, the present application adopts the following technical scheme:
an application of a yellow water branch alcohol extract in preparing a medicament for treating or protecting and regulating Alzheimer disease.
Further, the yellow water branch alcohol extract is used as a pharmaceutical preparation for preparing an effective component of a medicament for treating or protecting and regulating Alzheimer disease, and the pharmaceutical preparation is an oral preparation or an injection preparation.
Further, the preparation method of the yellow water branch alcohol extract comprises the following steps: pulverizing herba Vernoniae Dillecae into coarse powder, extracting with alcohol as solvent, filtering the extractive solution, concentrating under reduced pressure to recover solvent, and drying.
Further, the extraction is a heated reflux extraction.
Further, the alcohol is methanol or ethanol and an aqueous solvent containing methanol or ethanol as a main component.
Further, the specific method of the yellow water branch alcohol extract comprises the following steps: 200 parts of coarse powder of the yellow water branch is weighed, 1900-2100 parts of 75% ethanol is added for the first time, after soaking for 1 hour, the mixture is heated and refluxed for 1 hour, the ethanol with the same volume and concentration is added for the second time, the mixture is heated and refluxed for 1 hour, the two extracts are combined, filtered, the filtrate is taken, the reduced pressure concentration is carried out, the solvent is recovered, and the mixture is dried, thus obtaining the yellow water branch.
Further, the medicine for treating or protecting and regulating Alzheimer disease is prepared by combining a yellow water branch alcohol extract and pharmaceutically acceptable auxiliary materials.
Yellow water branches extracted by different solvents (water, methanol, 95% ethanol, 75% ethanol, 50% ethanol, 30% ethanol) were screened using AChE inhibitory activity model. The results show that the yellow water extract has stronger AChE inhibition activity than the water extract, and the methanol extract has the strongest effect, and the 75% ethanol extract (TPE) is selected for the anti-mouse Alzheimer's disease test in view of the small difference of chemical components of methanol and ethanol and the better water solubility of the 75% ethanol yellow water extract.
Because the application adopts the technical proposal, the application has the following beneficial effects:
(1) The application adopts 75% ethanol to extract the yellow water branch, and the ethanol extract is obtained after the solvent is removed; through establishing a mouse learning and memory disorder model, the yellow water branch alcohol extract is adopted to administer different doses of high, medium and low to the mouse learning and memory disorder model, and research discovers that the yellow water branch alcohol extract has a certain protection effect on SCOP-induced learning and memory disorder mice, and the effect is related to balancing a cerebral cholinergic system, relieving oxidative stress injury, improving inflammatory response and inhibiting over-expression of p-Tau, BACE1 and APP proteins.
(2) Cholinergic system injury is considered an important factor in the development of AD. The clinical symptoms of AD are closely related to the reduction of acetylcholine levels. ChAT is a key enzyme for synthesizing acetylcholine, and AChE can rapidly decompose acetylcholine, so that the content of acetylcholine can be increased by inhibiting AChE level and enhancing ChAT level, thereby improving cognitive function. The research result shows that TPE can obviously reduce the level of AchE in brain tissues of mice with learning and memory disorder models and improve the level of ChAT, and the TPE is possibly used for maintaining the function of a cholinergic system by regulating the levels of AchE and ChAT in the brain tissues.
(3) Research shows that inflammatory factors can promote amyloid plaques to be converted into neuroinflammatory plaques and spread in the AD progression process, and promote APP protein degradation to generate a large amount of Abeta; an elevated IL-6 level causes calcium ions to flow into the Tau protein and changes the phosphorylation level of the latter; TNF-alpha is secreted by glial cells activated by Abeta and is toxic to neurons, increasing the permeability of the blood brain barrier, promoting lymphocyte penetration through the blood brain barrier and thus causing a series of neuroinflammatory reactions in the central system. It follows that inflammatory factors play an important role in the development and progression of AD. The research result shows that TPE can obviously inhibit the increase of IL-6 and TNF-alpha levels in brain tissues of mice with learning and memory disorder models, and the TPE is capable of improving the learning and memory disorder of the mice by reducing inflammatory response.
(4) Oxidative stress is one of the important links in the development of AD. The brain is mainly composed of lipid, has higher oxygen consumption rate, and is easy to attack by active oxygen free radicals. MDA is an important lipid peroxide product, and the level of MDA can reflect the degree of lipid peroxidation in vivo; SOD can effectively remove active oxygen free radical, and its level can reflect the capability of organism to remove free radical. The research result shows that TPE can inhibit the reduction of SOD level and the increase of MDA level in brain tissue of a learning memory disorder model mouse, and the TPE can improve the learning memory disorder of the mouse by relieving oxidative stress.
(5) BACE1 is a key enzyme for cleaving aβ produced by APP, and neurofibrillary tangles caused by amyloid plaques formed by abnormal deposition of aβ in the brain and hyperphosphorylation of Tau protein are special pathological changes of AD, which are vital in the development process of AD. In order to further elucidate the action mechanism of TPE for protecting scopolamine-induced mice from learning and memory impairment, the effect of TPE on the expression of p-Tau, APP and BACE1 proteins in the hippocampal tissues of mice was explored by using the Westernblot method. The results show that TPE can inhibit the over-expression of p-Tau, APP, BACE1 protein in the hippocampal tissue of a learning memory disorder model mouse, which indicates that TPE can change the learning memory disorder by improving the over-expression of p-Tau protein.
Drawings
For a clearer description of an example of the application or of a technical solution in the prior art, the drawings required in the description of the embodiment or of the prior art will be briefly described, it being obvious that the drawings in the description below are only some examples of the application, from which, without the inventive development, other drawings can be obtained for a person skilled in the art:
FIG. 1 is a diagram showing the result of the Westernblot detection of the p-Tau, APP, BACE1 proteins of each group;
FIG. 2 is a graph showing the effect of HE-stained TPE of the application on histopathological changes in the hippocampus of learning-memory-impaired mice (x 400);
in the accompanying drawings: a represents a normal group; b represents a model group; c represents donepezil hydrochloride group; d represents TPE high dose group; e represents the dose group in TPE; f represents TPE low dose group.
Detailed Description
The following detailed description of the application is provided in further detail, but the application is not limited to these embodiments, any modifications or substitutions in the basic spirit of the present examples, which still fall within the scope of the application as claimed.
Example 1
The application of the yellow water branch alcohol extract in preparing a medicament for treating or protecting and regulating Alzheimer disease is characterized in that the yellow water branch alcohol extract is used as a medicinal preparation for preparing an effective component of the medicament for treating or protecting and regulating Alzheimer disease, and the medicament for preparing the medicament for treating or protecting and regulating Alzheimer disease is prepared by combining the yellow water branch alcohol extract and pharmaceutically acceptable auxiliary materials.
The preparation method of the yellow water branch alcohol extract (TPE) comprises the following steps: 200g of coarse powder of the yellow water branch is weighed, 2000mL of 75% ethanol is added for the first time, after soaking for 1h, heating reflux extraction is carried out for 1h, ethanol with the same volume and concentration is added for the second time, heating reflux extraction is carried out for 1h, the two extracting solutions are combined, filtration is carried out, filtrate is taken, the solvent is recovered after decompression concentration, and drying is carried out, thus obtaining the yellow water branch.
Example 2
The application of the yellow water branch alcohol extract in preparing a medicament for treating or protecting and regulating Alzheimer disease is characterized in that the yellow water branch alcohol extract is used as a medicinal preparation for preparing an effective component of the medicament for treating or protecting and regulating Alzheimer disease, and the medicament for preparing the medicament for treating or protecting and regulating Alzheimer disease is prepared by combining the yellow water branch alcohol extract and pharmaceutically acceptable auxiliary materials.
The specific method of the yellow water branch alcohol extract comprises the following steps: weighing 200 parts of coarse powder of the yellow water branch, adding 1900 parts of methanol for the first time, soaking for 1 hour, heating and reflux-extracting for 1 hour, adding methanol with the same volume and concentration for the second time, heating and reflux-extracting for 1 hour, combining the two extracting solutions, filtering, taking filtrate, concentrating under reduced pressure, recovering solvent, and drying to obtain the extract.
Example 3
The difference from example 2 is that: the concentration of methanol used was 95% with the other conditions unchanged.
Example 4
The difference from example 2 is that: the concentration of methanol used was 90% with the other conditions unchanged.
Example 5
The difference from example 2 is that: the concentration of methanol used was 75% with the other conditions unchanged.
Example 6
The difference from example 2 is that: the concentration of methanol used was 50% with the other conditions unchanged.
Example 7
The difference from example 2 is that: the concentration of methanol used was 30% with the other conditions unchanged.
Example 8
The difference from example 2 is that: the concentration of methanol used was 10% with the other conditions unchanged.
Example 9
The application of the yellow water branch alcohol extract in preparing a medicament for treating or protecting and regulating Alzheimer disease is characterized in that the yellow water branch alcohol extract is used as a medicinal preparation for preparing an effective component of the medicament for treating or protecting and regulating Alzheimer disease, and the medicament for preparing the medicament for treating or protecting and regulating Alzheimer disease is prepared by combining the yellow water branch alcohol extract and pharmaceutically acceptable auxiliary materials.
The specific method of the yellow water branch alcohol extract comprises the following steps: 200 parts of coarse powder of the yellow water branch is weighed, 2100 parts of ethanol is added for the first time, after soaking for 1 hour, the mixture is heated and refluxed for 1 hour, ethanol with the same volume and concentration is added for the second time, the mixture is heated and refluxed for 1 hour, the two extracts are combined, filtered, filtrate is taken, the solvent is recovered after decompression concentration, and the mixture is dried.
Example 10
The difference from example 1 is that: the concentration of ethanol used was 95% with the other conditions unchanged.
Example 11
The difference from example 1 is that: the concentration of ethanol used was 85% with the other conditions unchanged.
Example 12
The difference from example 1 is that: the concentration of ethanol used was 50% with the other conditions unchanged.
Example 13
The difference from example 1 is that: the concentration of ethanol used was 30% with the other conditions unchanged.
Example 14
The difference from example 1 is that: the concentration of ethanol used was 10% with the other conditions unchanged.
In order to further illustrate that the technical effect of the application can be achieved, the following experiment is carried out:
experiment 1
Establishing a model of SCOP-induced mouse learning and memory impairment.
1.1 laboratory animals and pharmaceutical preparations
1.1.1 animals
SPF-level male Kunming mice, 60, with body mass of 18-22 g, are fed by the university of medical science laboratory animal center of Guizhou, and are well ventilated, the temperature is 18-25 ℃, the relative humidity is 40-70%, and 12 hours of illumination and round-the-clock circulation are carried out. License number SCXK (black) 2018-0001. The relevant animal experiments were reviewed by the laboratory animal ethics committee of the university of Guizhou medical science (accession number 2000069).
1.1.2 reagents and apparatus
Drugs and reagents: yellow-leaved whole grass (Kunming Intelligence-picking biotechnology Co., ltd., collection site: enshishi Jia jun county Planta, hubei province, lot number: 202010), identified as yellow-leaved whole grass by the professor Long Qingde of Guizhou medical university; scopolamine hydrobromide (lot number M1112A, purity > 98%), donepezil hydrochloride (positive control drug, lot number D0918A, purity > 98%) were purchased from calico biotechnology limited; the microplate method for measuring superoxide dismutase (SOD), the colorimetric method for measuring Malondialdehyde (MDA) and AChE kit (batch numbers are 20210708, 20201230 and 20210519 respectively) are purchased from Nanjing's institute of biological engineering; acetylcholine transferase (ChAT) enzyme-linked immunosorbent assay kit (lot 2021061541M) was purchased from lithocarpus shanghai real company, inc; interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) ELISA kits (lot numbers L210413814 and L210423372, respectively) were purchased from Wuhan cloud cloning technologies Co., ltd; rabbit-derived phosphorylated Tau (p-Tau), beta amyloid precursor protein lyase 1 (BACE 1), amyloid Precursor Protein (APP) monoclonal antibodies (lot numbers 4000001573, 4000000696, 4000000465, respectively) were all purchased from martial arts, ibotake biotechnology limited; rabbit glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibody (lot 22w 8364) was purchased from Affinity corporation; horseradish peroxidase-labeled goat anti-rabbit immunoglobulin G (IgG) secondary antibody (lot G201217) was purchased from hangzhou long biotechnology limited; the rest reagents are analytically pure or chemically pure, and the water is purified water. Polyvinylidene fluoride membrane (PVDF) (Merck, germany, lot number: P2938).
Instrument apparatus: full-band multifunctional microplate reader, universal hood type II imaging analyzer (Bio-Tek Co., U.S.A.); a high-speed cryocentrifuge (Eppendorf, germany); CP124C electronic balance (ohus instruments, inc.); eclipse 100 was placed on an optical microscope (japanese nikon).
1.2 moulding, administration and handling
60 mice of the body type and body weight equivalent to Kunming species were randomly divided into 6 groups of 10 mice each. The low, medium and high doses of TPE (i.e. the dosing group) were normal, model, positive and TPE respectively, and the dosing group was the extract of yellow crotyl alcohol extracted in example 1. Referring to related documents, the positive group mice are irrigated with 4mg/kg of donepezil hydrochloride, each dosage group of TPE is irrigated with 150 mg/kg, 300 mg/kg of corresponding liquid medicine, 600mg/kg of corresponding liquid medicine respectively, the dosage is obtained by converting the body surface area of a human and the mice according to the clinical average administration dosage of yellow water branch in Chinese herbal medicine), and the normal group and the model group are irrigated with equal volume water. Dosing was performed 1 time a day in the morning for 22 days. Morris water maze experiments are carried out on the 17 th to 22 th days after the administration, and 3mg/kg scopolamine hydrobromide is injected into the abdominal cavity of mice in the administration group and the model group 30min before the water maze training to establish a learning and memory disorder model; normal mice were intraperitoneally injected with an equal volume of physiological saline.
After the behavioural experiment was completed, each group of mice was sacrificed for cervical dislocation. The brain tissue of the mice is taken and washed by normal saline, the right half brain tissue of 4 mice is randomly taken in each group and is fixedly stored in 4% paraformaldehyde solution, and the other hippocampus and brain tissue of the mice are all stored in a refrigerator at the temperature of minus 20 ℃. Right half brain tissue fixed in 4% paraformaldehyde solution is taken, paraffin embedded, sectioned, stained with hematoxylin-eosin, and placed under a microscope to observe the morphological changes of the hippocampal tissue cells of each group of mice.
Experiment 2
Influence of TPE on learning and memory in learning and memory impaired mice
Based on experiment 1, the learning and memory conditions of mice were observed, and the results are shown in tables 1 and 2.
The escape latency of each group of mice is shortened along with the extension of training days in the positioning navigation experiment. On training day 1, the escape latency of mice in each group was compared, and the differences were not statistically significant (P > 0.05); on the 2 nd to 5 th days of training, the escape latency of the mice in the model group is obviously prolonged (P is less than 0.05) compared with that of the mice in the normal group, and the mice caused by scopolamine are prompted to be successfully built with a learning and memory disorder model; on days 3-5 of training, the escape latency of mice in the positive group and the low, medium and high dose groups of TPE is obviously shortened (P < 0.05) compared with that of mice in the model group. The experimental results are shown in Table 1.
TABLE 1 escape latency results for mice of each groupn＝10)
| Group of | Day 1 | Day 2 | Day 3 | Day 4 | Day 5 |
| Normal group | 51.86±15.14 | 41.52±16.40 | 37.50±15.50 | 43.23±14.75 | 32.38±14.83 |
| Model group | 56.50±4.61 | 53.09±11.18 a | 50.68±13.86 a | 53.13±13.13 a | 50.91±13.54 a |
| Positive group | 52.10±11.45 | 40.31±16.23 b | 33.37±14.76 b | 38.88±16.05 b | 25.06±15.46 b |
| TPE high dose group | 49.10±14.93 | 49.27±15.92 | 40.12±16.53 b | 31.53±14.07 b | 26.88±12.11 b |
| Dose group in TPE | 49.91±14.31 | 54.19±13.11 | 58.40±4.11 | 40.89±14.73 b | 35.75±16.58 b |
| TPE low dose group | 53.82±12.15 | 48.31±15.01 | 53.80±9.07 | 26.91±13.06 b | 40.27±18.31 b |
Note that: a: p <0.05 compared with the blank group; b: p <0.05 compared to model group.
The spatial search experiment was compared with the normal group, and the number of times of passing through the platform and the percentage of the target quadrant movement distance of the mice in the model group were significantly reduced (P < 0.05). Both the number of platform crossings and the percentage of target quadrant movements were significantly increased (P < 0.05) in the positive and TPE medium and high dose mice compared to the model group. The experimental results are shown in Table 2.
TABLE 2 results of percent platform pass times and target quadrant movement distance for each group of mice [ ("C")n＝10)
| Group of | Number of passes through the platform | Target quadrant movement distance percentage/% |
| Normal group | 2.50±0.55 | 41.21±11.46 |
| Model group | 0.33±0.52 a | 28.53±4.68 a |
| Positive group | 1.33±0.52 b | 38.00±5.43 b |
| TPE high dose group | 1.67±0.82 b | 39.82±9.01 b |
| Dose group in TPE | 1.50±1.05 b | 35.84±5.55 b |
| TPE low dose group | 1.17±0.98 | 30.88±9.03 |
Note that: a: p <0.05 compared with the blank group; b: p <0.05 compared to model group.
The comprehensive directional navigation experiment and the spatial search experiment result show that the high and medium doses of TPE can improve the learning and memory capacity of the learning and memory disorder model mouse.
Experiment 3
Effects of TPE on AChE and ChAT levels in learning and memory impaired mouse brain tissue
Based on experiment 1, the AChE and Elisa methods in brain tissues were used to determine ChAT activity. The experimental results are shown in table 3.
TABLE 3 AChE and ChAT results in mice brain tissue of each group [ (]n＝10)
| Group of | AChE/(U/mgprot) | ChAT/(pmol/L) |
| Normal group | 8.76±1.21 | 318.96±16.91 |
| Model group | 11.30±1.71 a | 270.81±14.34 a |
| Positive group | 8.32±1.65 b | 297.38±23.38 b |
| TPE high dose group | 7.52±2.35 b | 296.83±9.62 b |
| Dose group in TPE | 9.07±2.01 b | 288.63±11.15 b |
| TPE low dose group | 10.00±2.57 | 279.92±11.74 |
Note that: a: p <0.05 compared with the blank group; b: p <0.05 compared to model group.
As shown by experimental results, compared with a normal group, AChE level in brain tissues of mice in a model group is obviously increased, and ChAT level is obviously reduced (P is less than 0.05); compared with the model group, AChE levels in brain tissues of mice in the positive group and the TPE group at high dose are significantly reduced, and ChAT levels are significantly increased (P < 0.05).
Experiment 4
Effects of TPE on SOD and MDA levels in learning and memory impaired mouse brain tissue
Based on experiment 1, the contents of SOD and MDA in brain tissue are detected by a microplate method and a colorimetric method. The experimental results are shown in table 4.
TABLE 4 results of SOD and MDA in brain tissue of mice of each group [ (]n＝10)
| Group of | SOD/(U/mgprot) | MDA/(nmol/mgprot) |
| Normal group | 10.33±1.11 | 1.52±0.24 |
| Model group | 8.39±0.61 a | 2.16±0.29 a |
| Positive group | 9.98±1.44 b | 1.49±0.30 b |
| TPE high dose group | 10.29±1.56 b | 1.78±0.37 b |
| Dose group in TPE | 9.35±0.80 b | 1.91±0.17 b |
| TPE low dose group | 8.38±2.43 | 2.10±0.32 |
Note that: a: p <0.05 compared with the blank group; b: p < 0.055 compared to model group.
As shown by experimental results, compared with a normal group, the SOD level in the brain tissue of a model group mouse is obviously reduced, and the MDA level is obviously increased (P is less than 0.05); compared with the model group, the SOD level in the brain tissue of mice in the positive group and the TPE group at high dosage is obviously increased, and the MDA level is obviously reduced (P is less than 0.05).
Experiment 5
Effects of TPE on IL-6, TNF-alpha levels in brain tissue of learning and memory impaired mice
Based on experiment 1, the content of IL-6 and TNF-alpha in brain tissue was detected by an Elisa method. The experimental results are shown in table 5.
TABLE 5 influence of TPE on IL-6, TNF-alpha in serum of mice with learning and memory impairmentn＝10)
| Group of | IL-6/(pg/mL) | TNF-α/(pg/mL) |
| Normal group | 22.95±7.40 | 71.42±7.10 |
| Model group | 46.35±6.90 a | 95.62±13.22 a |
| Positive group | 18.64±6.36 b | 72.15±10.54 b |
| TPE high dose group | 19.55±6.37 b | 66.71±10.13 b |
| Dose group in TPE | 21.67±7.79 b | 70.11±12.49 b |
| TPE low dose group | 27.02±7.79 b | 101.40±12.30 |
Note that: a: p <0.05 compared with the blank group; b: p <0.05 compared to model group.
As shown by the experimental results, compared with the normal group, the levels of IL-6 and TNF-alpha in the brain tissues of the mice in the model group are obviously increased (P is less than 0.05); IL-6 levels were significantly reduced (P < 0.05) in both the positive and TPE, in the high dose group, and in the low dose group, compared to the model group.
Experiment 6
Effect of TPME on learning and memory impaired mouse hippocampal tissue p-Tau, BACE1 and APP protein expression
Based on experiment 1, protein expression of p-Tau, BACE1 and APP in sea horse tissues is detected by adopting a Westernblot method.
Re-heating the sea Ma Zuzhi in a refrigerator at-80deg.C, weighing 100mg of each Hippocampus tissue sample, adding 1mLRIPA protein lysate, homogenizing on ice with a full-automatic grinder, centrifuging at 4deg.C and 10000r/min for 10min, and collecting the supernatant to obtain Hippocampus tissue proteins of each group. Protein concentration in hippocampal tissue samples was determined using BCA method. Adding 5 Xprotein loading buffer solution, heating at 100deg.C for 5min for denaturation, performing 10% SDS-PAGE electrophoresis, transferring protein onto polyvinylidene fluoride (PVDF) membrane by wet transfer method, sealing the membrane with 5% skimmed milk powder for 2h, washing with TBST buffer solution for 3 times, each time for 10min, respectively adding APP (1:1500), p-Tau (1:1500) and BACE1 (1:1000) antibody, incubating overnight at 4deg.C, incubating PVDF membrane at room temperature for 15min, washing with TBST buffer solution for 3 times, each time for 10min, incubating with secondary antibody at room temperature for 1h, washing with TBST buffer solution for 3 times, each time for 10min, developing with ECL luminescence kit, analyzing the grey value of the strip by imageJ1.8.0 software, and evaluating the relative expression of protein by the ratio of target protein grey value and internal reference GAPDH grey value. The experimental results are shown in FIG. 1 and Table 6.
TABLE 6 influence of TPME on the expression of BACE1, APP and p-Tau protein in hippocampal tissue of learning and memory impaired micen＝3)
| Group of | p-Tau | BACE1 | APP |
| Normal group | 0.52±0.29 | 2.29±0.25 | 1.40±0.25 |
| Model group | 1.16±0.20 a | 2.88±0.28 a | 2.13±0.15 a |
| Positive group | 0.68±0.31 b | 2.18±0.26 b | 1.76±0.44 b |
| TPE high dose group | 0.84±0.15 b | 1.98±0.23 b | 1.19±0.30 b |
| Dose group in TPE | 0.86±0.13 b | 2.00±0.38 b | 1.24±0.30 b |
| TPE low dose group | 0.94±0.34 | 2.64±0.20 | 1.54±0.24 b |
Note that: a: p <0.05 compared with the blank group; b: p <0.05 compared to model group.
Compared with the normal group, the relative expression amounts of P-Tau, BACE1 and APP proteins in the hippocampal tissues of the mice in the model group are obviously increased (P is less than 0.05) compared with the normal group; compared with the model group, the relative expression amount of the P-Tau, APP, BACE1 protein in the hippocampal tissues of the mice in the positive group and the TPE group and the high-dose group and the hippocampal tissues of the mice in the TPE low-dose group are obviously reduced (P < 0.05).
Experiment 7
Effects of TPE on pathological changes in hippocampal visceral tissue in learning and memory impaired mice
On the basis of experiment 1, hematoxylin-eosin (HE) staining was used to assess hippocampal histopathological changes in brain tissue.
Taking brain tissue fixed by 4% paraformaldehyde, dehydrating by ethanol, embedding by paraffin, slicing, respectively staining by HE and Masson, and observing pathological changes of liver tissue under a light microscope. The experimental results are shown in FIG. 2.
HE staining results show that the neurons of the CA1 region of the hippocampus of the normal mice are closely arranged, the morphology is complete, and the cell membranes and the cell nuclei are clear. Compared with the normal group, the number of neurons of the mice in the model group is reduced, the arrangement is loose, a small part of neurons are in a solidification atrophy state, a large amount of necrosis is visible, cytoacidophilic property is enhanced, and cell nuclei are contracted, dissolved and disappeared. Compared with the model group, the neurons of the mice in the positive group and the low, medium and high dose groups of TPE are increased, the neurons in the curing atrophy state are reduced, the arrangement of the neurons tends to be compact, and the TPE is suggested to be capable of improving the pathological changes of the neurons in the CA1 region of the hippocampus of the mice in the learning memory disorder model.
Similarly, experiments conducted one by adopting the experimental mode prove that the yellow water branch alcohol extract can be used for preparing medicines for treating or protecting and regulating Alzheimer disease diseases according to other alcohol concentrations adopted in the embodiments 2-14, and the differences of curative effects are not large, so the yellow water branch alcohol extract is not listed in the present application document.
Therefore, the yellow water branch alcohol extract has a better protection effect on scopolamine-induced mice with learning and memory impairment, and can be used for preparing medicines for treating or protecting and regulating Alzheimer disease diseases.
It should be noted that all the data from the above experiments were counted and analyzed using SPSS26.0 statistical software,
ˉ
the data are all metering data, expressed by mean value + -standard deviation (x + -s), the comparison among multiple groups adopts single-factor variance analysis, and P <0.05 represents that the difference has statistical significance.
It will be evident to those skilled in the art that the application is not limited to the details of the foregoing illustrative embodiments, and that the present application may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the application being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Claims (3)
1. The application of the yellow water branch alcohol extract in preparing a medicament for treating or protecting and regulating Alzheimer disease is characterized in that: the specific preparation method of the yellow water branch alcohol extract comprises the following steps: 200 parts of coarse powder of the yellow water branch is weighed, 1900-2100 parts of 75% ethanol is added for the first time, after soaking for 1 hour, the mixture is heated and refluxed for 1 hour, the ethanol with the same volume and concentration is added for the second time, the mixture is heated and refluxed for 1 hour, the two extracts are combined, filtered, the filtrate is taken, the reduced pressure concentration is carried out, the solvent is recovered, and the mixture is dried, thus obtaining the yellow water branch.
2. The use of the yellow water branch alcohol extract according to claim 1 for preparing a medicament for treating or protecting and regulating alzheimer's disease, characterized in that: the yellow water branch alcohol extract is used as an effective component for preparing a medicament for treating or protecting and regulating Alzheimer disease, and is prepared into an oral preparation or an injection preparation.
3. The use of the yellow water branch alcohol extract according to claim 1 for preparing a medicament for treating or protecting and regulating alzheimer's disease, characterized in that: the medicine for treating or protecting and regulating Alzheimer disease is prepared by combining a yellow water branch alcohol extract and pharmaceutically acceptable auxiliary materials.
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