CN111454303A - Cistanche phenylethanoid glycoside and application thereof in preparation of medicine for treating Alzheimer disease - Google Patents
Cistanche phenylethanoid glycoside and application thereof in preparation of medicine for treating Alzheimer disease Download PDFInfo
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- CN111454303A CN111454303A CN202010291031.2A CN202010291031A CN111454303A CN 111454303 A CN111454303 A CN 111454303A CN 202010291031 A CN202010291031 A CN 202010291031A CN 111454303 A CN111454303 A CN 111454303A
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Abstract
The invention discloses cistanche phenylethanoid glycoside and application thereof in preparation of a medicament for treating Alzheimer disease, and belongs to the technical field of medicaments. The invention discusses the specific action mechanism of cistanche phenylethanoid glycoside for preventing and treating Alzheimer disease through an APP/PS1 double-transgenic mouse AD model, and proves that cistanche phenylethanoid glycoside can improve the transduction disorder of an insulin signal channel and regulate the brain energy metabolism through regulating upstream and downstream key targets of the channel, thereby showing that cistanche phenylethanoid glycoside has obvious prevention and treatment effects on Alzheimer disease. The invention not only enriches the research data of preventing and treating the Alzheimer disease by the traditional Chinese medicine, but also lays the research foundation for developing the new drug of the desertliving cistanche for resisting the Alzheimer disease, which takes the phenylethanoid glycoside as the mother nucleus structure, and is suitable for popularization and application.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to cistanche phenylethanoid glycosides and a preparation method thereof, and application of the cistanche phenylethanoid glycosides in preparation of medicines for treating Alzheimer disease.
Background
Alzheimer's Disease (AD) is a degenerative disease of the central nervous system for which effective curative strategies are still lacking clinically. AD is clinically manifested primarily as progressive cognitive dysfunction and memory impairment. Because AD has hidden onset, high morbidity, long course of disease and high treatment cost, the traditional Chinese medicine brings huge harm and economic burden to families and society of patients.
Because the Alzheimer disease is difficult to treat and the pathological mechanism is complex, compared with the traditional Chinese medicine prevention and treatment principle of chemical medicines, the traditional Chinese medicine is developed from the overall view and multiple targets, and is more suitable for intervening in the treatment of AD with multiple pathological mechanisms. In recent years, the discovery of insulin signaling pathway has brought new inspiration for the study of AD. Insulin exists in memory-related hippocampus, and is widely distributed in other cells and tissues in brain, and plays a considerable role in protecting neurons, learning and memorizing and other advanced intelligent activities. Therefore, the detection of the expression of the key protein of the insulin signal pathway is of great importance to the search of new targets in the prevention and treatment of AD and the deep research of pathogenesis.
Although the traditional Chinese medicine cistanche has multiple effects of oxidation resistance, inflammation resistance, aging resistance and the like, the research on the pharmacological activity of the traditional Chinese medicine cistanche also proves that the cistanche phenylethanoid glycosides show better antagonistic AD potential, no research report for researching the effect of the cistanche phenylethanoid glycosides on AD through an insulin signal path is found at present. The invention discusses the specific action mechanism of cistanche phenylethanoid glycoside for preventing and treating AD through an APP/PS1 double-transgenic mouse AD model, and proves that cistanche phenylethanoid glycoside can improve the transduction disorder of an insulin signal channel and regulate the brain energy metabolism by regulating the upstream and downstream key targets of the channel, and the cistanche phenylethanoid glycoside has obvious prevention and treatment effects on AD.
Disclosure of Invention
In view of the above, the present invention aims to provide a cistanche phenylethanoid glycoside for preparing a medicament for preventing and treating alzheimer disease, which aims to solve the problems in the prior art.
In order to achieve the purpose, the invention adopts the following technical scheme:
the cistanche phenylethanoid glycoside is obtained by extracting cistanche phenylethanoid glycoside from succulent stems, and the content (purity) of the cistanche phenylethanoid glycoside extract is 87.6-94.1%.
The invention also claims an extraction process of the cistanche phenylethanoid glycoside, which specifically comprises the following steps:
(1) crushing and sieving the succulent stems of the cistanche, performing reflux extraction for 3-4 times by using 80% ethanol, combining filtrates, and performing reduced pressure concentration to obtain an extract;
(2) suspending the extract prepared in the step (1) with water, sequentially performing macroporous adsorption resin column chromatography and H2After gradient elution of O and 30-95% ethanol, collecting 30-50% ethanol elution fractions;
(3) concentrating the ethanol eluate of step (2) under reduced pressure, suspending with water, sequentially performing MCI column chromatography and H column chromatography2O-CH3After gradient elution of an OH system, collecting 30-50% of CH3An OH elution fraction section;
(4) and (4) concentrating the methanol elution fraction collected in the step (3) under reduced pressure to be thick paste, then suspending with water and performing ODS column chromatography to obtain the cistanche phenylethanoid glycoside.
Preferably, in the step (1), the volume ratio of the 80% ethanol to the succulent stems of cistanche is (3-4): 1.
specifically, the preparation method of the cistanche phenylethanoid glycoside comprises the following steps:
weighing a proper amount of dry succulent stems of cistanche deserticola, crushing, sieving, respectively reflux-extracting for 3-4 times by using 80% ethanol in an amount which is 3-4 times that of the dry succulent stems, combining filtrates, concentrating under reduced pressure to obtain an extract, suspending the extract by using a proper amount of water, performing SP-825 macroporous adsorption resin column chromatography, and sequentially performing H-column chromatography2Performing gradient elution on the O and the 30-95% ethanol, and then combining elution fractions of the 30-50% ethanol; concentrating under reduced pressure to obtain extract, suspending with appropriate amount of water, performing MCI column chromatography, and purifying with H column2O-CH3Gradient elution is carried out on an OH system, and 30-50% of CH is collected3And (4) OH eluting the flow section, finally concentrating under reduced pressure to obtain thick paste, suspending the extract by using a proper amount of water, and performing ODS repeated column chromatography to obtain the cistanche phenylethanoid glycoside.
The invention also aims to provide the application of the cistanche phenylethanoid glycoside in preparing the medicament for treating the Alzheimer disease.
In order to achieve the purpose, the technical scheme of the invention is as follows:
the application of cistanche phenylethanoid glycosides in preparing a medicament for treating Alzheimer disease is disclosed, wherein the effective dose range of the cistanche phenylethanoid glycosides in the medicament for treating Alzheimer disease is 62.5-250 mg/kg/d.
Further preferably, the effective dosage range of the cistanche phenylethanoid glycosides in the medicament for treating the alzheimer disease is 125-250 mg/kg/d.
The cistanche phenylethanoid glycosides play a role in preventing and treating alzheimer disease by regulating insulin signal pathway targets.
Wherein the target comprises insulin receptor (InR), insulin-like growth factor 1 receptor (IGF-1R), Insulin Receptor Substrate (IRS), phosphatidylinositol-3 hydroxykinase (PI3K), threonine protein kinase (AKT).
In recent years, the discovery of insulin signaling pathway has brought new inspiration for the study of AD. Insulin exists in memory-related hippocampus, is widely distributed in other cells and tissues in brain, and plays a considerable role in protecting neurons, learning memory and other high-level intelligent activities. After the insulin receptor (InR) and the insulin-like growth factor 1 receptor (IGF-1R) in AD brain are combined with the receptor, the phosphorylation of the autoreceptor is induced, the phosphorylation of an Insulin Receptor Substrate (IRS) protein is promoted, the growth and the metabolism of downstream molecules are regulated, further, p85 is enabled to regulate subunit phosphatidylinositol-3 hydroxykinase (PI3K) to stimulate the glucose conversion, threonine protein kinase (AKT) is activated, downstream and apoptosis-related proteins are inhibited, and neurons are enabled to survive (see figure 1). Thus, the insulin signaling pathway acts as an important neuroprotective signaling pathway in vivo, and its primary stimulatory signal not only regulates protein expression, but also its primary signaling pathway PI3K/AKT regulates synaptic plasticity and participates in learning and memory processes.
The traditional AD prevention and treatment mechanism research mostly aims at classical signal paths of nervous system diseases such as inflammation, immunity, antioxidant stress and the like, and has certain limitations. The evolution and development of AD are rapid, the pathogenesis is not clear up to now, and the effective research of the insulin signal pathway as a hotspot research pathway of AD called as 'type III diabetes' has great practical significance on the improvement of later-stage drug development and prevention and treatment means.
In view of the above, the invention is of great importance for the search of new drug targets in an AD prevention and treatment system and the deep research of pathogenesis by investigating the expression condition of key proteins of an insulin signal pathway after the intervention of cistanche phenylethanoid glycosides.
According to the technical scheme, compared with the prior art, the cistanche phenylethanoid glycoside and the application of the cistanche phenylethanoid glycoside in preparing the medicine for treating the Alzheimer disease have the following excellent effects:
the invention discusses the specific action mechanism of cistanche phenylethanoid glycoside for preventing and treating Alzheimer disease through an APP/PS1 double-transgenic mouse AD model, proves that the cistanche phenylethanoid glycoside can improve the transduction disorder of an insulin signal path and regulate the brain energy metabolism through regulating and controlling a key target of the path, thereby showing that the cistanche phenylethanoid glycoside has obvious prevention and treatment effects on the Alzheimer disease and providing a basis for developing a new cistanche AD-resistant medicament taking the phenylethanoid glycoside as a mother nucleus structure.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a diagram of the insulin signaling pathway in the brain.
FIG. 2 is an ultraviolet spectrum of phenylethanoid glycosides from Cistanchis herba.
FIG. 3 is a graph showing the result of expressing InR and IRS-1 proteins of AD mice with cistanche phenylethanoid glycosides (wherein, the graph (a) shows the expression of InR proteins of AD mice, and the graph (b) shows the expression of IRS-1 proteins of AD mice).
FIG. 4 is a graph showing the result of the expression of cistanche phenylethanoid glycosides on AD mouse IGF-1R protein.
FIG. 5 is a graph showing the results of the expression of cistanche phenylethanoid glycosides on the AD mouse PI3K and p-PI3K proteins (wherein, the graph (a) shows the expression on the AD mouse PI3K protein, and the graph (b) shows the expression on the AD mouse p-PI3K protein).
FIG. 6 is a graph showing the results of the expression of cynamorloside on AKT and p-AKT proteins in AD mice (wherein, the graph (a) shows the expression on AKT proteins in AD mice, and the graph (b) shows the expression on p-AKT proteins in AD mice).
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The embodiment of the invention discloses a preparation method of cistanche phenylethanoid glycosides and application of the cistanche phenylethanoid glycosides in preparing a medicament for preventing and treating Alzheimer disease.
The present invention will be further specifically illustrated by the following examples for better understanding, but the present invention is not to be construed as being limited thereto, and certain insubstantial modifications and adaptations of the invention by those skilled in the art based on the foregoing disclosure are intended to be included within the scope of the invention.
The invention discloses cistanche phenylethanoid glycosides, which are extracted from cistanche succulent stems, and the content (purity) of the cistanche phenylethanoid glycoside extract is 87.6-94.1%.
In addition, the invention also discloses a preparation method of the cistanche phenylethanoid glycoside, which specifically comprises the following steps:
weighing a proper amount of dry succulent stems of cistanche deserticola, crushing, sieving, respectively performing reflux extraction for 3-4 times by using 80% ethanol in an amount which is 3-4 times that of the dry succulent stems, combining filtrates, performing reduced pressure concentration to obtain an extract, and soakingSuspending the paste with appropriate amount of water, performing SP-825 macroporous adsorbent resin column chromatography, and sequentially purifying with H2Performing gradient elution on the O and the 30-95% ethanol, and then combining elution fractions of the 30-50% ethanol; concentrating under reduced pressure to obtain extract, suspending with appropriate amount of water, performing MCI column chromatography, and purifying with H column2O-CH3Gradient elution is carried out on an OH system, and 30-50% of CH is collected3And (4) OH eluting the flow section, finally concentrating under reduced pressure to obtain thick paste, suspending the extract by using a proper amount of water, and performing ODS repeated column chromatography to obtain the cistanche phenylethanoid glycoside.
Wherein, the preferable experiment of the extraction process is as follows:
experiment one: preference of macroporous resin type
Placing 1g of pretreated SP-825 resin, AB-8 resin and D301 resin in conical flask, respectively adding 10m L sample solution, sealing, and placing in constant temperature culture shaker (25 deg.C, 60r min)-1) Oscillating for 24h, adsorbing, vacuum filtering, diluting the filtrate with distilled water to 10m L, measuring content, calculating the adsorption rate of filler, placing the above adsorbed filler in a conical flask, adding 50% ethanol 10m L, sealing, and placing in a constant temperature culture oscillator (25 deg.C, 60r min)-1) And after 24 hours, carrying out suction filtration, fixing the volume of the filtrate to 10m L by using 50% ethanol, taking the liquid after fixing the volume, measuring the content of the sample, and calculating the desorption rate.
Adsorption rate ═ C0–C1)/C0×100％
Desorption rate ═ C2/(C0-C1)]×100％
C0The mass concentration of the stock solution is mg.m L-1；C1For the post-adsorption mass concentration, mg · m L-1；C2As the mass concentration of the eluent, mg · m L-1. The results show that the adsorption effect of the SP-825 macroporous adsorption resin is optimal (see Table 1).
TABLE 1 comparison of adsorption and elution performance of echinacoside and verbascoside with different types of macroporous resin
Experiment two: preference of the eluent
Weighing the treated SP-825 resin 8g, loading into column by wet method, and accurately weighing Cistanchis herba phenylethanoid glycosides extract 0.01g to obtain 2m L sample solution at 0.5m L min with diameter-height ratio l:10(1BV ═ 8ml)-1Loading, adsorbing for 3 hr, washing with 4BV water, eluting with 10%, 30%, 50%, 75%, 95% ethanol at 2 BV.h-1And 3BV of alcohol eluent is collected and concentrated to 10m L L C, and the mass concentration of echinacoside and verbascoside in each alcohol eluent is determined by the method, the result shows that ethanol is selected as the eluent, the concentration range of the eluent is 10-95%, and the concentration range of the effective eluent is 30-50% (see table 2).
Table 2 eluent investigation (n ═ 3)
Experiment three: preference of extraction process
Weighing a proper amount of dry succulent stems of cistanche deserticola, crushing, sieving, respectively reflux-extracting for 3-4 times by using 80% ethanol in an amount which is 3-4 times that of the dry succulent stems, combining filtrates, concentrating under reduced pressure to obtain an extract, suspending the extract by using a proper amount of water, performing SP-825 macroporous adsorption resin column chromatography under different conditions, and sequentially performing H-column chromatography by using H2Performing gradient elution on the O and the 30-95% ethanol, and then combining elution fractions of the 30-50% ethanol; concentrating under reduced pressure to obtain extract, suspending with appropriate amount of water, performing MCI column chromatography under different conditions, and purifying with H column chromatography2O-CH3Gradient elution is carried out on an OH system, and 30-50% of CH is collected3OH elution fraction, decompression concentrating to obtain thick paste, suspending the extract with proper amount of water, and performing ODS repeated column chromatography under different conditions to obtain the cistanche phenylethanoid glycoside, wherein the content of the cistanche phenylethanoid glycoside prepared under different conditions is 87.6-94.1% as measured by high performance liquid chromatography (HP L C).
TABLE 3 Phenylethanoid glycoside content obtained with different extraction process preferences
The technical scheme of the invention will be further explained and demonstrated by combining specific experiments.
Experiment 1: content determination of cistanche phenylethanoid glycoside
The extraction process of the cistanche phenylethanoid glycoside comprises the following steps:
(1) weighing appropriate amount of dry succulent stem of Cistanchis herba, pulverizing, sieving, reflux-extracting with 3 times of 80% ethanol for 3 times, mixing filtrates, concentrating under reduced pressure to obtain extract, suspending the extract with appropriate amount of water, subjecting to SP-825 macroporous adsorbent resin column chromatography, and sequentially subjecting to H-column chromatography2Gradient eluting with 30%, 50%, 70% and 95% ethanol, and combining the elution fractions with 30% -50% ethanol;
(2) concentrating the ethanol eluate of step (1) under reduced pressure to obtain extract, suspending the extract with appropriate amount of water, performing MCI column chromatography, and performing H column chromatography2O-CH3Gradient elution of OH system, collecting 30-50% CH3An OH elution fraction section;
(3) and (3) decompressing and concentrating the methanol elution fluid collected in the step (2) into a thick paste, suspending the extract by using a proper amount of water, and performing ODS repeated column chromatography to obtain the cistanche phenylethanoid glycoside.
It should be noted that, as a result of the ultraviolet spectrograms of the test solution, the verbascoside reference solution and the echinacoside reference solution, the ultraviolet spectrograms are found to be basically consistent (see fig. 2), and the maximum absorption wavelength is 333nm, therefore, the content of the phenethyl alcohol total glycoside in the purified product can be determined by adopting an ultraviolet spectrophotometry by taking the verbascoside or the echinacoside as the reference. The experiment uses echinacoside as a reference substance to measure the content of the phenethyl alcohol total glycosides of cistanche, and the result shows that the content of the phenethyl alcohol total glycosides in the extracted and separated product reaches 87.6 percent.
Experiment 2: effective dosage range of cistanche phenylethanoid glycosides for preventing and treating Alzheimer disease
The applicant has proved that cistanche salsa is safe and nontoxic by combining a large amount of literature research and the related pharmacodynamics research results of cistanche salsa at the previous stage, so that the administration dosage is not obviously limited, and the specific effective dosage needs to be screened and determined according to the research purpose. Based on the above, the experiment adopts PC12 nerve cells to replicate a classical Alzheimer disease in vitro cell model, and carries out the experimental study of the effective dose of cistanche phenylethanoid glycosides for preventing and treating Alzheimer disease. Research results show that 62.5-250mg/kg of cistanche phenylethanoid glycosides is dried, the proliferation rate of model cells is more than 80%, the proliferation rate is stable, the influence on cell morphology is small, the safety is good, and the dry preparation method can be used as a reference dose range for later-stage drug intervention (see table 4).
TABLE 4 dosage screening results for cistanche phenylethanoid glycosides for preventing and treating Alzheimer's disease
Experiment 3: effect of cistanche phenylethanoid glycosides on the expression of InR, IRS-1, IGF-1R and downstream PI3K, p-PI3K, AKT, p-AKT in AD mice
1. Materials and methods
1.1 laboratory animals and reagents
60 APP/PS1 double-transgenic AD model mice and 10 littermate negative mice with weight of 20-30g are purchased from the institute of Nanjing model animals, Jiangsu, China, the mouse feed is clean maintenance mouse feed, drinking water is disinfectant water, the mouse padding is disinfectant padding, animals can freely eat and drink water during the experiment, cistanche phenylethanoid glycosides (self-made, the content of phenylethanol total glycosides measured by a UV method reaches 87.6%), Anrishengshi (health care product pharmaceutical industry), SDS-PAGE gel preparation kit, 4 × protein sample buffer solution, 10 × TBST, SDS, glycine (Solarbio), BCA protein quantification kit (Nanjing Kai biology), A β1-40、Aβ1-42β -actin (CST company), alkali phosphatase labeled rabbit anti-goat IgG (H + L), secondary antibody (China fir bridge), PVDF membrane (Roche), Tris (VETEC), skimmed milk powder (BD), BCIP/NBT Kit (invitrogen), methanol and other reagents are analytically pure.
1.2 instruments
Gel imager (BIO-RAD, USA), RM2016 pathological microtome (L eica, Germany), SIM-F124 ice maker (SANYO, Japan), gel electrophoresis, andmembrane transfer equipment (BIO-RAD, USA), full-wavelength microplate reader (Thermo Scientific, USA), CO2A constant temperature incubator (Thermo company, USA), a low temperature freezing high speed centrifuge (Thermo Scientific company, USA), a DVKW-D-2 digital display electric heating constant temperature water bath (Youging medical instrument factory, Beijing), a WD-9405A type decoloring shaker (six instruments factory, Beijing), an adjustable constant voltage and constant current power supply (BIO-RAD company, USA), and a DM4000 fluorescence inverted microscope (L eica company, Germany).
1.3 Experimental groups and methods of administration
According to random numbers generated by SPSS18.0 statistical software, AD model mice are randomly divided into a model group, a donepezil group (the daily gavage dose is 0.65mg/kg), a cistanche phenylethanoid glycoside high, medium and low dose groups (the daily gavage doses are respectively 250, 125 and 62.5mg/kg), and 10 mice in each group. The mice were subjected to intragastric administration for 3 months and 1 time daily starting at 6 months of age. The normal group was littermate wild type mice, and the model group and the normal group were gavaged with double distilled water of equal volume.
1.4 immunohistochemical staining
Sections were immunohistochemically stained according to the following procedure: dewaxing → antigen retrieval → blocking endogenous catalase → serum sealing → primary antibody incubation → secondary antibody incubation → DAB coloration → dehydration → sealing piece → Image J software Image analysis.
1.5 Western blot analysis of protein expression
The method comprises the steps of killing each group of selected mice by quickly cutting off heads on ice, immediately placing stripped fresh brain hippocampus tissues in an EP tube, adding lysis solution for tissue homogenate, sucking supernate into a new centrifugal tube with a label, measuring the content of protein extracted from the brain tissue hippocampus according to a BCA protein quantification method, fully and uniformly mixing the quantified protein and 4 × protein sample buffer solution according to the ratio of 4: 1, heating for 10min at 95 ℃, naturally cooling at room temperature, and storing in a refrigerator at-20 ℃ for later use.
Preparing SDS-PAGE gel, removing upper ultrapure water after gel polymerization is finished, adding concentrated gel after filter paper is dried by suction, lightly and vertically inserting a proper comb, removing the comb after gel layers are completely polymerized, adding prepared electrophoresis liquid, sequentially adding each group of proteins such as a normal group, a model group, a positive control group and a drug drying pre-group, adding 5 mu L pre-dyed protein marker into glue holes on two sides, performing constant voltage electric rotation in an ice bath box, performing electrophoresis at 100V for 30min, increasing the voltage to 120V after a sample runs through the concentrated gel, stopping electrophoresis until the bottom of a separation gel after electrophoresis, washing a gel plate with ultrapure water after electrophoresis is finished, placing the gel plate into pre-cooled transfer liquid at 4 ℃, balancing for 5min, shearing 1 piece of PVDF membrane and 6 pieces of thick filter paper according to the size of the gel, taking a picture of the PVDF membrane in methanol, placing the activated membrane liquid and the filter paper into the transfer liquid, placing the membrane into a black fiber pad → 3 layer of PVDF membrane → 3 layer → a black fiber pad, removing the PVDF membrane, removing the membrane between the PVDF membrane, removing the PVDF membrane, placing the membrane and completely in a constant voltage conversion tank, placing the filter paper into a constant voltage shaking and performing constant voltage electric washing, placing the light-shaking, and performing constant voltage direct imaging on a constant voltage electric washing machine, placing the membrane for 2 times of a black fiber pad → the membrane, placing the membrane for the membrane, performing constant voltage electric shock-shaking, and performing.
1.6 statistical analysis of data
Statistical analysis of data was performed using SPSS18.0
As indicated, data comparisons between groups were analyzed by One-Way ANOVA test, and multiple comparisons between data were analyzed by L SD-t test (least squares interference, L SD) as p<0.05 or p<0.01 indicates that the difference is statistically significant.
2. Results
2.1 Effect of Rongcong cistanche phenylethanoid glycosides on InR and IRS-1 expression in AD mice
The prognosis of cistanche phenylethanoid glycoside is carried out, APP/PS1 double-transgenic mouse brain tissue slices of 9-month-old are taken, InR and IRS-1 immunohistochemical staining is carried out on a hippocampal CA1 area, and the result shows that: compared with the normal group, the number of InR and IRS-1 positive cells in the CA1 area of the hippocampus of the model group mice is obviously increased (p is less than 0.01); compared with the model group, the number of positive cells of each dose group of cistanche phenylethanoid glycosides is obviously reduced (p is less than 0.01) (see table 5).
TABLE 5 Hippocampus CA1 regions InR, IRS-1 Positive nerve cell number (II)
n＝10)
Note: in comparison with the normal group,##p<0.01，#p<0.05; in comparison to the set of models,**p<0.01，*p<0.05。
the expression of the mouse hippocampal InR and IRS-1 proteins is detected by a Western-blot method, and the result shows that the protein expression of the mouse hippocampal InR and IRS-1 in a model group is obviously up-regulated compared with that in a normal group, and has significant difference (p is less than 0.05). Compared with the model group, the protein expression of the InR and IRS-1 of the hippocampi of mice of each dose intervention group of the cistanche phenylethanoid glycosides is obviously reduced, and the difference has statistical significance (p is less than 0.01) (see figure 3).
2.2 Effect of Cistanchis herba Phenylethanoid glycosides on IGF-1R expression in AD mice
After the prognosis of cistanche phenylethanoid glycoside, 9-month-old APP/PS1 double-transgenic mouse brain tissue slices are taken, IGF-1R immunohistochemical staining is carried out on a hippocampal CA1 region, and the result shows that: compared with the normal group, the mouse hippocampal CA1 area IGF-1R positive cell number of the model group is obviously increased (p is less than 0.01); compared with the model group, the number of positive cells of each dose group of the cistanche phenylethanoid glycosides is obviously reduced, and the difference has statistical significance (p is less than 0.01) (see table 6).
TABLE 6 Hippocampus CA1 region IGF-1R Positive neural cell number ((II))
n＝10)
Note: in comparison with the normal group,##p<0.01，#p<0.05; in comparison to the set of models,**p<0.01，*p<0.05。
the expression of mouse hippocampal IGF-1R protein is detected by a Western-blot method, and the result shows that the protein expression of mouse hippocampal IGF-1R in a model group is obviously up-regulated compared with that in a normal group, and has significant difference (p is less than 0.05). Compared with the model group mice, the hippocampal IGF-1R protein expression of the mice of the donepezil group and the cistanche phenylethanoid glycoside dose intervention group is obviously reduced, and the difference is statistically different (p <0.01) (see figure 4).
2.3 Effect of Cistanchis herba Phenylethanoid glycosides on the expression of PI3K and p-PI3K in AD mice
After the intervention of cistanche phenylethanoid glycosides for 3 months, the Western-blot method is adopted to detect the expression of mouse hippocampal PI3K and p-PI3K proteins, and the results show that the protein expression of the mouse hippocampal PI3K and p-PI3K in a model group is reduced to different degrees compared with that in a normal group, and the significant difference is achieved (p is less than 0.05). Compared with the model group, the expressions of the mouse hippocampal PI3K and p-PI3K proteins of the intervention group with various doses of the cistanche phenylethanoid glycosides are obviously up-regulated, and the difference has statistical significance (p is less than 0.01) (see figure 5).
2.4 Effect of Cistanchis herba Phenylethanoid glycosides on AKT and p-AKT expression in AD mice
After the intervention of cistanche phenylethanoid glycosides for 3 months, the Western-blot method is adopted to detect the expression of mouse hippocampal AKT and p-AKT proteins, and the results show that the protein expression of mouse hippocampal AKT and p-AKT in a model group is reduced to different degrees compared with that in a normal group, and the significant difference is achieved (p is less than 0.05). Compared with the model group, the expressions of the hippocampal AKT and p-AKT proteins of mice in each dose intervention group of cistanche phenylethanoid glycoside are obviously up-regulated, and the difference has statistical significance (p is less than 0.01) (see figure 6).
It is known from the intracerebral insulin signal transduction pathway shown in fig. 1 that AD intracerebral insulin receptor (InR), insulin-like growth factor 1 receptor (IGF-1R) and receptor bind to induce self-receptor phosphorylation, promote phosphorylation of Insulin Receptor Substrate (IRS) protein, regulate the growth and metabolism of downstream molecules, further make p85 regulate subunit phosphatidylinositol-3 hydroxykinase (PI3K) to stimulate glucose conversion, activate threonine protein kinase (AKT), inhibit downstream and apoptosis-related proteins, and allow neurons to survive, meanwhile, excessive accumulation of a β disrupts synaptic signal transmission, causing InR damage, causing neuronal insulin resistance and reduction of synaptic plasticity, which abnormalities may lead to damage of insulin-stimulating signals in neurons, inhibit the function of insulin in the central nervous system, and further allow neurodegenerative diseases to be accumulated, thus it is clear that the insulin signal pathway is an important neuroprotective signal pathway in vivo, which mainly stimulates signal-regulating protein expression, and its main signal-regulating pathway 3K can regulate the upstream and downstream signal transduction pathways of insulin, thus it is able to detect the key metabolic mechanisms of insulin, and to prevent and treat diseases caused by ethanol-induced AD diseases.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (7)
1. The cistanche phenylethanoid glycoside is characterized in that the cistanche phenylethanoid glycoside is obtained by extracting cistanche succulent stems, and the content (purity) of the cistanche phenylethanoid glycoside extract is 87.6-94.1%.
2. The extraction process of the cistanche phenylethanoid glycosides as claimed in claim 1, which is characterized by comprising the following steps:
(1) crushing and sieving the succulent stems of the cistanche, performing reflux extraction for 3-4 times by using 80% ethanol, combining filtrates, and performing reduced pressure concentration to obtain an extract;
(2) suspending the extract prepared in the step (1) with water, sequentially performing macroporous adsorption resin column chromatography and H2After gradient elution of O and 30-95% ethanol, collecting 30-50% ethanol elution fractions;
(3) concentrating the ethanol eluate of step (2) under reduced pressure, suspending with water, sequentially performing MCI column chromatography and H column chromatography2O-CH3After gradient elution of an OH system, collecting 30-50% of CH3An OH elution fraction section;
(4) and (4) concentrating the methanol elution fraction collected in the step (3) under reduced pressure to be thick paste, then suspending with water and performing ODS column chromatography to obtain the cistanche phenylethanoid glycoside.
3. The extraction process of phenylethanoid glycosides from cistanche as claimed in claim 2, wherein in the step (1), the volume ratio of 80% ethanol to the fleshy stems of cistanche is (3-4): 1.
4. the use of the cistanche phenylethanoid glycosides as claimed in claims 1-3, in the preparation of a medicament for treating alzheimer's disease, wherein the effective dose of the cistanche phenylethanoid glycosides in the medicament for treating alzheimer's disease is 62.5-250 mg/kg/d.
5. The application of the cistanche phenylethanoid glycoside in preparing a medicine for treating alzheimer disease according to claim 4, wherein the effective dose range of the cistanche phenylethanoid glycoside in the medicine for treating alzheimer disease is 125-250 mg/kg/d.
6. The use of the cistanche phenylethanoid glycoside according to claim 4 or 5 in the preparation of a medicament for treating alzheimer's disease, wherein the cistanche phenylethanoid glycoside acts by modulating an insulin signaling pathway target.
7. The use of the cistanche phenylethanoid glycosides in the preparation of a medicament for the treatment of alzheimer's disease as claimed in claim 6, wherein the targets include insulin receptor (InR), insulin-like growth factor 1 receptor (IGF-1R), Insulin Receptor Substrate (IRS), phosphatidylinositol-3 hydroxy kinase (PI3K), threonine protein kinase (AKT).
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Application publication date: 20200728 |