CN102755341A - Application of acteoside in preparing medicine for treating alzheimer disease - Google Patents
Application of acteoside in preparing medicine for treating alzheimer disease Download PDFInfo
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Abstract
The invention relates to an application of acteoside in preparing a medicine for treating alzheimer disease. The acteoside is used for preventing the alzheimer disease by multi-target-point action. The acteoside is synthesized artificially or is obtained from plants. Verified by in vivo and vitro experiments, the acteoside has the advantages that the central cholinergic nervous system can be improved, the level of glutamic acid can be adjusted, the formation and deposition of beta amyloid protein (Abeta) can be inhibited, the action of neuroprotection is achieved, the hyperphosphorylation of tau protein is inhibited, the antioxidant action is achieved, the cerebral circulation is improved, and cholesterol can be reduced, so that the multi-target-point prevention and treatment of the alzheimer disease is realized.
Description
Technical field
The present invention relates to the purposes of the many target spot controls of acteoside Alzheimer, belong to the field of medicaments category.
Background technology
Alzheimer is a kind of constitutional grey matter encephalopathy.Be characterized in the cerebral cortex atrophy, and with neurofibrillary tangles and senile plaque, the course of disease is carries out sexual development, cause dementia at last, but the cause of disease is unclear.Vascular dementia is then relevant with cerebral hemorrhage, cerebral thrombosis, cerebral arteriosclerosis etc., and is at present, though clinical institute medicament can be improved some symptoms that cerebral hemorrhage or cerebral embolism etc. bring, all invalid to improving memory or dysnoesia.Estimate that according to the United Nations the old people will reach 5.9 hundred million, 2025 and increase to 1,100,000,000 after 10 years.AD is not only the maximum healthy killer of elderly population; Also become main one of disease that causes death of modern society old people; Serious threat Mental Health in human old age and quality of life; Bring white elephant and pressure for society, family, caused social common concern, become a very important social public health difficult problem.Therefore the research that is directed against the medicine of senile dementia has important medical and sociology meaning.
The pathogenesis of Alzheimer is not fully aware of as yet at present, mainly contains 3 kinds of hypothesis: amyloid-beta cascade hypothesis, Tau albumen hypothesis and neural blood vessel hypothesis.In addition, also have some other relevant theories, like cholinergic theory, gene mutation theory, neuronal apoptosis theory, dysimmunity theory, oxidative stress theory, inflammatory reaction theory etc., but the pathological phenomenon of all AD of annotation that every kind of theory all can not be satisfactory.In sum, the pathogenesis of Alzheimer is complicated unusually, relates to many signaling pathway molecules, comprises that A β, Tau albumen and synapse are unusual etc., connects each other between each mechanism, impels neuronal degeneration jointly.All to a certain target spot, clinical efficacy is limited for existing so far Therapeutic Method.
Acteoside is the representative composition of phenethyl alcohol glycosides; Have and regulate immunity, antiinflammatory, inhibition tumor growth, antiviral and pharmacological activity widely such as antibiotic; Acteoside also is one of active component of multiple medicinal plants; Thereby having received the common concern of Chinese scholars, the effect of its many target spots and the discussion of mechanism of action have become the hot subject of Recent study.At present; Discover that acteoside has the potential of anti-senile dementia; Like anti-apoptosis, suppress cell migration, slow down aging, antioxidation, the recovery of atherosclerosis, promotion function of nervous system; Improve memory, suppress elastin laminin enzyme activity and platelet aggregation, suppress the increase of Endothelin etc.Up to now, do not see the relevant report that many target treatments of acteoside Alzheimer purposes is arranged.
The present invention is directed to the characteristics of Alzheimer frequently-occurring disease mechanism,, confirm that finally acteoside has the purposes of many target spot control Alzheimer the correlational study that acteoside carries out the effect of many target spots.
Summary of the invention
The object of the invention is to provide the purposes of acteoside in preparation control Alzheimer medicine, and wherein acteoside is through synthetic or from plant, prepare.Show that through test the relevant pharmacology test of employing carries out the pharmacodynamics evaluation of acteoside treatment Alzheimer; From improving the cholinergic nerve of centrum system, regulate the glutamic acid level, suppress amyloid beta (A β) formation and deposition, neuroprotective, the unusual phosphorylation of inhibition Protein tau, antioxidation, improving cerebral circulation or cholesterol reducing aspect, illustrate the purposes of acteoside multipath, many target treatments Alzheimer respectively.
The purposes of a kind of acteoside of the present invention in preparation control Alzheimer medicine, such leaf cimicifugoside is through many target spots effect control Alzheimer.
The source of described acteoside is from plant, to prepare or obtain through the synthetic approach.Wherein from plant, preparing acteoside, is to adopt organic reagent extraction, macroporous resin or column chromatography for separation, polydextran gel purification or methods such as preparative hplc preparation or recrystallization from the plant that contains acteoside, to obtain.Through the synthetic approach be adopt cheap, be easy to get, hypotoxicity, oligosaprobic raw material, obtains through the methods such as conversion between esterification, functional group are synthetic.
Acteoside control Alzheimer of the present invention; Be through improving the cholinergic nerve of centrum system, regulate the glutamic acid level, suppress amyloid beta (A β) formation and deposition, neuroprotective, the unusual phosphorylation of inhibition Protein tau, antioxidation, improve approach such as cerebral circulation and cholesterol reducing, thereby realize many target spots control Alzheimer.Wherein said medicine is oral liquid, powder, tablet, capsule, granule, pill.
The structural formula of acteoside of the present invention is:
Acteoside is meant with dry product weight and calculates acteoside content at 90-99.9%.
The purposes of acteoside control Alzheimer of the present invention; Show that through test acteoside can improve the cholinergic nerve of centrum system, regulates the glutamic acid level, suppresses amyloid beta (A β) formation and deposition, neuroprotective, the unusual phosphorylation of inhibition Protein tau, antioxidation, improve cerebral circulation and cholesterol reducing effect, thereby reach the purpose of many target treatments of multipath Alzheimer.
Description of drawings
Fig. 1 observes figure (* 10) figure down for each treated animal cerebral tissue hippocampus mirror of the present invention.
Fig. 2 observes figure (* 40) figure down for each treated animal cerebral tissue hippocampus mirror of the present invention.
Fig. 3 is the influence figure of acteoside of the present invention to SK-N-SH morphocytology due to the OA, and wherein 1 is the OA model group, and 2 is 5 μ gmL
-1, 3 is 10 μ gmL
-1, 4 is 20 μ gmL
-1, 5 is the blank group.
Fig. 4 causes the protective effect figure of neurocyte skelemin phosphorylation to OA for acteoside of the present invention.
The specific embodiment
Following specific embodiment is further described the present invention, rather than restriction the present invention, should this be interpreted as that the range of application of the above-mentioned theme of the present invention only limits to following embodiment.
Embodiment
The purposes of a kind of acteoside of the present invention in preparation control Alzheimer medicine, such leaf cimicifugoside is through multipath, the effect of many target spots, concrete experiment is:
Acteoside is to the effect of APP695V717I transgene mouse model:
Experimental technique: get with the monthly age be the normal control group with background C57BL/6J mice, as negative control group, 4 monthly age APP695V717I transgenic mices are divided into model group and medication group with the negative mice of monthly age APP transgenic, continue raising 6 months to 10 monthly ages; Adopt Morris water maze and object identification experiment that the behavioristics of mice space learning memory ability is studied.Adopt radioimmunology, SABC method, immunoblotting detection Hippocampus and cortex synaptophysin, PDM-95, alpha-synapse nucleoprotein (α-syn; Another nucleus of senile plaque in patient's AD brain) expression; With mitochondrion, cytoskeletal protein, synapse number and synapse intracellular vesicle Change of Ultrastructure in neural 4 projections of transmission electron microscope observing Hippocampus; The two mark methods of immunofluorescence detect cortexes and hippocampal neuron α-syn respectively with A β, ubiquitin (Ub) and amyloid fiber co, parkin albumen respectively with and α-syn in ubiquitin ude co.
Experimental result: 4 monthly age APP transgenic mices obviously prolong the incubation period in the Morris water maze laboratory; Ultrastructure is normal basically with synaptic structure in the Hippocampus CA1 district neurite, but mitochondrion light in structure degree swelling and rupture in the projection with ridge and adventitia in the part, Synaptic connections decreased, number of synapses density has reduction trend, and synaptic vesicle obviously reduces; The synaptophysin of Hippocampus and cortex and PDM-95 (PSD-95) expressed has reduction trend; 4 monthly age transgenic mice cerebral hippocampus and cortex α-syn expresses obviously to be increased; 10 monthly age APP transgenic mices all obviously prolong with the swimming distance incubation period in the Morris water maze laboratory; Resolving index also obviously descends in the object identification experiment; Mitochondrial swelling in the Hippocampus CA1 district projection, ridge fracture even vacuolation, synapse quantity reduces, and presynaptic caudacoria and synaptic space structural fuzzy are unclear, the PDM attenuation, most synaptic vesiclees reduce even disappearance in the neuropil; The synaptophysin of cortex and Hippocampus CA1 district, CA3 district and dentate gyrus is expressed obviously and is reduced, and cortex and Hippocampus PSD-95 protein expression obviously reduce; α in 10 monthly age transgenic mice cerebral hippocampus and the cortex-syn expresses obviously to be increased; The two mark of immunofluorescence is results show, 10 the monthly age transgenic mice Hippocampus have co with amyloid fiber respectively with the interior α-syn of parietal lobe of cortex neuron, ubiquitin; There is co in parkin with α-syn and ubiquitin respectively, and prompting α-syn is unusual via Ubiquitin-Proteasome Pathway (UPP) degraded, assembles, and participates in amyloid plaques and forms.The acteoside gastric infusion of various dose can obviously shorten the incubation period and swimming distance of 10 monthly age APP transgenic mice Morris water mazes in 6 months; Improve its resolving index in the object identification experiment, alleviate the damage of model mice synapse, make mitochondrion clear-cut in the model mice Hippocampus CA1 district neuropil projection; Ridge is many and obvious; Synapse connects abundant, and structure is normal basically, presynaptic caudacoria and synaptic space clear in structure; Postsynaptic, fine and close thing thickened, and vesicle quantity increases; Increase APP transgenic models mouse cortex and Hippocampus synaptophysin and the proteic expression of PSD-95.Acteoside can suppress in 10 monthly age APP transgenic mice cortexes and the Hippocampus α-syn albumen and cross and express and assemble.Acteoside makes the key component in the main path-Ubiquitin-Proteasome Pathway of α-syn degraded: the expression of ubiquitin, parkin, UCH-L1, HSP70 all increases, and the prompting acteoside is through promoting α-syn degraded with the performance neuroprotective.
Mitochondrial respiratory chain complex IV inhibitor sodium azide is intended the effect of AD cell model:
Experimental technique: cultivate adherent SH-SY5Y cell and use 0.25% trypsinization, behind the cell counting with every hole 1 * 10
6Individual/ml is added on the cell plates, and overnight incubation is waited to be paved into and absorbed former culture medium behind the monolayer, after establishing blank, normal control, positive control respectively and adding variable concentrations test sample hole and SH-SY5Y cell preincubate 24h, discards culture fluid, adding 50mmolL
-1Sodium azide damage 4h measures each item index.
Experimental result: adopt mtt assay and lactic acid dehydrogenase (LDH) to spill the influence that the rate algoscopy is measured mitochondrial membrane potential; After the SH-SY5Y cell of sodium azide and cultivation is as a result hatched altogether; The cell mitochondrial cytochrome C oxidase is active to descend, and cell survival rate descends, and is time and dose dependent; The fluorescence intensity level of cell mitochondrial transmembrane potential descends, and especially cell process reduces the most remarkable.After giving the acteoside of various dose, degree of impairment is significantly improved.
Acteoside is to active oxygen hydrogen peroxide (H
2O
2) cause neural cell injury model influence:
Experimental technique: adopt the MEM culture medium, at 5%CO
2, cultivating people's neuroblastoma SH-SY5Y cell is 1 * 10 with the SYSY cell with density in 37 ℃ the incubator
5Ml
-1Be inoculated in the culture bottle, treat that cell grows at 90% o'clock, processes 2 * 105ml
-1Cell suspension, change serum-free medium, be inoculated in the culture dish, divide into groups (blank group, normal control group, positive controls and administration group), after the test sample that administration group cell adds variable concentrations is hatched 24h, inhale and go culture medium, PBS to wash 1 time, add H
2O
2(500 μ molL
-1) damage 18h, discard culture medium, process single-cell suspension liquid, carry out follow-up test.
Experimental result: adopt ELIASA and flow cytometer to detect the influence of SOD activity and the interior ROS of cell, NO and GSH content in the test sample pair cell, as a result concentration be 10,20,40mgkg
-1Acteoside make the release of active oxygen reduce by 40% (P＜0.05), 32% (P＜0.01) and 25% (P＜0.05) respectively; The release of NO has reduced by 29% (P＜0.01), 37% (P＜0.05) and 51% (P＜0.01) respectively; The acteoside of various dose has the effect that obvious inhibition GSH reduces simultaneously; Show that acteoside can suppress the SH-SY5Y cellular oxidation damage of hydrogen peroxide-induced, has neuroprotective.Adopt the Western-blot method through detecting the expression of caspase-3, caspase-9, Bcl-2 and Bax; Three dose groups of acteoside are compared with model group as a result; The content of caspase-3 has reduced 30% (P＜0.01), 89% (P＜0.01) and 95% (P＜0.01) respectively; The content of caspase-9 has reduced 77% (P＜0.01), 101% (P＜0.01) and 85% (P＜0.01) respectively; Bcl-2 has increased by 76% (P＜0.01), 117% (P＜0.01) and 94% (P＜0.01) respectively; To not influence of Bax, show that acteoside possibly suppress the neuronal apoptosis of hydrogen peroxide-induced through antioxidation, thereby play the effect of neuroprotective.
Acteoside causes the improvement effect of memory dysfunction to scopolamine:
Experimental technique: after the Kunming mouse adaptability raised a week, remove ineligible mice, comprising congenital stupid with clever especially through the test of mice diving tower; Then to 72 qualified mices of process behavioristics's experiment screening; Adopting PEMS 3.1 medical statistical softwares, is index with the body weight, is divided into 6 groups at random; Every group each 12, administration group orally give dosage is respectively 30,60,120mgkg
-1Acteoside and huperzine A sheet, blank group, model group give the normal saline of equivalent, before carrying out mice behavioristics experiment, except that the blank group, all the other respectively organize mice lumbar injection scopolamine 3mgkg respectively
-1, the lumbar injection volume is 0.1mL (10g)
-1, capacity normal saline such as blank group lumbar injection.Carry out mice step down test and water maze test by EE behind the 15min.
Experimental result: model group mice escape latency is starkly lower than blank group (P＜0.01); Huperzine A sheet positive controls can prolong mice escape latency (P＜0.05), but the equal significant prolongation mice escape latency (P＜0.01) of the basic, normal, high dose groups of acteoside; The basic, normal, high dose groups of acteoside all can make mice because the diving tower errors number reduction (P＜0.01) that ability of learning and memory descends and causes in addition.The prompting scopolamine has destroyed mice passive avoidance ability of learning and memory, and the acteoside damage of scopolamine to mice passive avoidance ability of learning and memory of can overturning.
Table 1 acteoside is to the influence (
n=12) of mice diving tower achievement
Annotate: compare * * P＜0.01 with the blank group; Compare #P＜0.05, ##P＜0.01 with the scopolamine group
The model group mice seeks platform and compares with the blank group incubation period; All in various degree prolongations of average latency; Significant difference (P＜0.05) was arranged from the 3rd day, and the prompting scopolamine can cause mice space identification ability to descend, and compares with model group; Acteoside administration group and positive drug group average latency all shorten than model group; Wherein, low, middle dosage of acteoside and huperzine A sheet positive controls all reached significant difference (P＜0.05) at the 4th, 5 day, and prompting low, middle dosage of acteoside and huperzine A sheet all can improve the mice space cognitive disorders that scopolamine causes; And the acteoside high dose group does not have significant difference, and curative effect is not obvious.Point out scopolamine to destroy mice space identification ability thus, the damage of scopolamine to mice space identification ability and acteoside can overturn.
Table 2 acteoside is to the influence (
n=12) of mice space identification ability
Annotate: compare * P＜0.05 with the blank group; Compare #P＜0.05 with the scopolamine group
To mouse brain histone Determination on content result, show that histone content decreases in the scopolamine model group mouse brain cortex, but compare no difference of science of statistics (P＞0.05) with the blank group.The mouse brain inner tissue protein content that gives acteoside prevention in advance raises, wherein low, the middle dose groups histone content (P＜0.05, P＜0.01) that can obviously raise.
Table 3 acteoside is to the influence (
n=12) of protein content in the mouse brain tissue
Annotate: compare * * P＜0.01 with the blank group; Compare #P＜0.05, ##P＜0.01 with the scopolamine group
Mouse brain is organized LPO Determination on content result; Show in the scopolamine model group mouse brain cortex LPO content and blank group significantly raise (P＜0.01); The interior LPO content of mouse brain that gives the acteoside prevention in advance descends; Wherein low, middle dose groups all can obviously reduce LPO level (P＜0.05), the not obvious difference (P＞0.05) but high dose is compared with model group.
Table 4 acteoside is to the influence (
n=12) of LPO content in the mouse brain tissue
Annotate: compare * * P＜0.01 with the blank group; Compare #P＜0.05, ##P＜0.01 with the scopolamine group
Mouse brain is organized the testing result of AChE vigor, show that AChE vigor and blank group significantly raise (P＜0.01) in the scopolamine model group mouse brain cortex, can point out scopolamine to cause one of amnemonic mechanism is the cholinergic damage.The interior AChE vigor of mouse brain that gives the acteoside prevention in advance reduces, and wherein basic, normal, high dose groups all can obviously reduce AChE level (P＜0.01).
Table 5 acteoside is to the influence (
n=12) of AChE vigor in the mouse brain tissue
Annotate: compare * * P＜0.01 with the blank group; Compare #P＜0.05, ##P＜0.01 with the scopolamine group.
Relevant oxidation index is measured the result, show that the SOD vigor relatively has significance decline (P＜0.05) with the blank group in the scopolamine model group mouse brain cortex, can point out scopolamine to cause one of amnemonic mechanism is oxidative damage.Give in advance that SOD is active in the mouse brain of acteoside prevention significantly raises, wherein the obvious increased SOD level (P＜0.05) of high dose group.GSH-Px vigor and blank group relatively have significance to descend (P＜0.05) in the scopolamine model group mouse brain cortex, and can point out scopolamine to cause one of amnemonic mechanism is oxidative damage.Give in advance that GSH-Px is active in the mouse brain of acteoside prevention significantly raises, the basic, normal, high dose groups GSH-Px level (P＜0.05, P＜0.01, P＜0.01) that all can obviously raise.
Table 6 acteoside is to the influence (
n=12) of SOD vigor in the mouse brain tissue
Annotate: compare * P＜0.05 with the blank group; Compare #P＜0.05 with the scopolamine group
Table 7 acteoside is to the influence (
n=12) of GSH-Px vigor in the mouse brain tissue
Annotate: compare * P＜0.05 with the blank group; Compare #P＜0.05, ##P＜0.01 with the scopolamine group
Mensuration result to mouse brain tissue acetylcholinesterase vigor; Show that ChAT vigor and blank group relatively have significance to reduce (P＜0.05) in the scopolamine model group mouse brain cortex, can point out scopolamine to cause one of amnemonic mechanism is the cholinergic damage.Give in advance that the ChAT vigor raises in the mouse brain of acteoside prevention, the wherein basic, normal, high dose groups ChAT level (P＜0.01) that all can obviously raise.
Table 8 acteoside is to the influence (
n=12) of ChAT vigor in the mouse brain tissue
Annotate: compare * P＜0.05 with the blank group; Compare #P＜0.05 with the scopolamine group
Mouse brain is organized the testing result of ACh vigor; Show that ACh vigor and blank group relatively have significance to reduce (P＜0.05) in the scopolamine model group mouse brain cortex, equally also can point out scopolamine to cause one of amnemonic mechanism is the cholinergic damage.Give in advance that the ACh vigor raises in the mouse brain of acteoside prevention, the wherein basic, normal, high dose groups ACh level (P＜0.01) that all can obviously raise.
Table 9 acteoside is to the influence (
n=12) of ACh vigor in the mouse brain tissue
Annotate: compare * P＜0.05 with the blank group; Compare #P＜0.05, ##P＜0.01 with the scopolamine group
Acteoside is organized the influence of ATPase vigor to mouse brain:
Na-K-ATPase, Ca in the scopolamine model group mouse brain cortex
2+-ATPase vigor and blank group relatively have significance to reduce (P＜0.05), give the interior Na-K-ATPase of mouse brain, the Ca of acteoside prevention in advance
2+-ATPase vigor raises, wherein low, middle dose groups all can obviously raise Na-K-ATPase, Ca
2+-ATPase level (P＜0.05).
Table 10 acteoside is to the influence (
n=12) of ATPase vigor in the mouse brain tissue
Annotate: compare * P＜0.05 with the blank group; Compare #P＜0.05, ##P＜0.01 with the scopolamine group.
Acteoside causes the experimentation of brain aging model to the D-galactose:
Experimental technique: after the Kunming mouse adaptability raised a week; Remove ineligible mice through mice diving tower test, comprising congenital stupid, then to through 72 qualified mices of behavioristics's experiment screening with clever especially; Adopt PEMS 3.1 medical statistical softwares; With the body weight is index, is divided into 6 groups at random, every group each 12.70 days laggard capable behavioristicss of successive administration are detected, and to the activity that mouse orbit is got blood sampling Detection mice serum SOD, intend the Model of Dementia success or not to confirm mice.D-galactose solution compound method: precision takes by weighing D-galactose 0.3g and is dissolved in the 20mL0.9% normal saline, shakes up it to be dissolved promptly fully get, and is existing with join at present.After the modeling success, basic, normal, high three dose groups of acteoside give respectively that concentration is 30,60,120mgkg
-1, huperzine A sheet concentration is 0.051mgkg
-1, successive administration 20 days.
Experimental result: observe through the mice ordinary circumstance, blank group mice chaeta is dense bright and clean as a result, and activity is normal, and reaction is quick, and action is rapid, honey stomach, and eyes have god, and are responsive to external irritant, are the attack state, and diet drinking-water is normal.Visible chaeta was sparse, lackluster after other respectively organized the mice modeling, arched in the back, and voluntary activity reduces, and is drowsiness, and the reaction action all becomes slow, and appetite is not prosperous, and eyes are narrowed little, and accidental gum is insensitive to external irritant, was to hide state, and diet drinking-water reduces.Treatment group mice state takes a turn for the better with comparing in the past to some extent, and diet drinking-water increases, and voluntary activity strengthens to some extent, makes moderate progress before the reaction action.
Mice passive avoidance property response learning memory ability test (test of mice diving tower) result; The model group mice goes up the leave from office escape latency and is starkly lower than blank group (P＜0.01); The electric shock errors number obviously increases (P＜0.01), and huperzine A sheet positive controls can prolong mice and go up leave from office escape latency (P＜0.05).But the basic, normal, high dose groups of acteoside all significant prolongation mice goes up leave from office escape latency (P＜0.01); The basic, normal, high dose groups of acteoside all can make mice because the diving tower errors number reduction (P＜0.01) that ability of learning and memory descends and causes in addition.Point out the D-galactose to destroy mice passive avoidance ability of learning and memory thus, the damage of D-galactose to mice passive avoidance ability of learning and memory and acteoside can overturn.
Table 14 acteoside is to the influence (
n=12) of mice diving tower achievement
Annotate: compare * * P＜0.01 with the blank group; Compare #P＜0.05, ##P＜0.01 with model group
Mice space identification ability test (Morris water maze test) results model group mice seeks platform and compares with the blank group incubation period; All in various degree prolongations of average latency; Significant difference (P＜0.05) was arranged in the time of the 5th day, and prompting D-galactose can cause mice space identification ability to descend.Compare with model group; Acteoside administration group and positive drug group average latency all shorten than model group; Wherein, low, middle dosage of acteoside and huperzine A sheet positive controls all reached significant difference (P＜0.05) at the 5th day, and prompting low, middle dosage of acteoside and huperzine A sheet all can improve the mice space cognitive disorders that the D-galactose causes; And the acteoside high dose group does not have significant difference, and curative effect is not obvious.Point out the D-galactose to destroy mice space identification ability thus, the damage of D-galactose to mice space identification ability and acteoside can overturn.
Table 15 acteoside is to the influence (
n=12) of mice space identification ability
Annotate: compare * P＜0.05 with the blank group; Compare #P＜0.05 with model group
Acteoside is compared with the blank group the result that influences of D-galactose model mice SOD vigor, and model group SOD vigor significance reduces (P＜0.05).Compare with model group, low, middle dose groups of acteoside and huperzine A positive drug group SOD vigor significantly raise (P＜0.05), and prompting low, the middle dosage of acteoside and huperzine A all can improve the vigor of mouse aging SOD due to the D-galactose.The acteoside high dose group all can significantly improve the SOD vigor except SOD vigor in ♀ mouse brain tissue does not have the remarkable rising, points out this maybe be relevant with ♀ mice hormone in vivo.
Table 16SOD measures result (
n=6)
Annotate: compare * P＜0.05, * * P＜0.01 with the blank group; Compare #P＜0.05, ##P＜0.01 with model group
Acteoside is to the result that influences of D-galactose model mice LPO content; D-galactose model group mice LPO content and blank group significantly raise (P＜0.01); Low, middle dose groups of acteoside and huperzine A positive drug group LPO content significantly reduce (P＜0.05), but acteoside high dose group obvious difference not in the mouse brain tissue.
Table 17LPO measures result (
n=6)
Annotate: compare * P＜0.05, * * P＜0.01 with the blank group; Compare #P＜0.05, ##P＜0.01 with model group
Acteoside is to the influence of D-galactose model mice GSH-Px vigor, and D-galactose model group mice GSH-Px vigor and blank group significantly reduce (P＜0.05) as a result, and can point out the D-galactose to cause one of old and feeble mechanism is oxidative damage.Basic, normal, high dose groups of acteoside and huperzine A positive drug group GSH-Px vigor be significantly rising (P＜0.05) all;
Table 18GSH-Px measures result (
n=6)
Annotate: compare * P＜0.05, * * P＜0.01 with the blank group; Compare #P＜0.05, ##P＜0.01 with model group
Acteoside is to the influence of D-galactose model mice AChE vigor, and D-galactose model group mice AChE vigor and blank group significantly raise (P＜0.05) as a result, and can point out the D-galactose to cause one of old and feeble mechanism possibly be the cholinergic damage.Basic, normal, high dose groups of acteoside and huperzine A positive drug group AChE vigor all significantly reduce (P＜0.05);
Table 19AChE measures result (
n=6)
Annotate: compare * P＜0.05, * * P＜0.01 with the blank group; Compare #P＜0.05, ##P＜0.01 with model group
Acteoside is to the influence of D-galactose model mice cerebral tissue ChAT vigor; D-galactose model group mice ChAT vigor and blank group significantly reduce (P＜0.05) as a result, and can point out the D-galactose to cause one of old and feeble mechanism possibly be the cholinergic damage.Basic, normal, high dose groups of acteoside and huperzine A positive drug group ChAT vigor be significantly rising (P＜0.05) all;
Table 20ChAT measures result (
n=6)
Annotate: compare * P＜0.05 with the blank group; Compare #P＜0.05 with model group
Acteoside is to the influence of D-galactose model mice cerebral tissue ATPase vigor; D-galactose model group mice Na-K-ATPase vigor and blank group relatively have significance to reduce (P＜0.05) as a result, low, middle dose groups of acteoside and huperzine A positive drug group Na-K-ATPase vigor rising (P＜0.05).
Table 21ATPase measures result (
n=6)
Annotate: compare * P＜0.05, * * P＜0.01 with the blank group; Compare #P＜0.05, ##P＜0.01 with model group
Acteoside is to the influence of D-galactose model mice cerebral tissue MAO vigor, and D-galactose model group mice MAO vigor and blank group significantly raise (P＜0.05) as a result, and can point out the D-galactose to cause one of old and feeble mechanism is oxidative damage.Basic, normal, high dose groups of acteoside and huperzine A positive drug group MAO vigor all significantly reduce (P＜0.05).
Table 22MAO measures result (
n=6)
Annotate: compare * P＜0.05 with the blank group; Compare #P＜0.05, ##P＜0.01 with model group
Acteoside is to the influence of mouse thymus index, index and spleen index, cerebral index, body weight gain rate; D-galactose model group mouse thymus index, index and spleen index, cerebral index, body weight gain rate and blank group significantly reduce (P＜0.05) as a result, and low, middle dose groups of acteoside and huperzine A positive drug group thymus index, index and spleen index, cerebral index, body weight gain rate be significantly rising (P＜0.05) all.
Table 23 acteoside is to the influence (
n=12) of mouse thymus index, index and spleen index, cerebral index, body weight gain rate
Annotate: compare * P＜0.05, * * P＜0.01 with the blank group; Compare #P＜0.05, ##P＜0.01 with model group
Acteoside is to the influence of mice hippocampal cell form, and the low power light microscopic is observed down as a result, and model group neurocyte band is obviously thinning dredges basic, normal, high dose groups of acteoside and the dense relatively (see figure 1) of positive drug group neurocyte band; Light microscopic is observed down, and a large amount of pycnotic cells appear in the pyramidal layer of the visible cerebral hippocampus district of model group tissue, and cell volume is little; Differ in size, fall into disarray, kernel is unintelligible; Karyopycnosis phenomenon in various degree also appears in PCL layer and cerebral cortex cell, and all the other cells show mild swelling; After the basic, normal, high dosage treatment of acteoside, the most of structure of visible low dose group pyramidal layer is clear, queueing discipline; Kernel is clear; Pycnotic cell quantity obviously reduces, in the above-mentioned change dose groups than low dose group still less, high dose group is then slightly heavy than low, middle dose groups; Huperzine A positive drug group pathological changes and the similar basically (see figure 2) of middle dose groups.
Acteoside is to the protective effect research of SK-N-SH cell injury due to the okadaic acid:
Experimental technique:
Acteoside is to the take the logarithm SK-N-SH cell of trophophase of the influence of SK-N-SH cel l proliferation, 1mL 0.25% trypsinization, adjustment cell concentration, 100uL hole
-1, spread 96 orifice plates, cultivate 3-4d, be divided into blank group and administration group for 37 ℃.The blank group adds the MEM complete medium, the 200uL hole
-1, normal cultured, each leu of administration group adds final concentration and is respectively 5,10,20,40,60,80 μ gmL
-1Acteoside, the 200uL hole
-1, 8 in multiple hole, temperature is cultivated 24h for 37 ℃, and every hole adds 5mgmL
-1MTT solution 20uL continues to cultivate 4h, discards culture fluid, adds DMSO 150uL hole
-1, the OD value (OD value) of mensuration 490nm wavelength on ELIASA, experiment repetition 3 times;
Cell survival rate (%)=(administration group OD/ normal group OD) * 100%.
The influence that OA grows to the SK-N-SH cell:
The take the logarithm SK-N-SH cell of trophophase, 1mL 0.25% trypsinization, adjustment cell concentration, 100uL hole
-1, spread 96 orifice plates, temperature is cultivated 3-4d for 37 ℃, is divided into blank group and OA group.Each leu of OA group adds that final concentration is respectively 5,10,20,40,60,80nML
-1OA, the 200uL hole
-1, 8 in multiple hole, temperature is cultivated 24h for 37 ℃, blank group normal cultured, Taking Pictures recording, every hole adds 5mgmL
-1MTT solution 20uL continues to cultivate 4h, discards culture fluid, adds the DMSO150uL hole
-1, the OD value (OD value) of mensuration 490nm wavelength on ELIASA, experiment repetition 3 times;
Cell survival rate (%)=(administration group OD/ normal group OD) * 100%.
The protective effect that acteoside descends to SK-N-SH cell survival rate due to the OA:
The take the logarithm SK-N-SH cell of trophophase, 1mL 0.25% trypsinization, adjustment cell concentration, 100uL hole
-1, spread 96 orifice plates, temperature is cultivated 3-4d for 37 ℃, is divided into normal control group and administration group, and the administration group adds 20nML
-1OA, the 200uL hole
-1, to inhale behind the cultivation 24h and abandon culture fluid, each leu adds final concentration 5,10,20,40,60,80 μ gmL respectively
-1Acteoside, the 200uL hole
-1, 8 in multiple hole, temperature is cultivated 24h, blank group normal cultured, Taking Pictures recording for 37 ℃.Every hole adds 5mgmL-1MTT solution 20uL, continues to cultivate 4h, discards culture fluid, adds DMSO 150uL hole
-1, the OD value (OD value) of mensuration 490nm wavelength on ELIASA, experiment repetition 3 times.
Cell survival rate (%)=(administration group OD/ normal group OD) * 100%.
Statistical procedures:
Adopt SPASS 13.0 statistics softwares that experimental data is carried out statistical analysis; Data result is with " mean ± standard deviation "
expression; Relatively adopt one factor analysis of variance and t check between group, there is statistical significance P＜0.05 for difference.
Experimental result
Acteoside to the influence of SK-N-SH cel l proliferation and blank group relatively, all growth plays a driving role to the SK-N-SH cell to give the acteoside of variable concentrations, and is dose-dependent relation, has significant difference (P＜0.05).Show growth unrestraint or the toxic action of acteoside to the SK-N-SH cell, on cell proliferation plays obvious facilitation.
Table 24 acteoside is to the influence (
n=8) of SK-N-SH cell growth
Annotate: compare * P＜0.05, * * P＜0.01 with the blank group
The influence that OA grows to the SK-N-SH cell: increase with OA concentration, cell survival rate also decreases, and is dose-dependent relation.Compare 10nML with the blank group
-1OA can make the SK-N-SH survival rate extremely significantly descend, but OA concentration too big (40,60,80nML
-1) cell survival rate is reduced, this moment, cell was dead mostly.Take all factors into consideration, 20nML-1OA makes the extremely significantly reduced while of cell survival rate, and dead cell quantity is less again, can be used for subsequent experimental and gets modeling dosage.
Table 25OA is to the influence (
n=8) of SK-N-SH cell growth
Annotate: compare * P＜0.05, * * P＜0.01 with the blank group
Acteoside is to the morphologic influence of SK-N-SH cell due to the OA: normal SK-N-SH cell outline level is clearly more demarcated, and it is stronger that the cell third dimension reaches refractivity by force, and synapse cell is obvious and more.And the cell outline level of model group that gives OA is relatively poor, the pericaryon rough surface, and synapse cell reduces, and cell space becomes circle.Give each dose groups of acteoside, it is for model group, and the third dimension of neuronal cell, refractivity strengthen; Cell process increases and is woven into netted; Endochylema endoparticle thing reduces, and the dead neurocyte of degenerating obviously reduces, and returns to normal level basically.The prompting acteoside has cytoprotection to a certain degree, and protection intensity has certain concentration dependent.See Fig. 3.
Acteoside is to the influence of SK-N-SH cell survival rate due to the OA: with model group relatively, the acteoside of variable concentrations all can make the survival rate of SK-N-SH cell significantly improve (P＜0.01).With the increase of acteoside concentration, cell survival rate also increases thereupon, is dose-dependent relation.Show that acteoside has protective effect to SK-N-SH cell due to the OA.
Table 26 acteoside is to the influence (
n=8) of SK-N-SH cell survival rate due to the OA
Annotate: compare * * P＜0.01 with the blank group; Compare ##P＜0.01 with model group
Acteoside causes the protective effect of neurocyte skelemin phosphorylation to OA: compare with the normal control group; Its tau of SK-N-SH cell (pSer-199/202) after OA induces and tau (pSer-404) protein expression obviously raise, and tau (paired 202) and tau (paired 404) expressing quantity decrease; After giving each dose groups effect of acteoside, the phosphorylation Protein tau obviously reduces in the expression in pSer-199/202 site, and the expression in the pSer-404 site also decreases; Each dose groups of acteoside all has the trend (see figure 4) that increases tau (paired 202) and the expression of tau (paired 404) site.
In vitro study shows that OA can make the excessive phosphorylation of tau; Show also that in body research OA can make A β deposition; Neuronal degeneration and decrease of synapses, these change all consistent with the pathology performance of AD, therefore; OA has critical role in this approach of research AD pathogenesis Protein tau phosphorylation; The SK-N-SH neurocyte is HNB's cell, has neuronic characteristics, and Protein tau content and protein kinase and phosphoprotein phosphatase content that the Protein tau phosphorylation is played regulating action are all than higher.
This experiment adopts OA to induce the SK-N-SH cell to set up the AD cell model; After give acteoside, inquire into it to the therapeutic effect that OA causes cell injury, show that mainly it can suppress the excessive phosphorylation of Protein tau; Keep the normal morphology of microtubule; Improve cell survival rate, reduce the seepage of cell LDH, the prompting acteoside is having good prospects for application aspect the treatment AD.
The effect research of acteoside cholesterol reducing:
Experimental technique: rat is divided into normal diet matched group and high cholesterol diet enteral feeding group at random, gets tail blood behind the 2wk that feeds, and measures serum cholesterol and triglyceride.With body weight and cholesterol value is foundation, high lipid food is fed Mus be divided into hyperlipidemia model group, various dose administration group, positive control drug group, continue to feed by preceding method, and the beginning gastric infusion, matched group and model group are irritated stomach with the volume normal saline.10 weeks back fasting 16h carries out subsequent detection then.
Experimental result: high cholesterol diet ability of learning and memory decline occurred after feeding; The object identification experimental result shows; The new and old object resolving index of rat obviously descends; Water maze laboratory is the result show, the rat escape latency obviously prolongs, and target quadrant swimming time accounts for the percentage ratio of total time obviously to be shortened.After the administration, find that acteoside can obviously shorten the escape latency of rat model and swim out of distance; Prolong the quadrant swimming time, a expression and A β content reduce in all visible brain of each administration group, and serum and cerebral tissue CHO and LDL-C content obviously reduce; WBV meter erythrocyte aggregation reduces.Show that acteoside can improve the learning memory disorder degree of rat model, reduce the generation of brain A β, reduce serum cholesterol, low-density lipoprotein cholesterol level, improve hemorheology.
Claims (1)
1. the purposes of an acteoside in preparation control Alzheimer medicine is characterized in that acteoside is through many target spots effect control Alzheimer.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103816168A (en) * | 2014-03-03 | 2014-05-28 | 新疆维吾尔自治区维吾尔医药研究所 | Application of acteoside in preparing medicine protecting cranial nerve cells damaged by ingestion and restraint of glutamate transporter |
| CN104147021A (en) * | 2014-07-28 | 2014-11-19 | 新疆维吾尔自治区维吾尔医药研究所 | Application of acteoside to preparation of drugs for preventing nerve cell injuries caused by neurotrophic factors (NTFs) and receptors thereof |
| CN105343114A (en) * | 2015-11-24 | 2016-02-24 | 上海中医药大学 | Medical application of verbascoside |
| CN106581019A (en) * | 2016-12-13 | 2017-04-26 | 新疆维吾尔自治区维吾尔医药研究所 | Application of acetoside in preparation of anti-anxiety drugs |
| CN108785318A (en) * | 2017-04-28 | 2018-11-13 | 江苏凯吉生物科技有限公司 | Purposes of the Phenylpropanoid Glycosides glycosides compound in preparing IDO inhibitor |
| CN110117301A (en) * | 2018-02-06 | 2019-08-13 | 上海东西智荟生物医药有限公司 | For preventing and treating noval chemical compound and its application of neurodegenerative disease |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103816168A (en) * | 2014-03-03 | 2014-05-28 | 新疆维吾尔自治区维吾尔医药研究所 | Application of acteoside in preparing medicine protecting cranial nerve cells damaged by ingestion and restraint of glutamate transporter |
| CN104147021A (en) * | 2014-07-28 | 2014-11-19 | 新疆维吾尔自治区维吾尔医药研究所 | Application of acteoside to preparation of drugs for preventing nerve cell injuries caused by neurotrophic factors (NTFs) and receptors thereof |
| CN105343114A (en) * | 2015-11-24 | 2016-02-24 | 上海中医药大学 | Medical application of verbascoside |
| CN106581019A (en) * | 2016-12-13 | 2017-04-26 | 新疆维吾尔自治区维吾尔医药研究所 | Application of acetoside in preparation of anti-anxiety drugs |
| CN108785318A (en) * | 2017-04-28 | 2018-11-13 | 江苏凯吉生物科技有限公司 | Purposes of the Phenylpropanoid Glycosides glycosides compound in preparing IDO inhibitor |
| CN110117301A (en) * | 2018-02-06 | 2019-08-13 | 上海东西智荟生物医药有限公司 | For preventing and treating noval chemical compound and its application of neurodegenerative disease |
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