CN106176696A - Asaricin or the purposes in preparation promotes cell proliferation of nerve cord medicine of the material containing asaricin - Google Patents

Abstract

The invention belongs to Field of Drug Discovery, particularly relate to asaricin or the purposes in preparation promotes cell proliferation of nerve cord medicine of the material containing asaricin.Present invention firstly discovers that, oral Rhizoma Acori Graminei can promote that the hippocampal neural of wild-type mice, transgenic AD model mice and aged mice regenerates.In vitro and in vivo in experiment, Rhizoma Acori Graminei and active component asaricin thereof promote cell proliferation of nerve cord.Rhizoma Acori Graminei and asaricin activate ERK cascade reaction and do not affect Akt cascade reaction.The research of the present invention shows, oral Rhizoma Acori Graminei and asaricin can not only prevent, moreover it is possible to as a kind for the treatment of means promoting neuranagenesis, carry out the decrease of cognitive function relevant with neurodegenerative diseases to defying age.

Description

Asaricin or the purposes in preparation promotes cell proliferation of nerve cord medicine of the material containing asaricin

Technical field

The invention belongs to Field of Drug Discovery, particularly relate to asaricin or the purposes in preparation promotes cell proliferation of nerve cord medicine of the material containing asaricin.

Background technology

In mammalian brain, propagation and the self renewal of neural stem cell (neural progenitor cells, NPC) continue at whole life process, are the important steps of neuranagenesis (neurogenesis).At old and feeble, chronic stress and nervous system disease, in the case of such as alzheimer's disease (Alzheimer ' s disease, AD) occurs, the propagation of neural stem cell and self-renewal capacity decline, and cause cognitive impairment.Promote that neuranagenesis is considered as the potential treatment means of the old and feeble and old and feeble related neurodegenerative disease of opposing.Therefore, a kind of possible scheme is that transplanting embryo neural stem cell or external evoked neural stem cell are to carry out cell replacement therapy.But, the complex technology of this new wound there is also certain dispute at present, especially the problem such as their safety issue and cell derived.Another kind of scheme is to activate endogenous neural stem cell with pharmacological tool, to reach to treat the purpose of neurodegenerative diseases.Pharmacological tool is easy and simple to handle, and can be with the specific function of selectively targeted neural stem cell, therefore, activates endogenous neural stem and is not only a kind of feasible treatment means, moreover it is possible to as preventive means.

The propagation of neural stem cell is regulated and controled by many extracellular signals and intracellular signaling pathway.Under the stimulation of somatomedin, neurotrophic factor or other morphogenesis factors, the signal path of intracellular key, as ERK and Akt cascade reaction is activated.Research shows, the compound regulating and controlling these signal paths can promote cell proliferation of nerve cord and self renewal.Research in recent years finds, the natural product of some resources of Chinese medicinal herb promotes the propagation of neural stem cell, discloses the mechanism of these Chinese medicine nootropic effects.Chinese medicine Rhizoma Acori Graminei (AT) derives from the root of phytobezoar Rhizoma Acori Graminei (Acori tatarinowii), cures mainly the cerebral disorders such as senile dementia, forgetful, apoplexy, is used so far as " monarch " medicine in Chinese medicine compound.Modern pharmacology research shows, Rhizoma Acori Graminei has neuroprotective function, improves old and feeble, the forgetful and ability of learning and memory of cerebral ischemic model animal.But, whether and how Rhizoma Acori Graminei affects the propagation of neural stem cell and self renewal has no knowledge about.

Summary of the invention

In order to overcome defect of the prior art, the invention provides asaricin or the purposes in preparation promotes cell proliferation of nerve cord medicine of the material containing asaricin.

The present invention is achieved by the following technical solutions:

A first aspect of the present invention, it is provided that asaricin or the purposes in preparation promotes cell proliferation of nerve cord medicine of the material containing asaricin.

Preferably, described asaricin combination of any one or more in α-asaricin, beta-Asarone, γ-asaricin.

Preferably, described asaricin is one of active ingredient promoting cell proliferation of nerve cord medicine or unique active ingredient.

Further, described asaricin can be obtained by commercial approach, it is also possible to extracts from the material containing asaricin and obtains.

Preferably, the described material containing asaricin is Rhizoma Acori Graminei or Rhizoma Acori Graminei effective component extracts.

Further, described Rhizoma Acori Graminei derives from the root of phytobezoar Rhizoma Acori Graminei (Acori tatarinowii).Described Rhizoma Acori Graminei can be obtained by commercial approach.

Those skilled in the art can use various usual manner directly using Rhizoma Acori Graminei or Rhizoma Acori Graminei effective component extracts as the medicine being used for promoting cell proliferation of nerve cord or the raw material producing such medicine.

Further, described Rhizoma Acori Graminei effective component extracts can use following method to prepare: by Rhizoma Acori Graminei dried roots alcohol reflux, and after the extract obtained merges, evaporation and concentration obtains Rhizoma Acori Graminei effective component extracts.

Preferably, described ethanol is 60% ethanol.Described reflux, extract, is specially reflux, extract, 3 times, each 2h.

Preferably, Rhizoma Acori Graminei or Rhizoma Acori Graminei effective component extracts are one of the active ingredient of described promotion cell proliferation of nerve cord medicine or unique active ingredient.

Preferably, described promotion cell proliferation of nerve cord medicine is the medicine to defying age and neurodegenerative diseases.

A third aspect of the present invention, it is provided that a kind of for promoting the pharmaceutical preparation of cell proliferation of nerve cord, its main pharmacodynamics composition is asaricin, Rhizoma Acori Graminei or Rhizoma Acori Graminei effective component extracts.

Further, the present invention for promoting that the pharmaceutical preparation of cell proliferation of nerve cord also includes the pharmaceutically acceptable adjuvant of one or more routines.Principal agent active ingredient can be 1: 0.1～10 with the gross weight ratio of pharmaceutically acceptable adjuvant.

Pharmaceutically acceptable adjuvant includes (but being not limited to): pharmaceutically acceptable carrier, diluent, filler, bonding agent and other excipient.Treat inert inorganic or organic carrier known to the branch art personnel of this area and include, but is not limited to lactose, corn starch or derivatives thereof, Talcum, vegetable oil, wax, fat, many antelopes based compound such as Polyethylene Glycol, water, sucrose, ethanol, glycerol, like this, various preservative, lubricant, dispersant, correctives.Wetting agent, antioxidant, sweeting agent, coloring agent, stabilizer, salt, buffer are like this also can be added thereto, and these materials are adapted to assist in the stability of formula as required or are favorably improved activity or its biological effectiveness or produce acceptable mouthfeel or abnormal smells from the patient in the case of oral.

The pharmaceutical preparation of the present invention can be oral formulations, such as capsule, tablet, dispersible tablet, buccal tablet, chewable tablet, effervescent tablet, slow releasing tablet, granule etc..

The pharmaceutical preparation of the present invention can use conventional method to prepare, as each effective ingredient is mixed, or the routine fashion according to various preparations, active ingredient is prepared after corresponding adjuvant mixture.The pharmaceutical preparation of the present invention also can be used together with other therapeutic agents.

The pharmaceutical preparation of the present invention can be used for promoting cell proliferation of nerve cord.Preferably, the pharmaceutical preparation of the present invention can be used for defying age and neurodegenerative diseases.

The dose therapeutically effective of the pharmaceutical preparation of the present invention is considered as the factor such as route of administration, patient health situation, within the scope of these are all skilled practitioners technical ability.

The beneficial effects of the present invention is:

Present invention firstly discovers that, oral Rhizoma Acori Graminei can promote that the hippocampal neural of wild-type mice, transgenic AD model mice and aged mice regenerates.In vitro and in vivo in experiment, Rhizoma Acori Graminei and active component asaricin thereof promote cell proliferation of nerve cord.Rhizoma Acori Graminei and asaricin activate ERK cascade reaction and do not affect Akt cascade reaction.The research of the present invention shows, oral Rhizoma Acori Graminei and asaricin can not only prevent, moreover it is possible to as a kind for the treatment of means promoting neuranagenesis, carry out the decrease of cognitive function relevant with neurodegenerative diseases to defying age.

Accompanying drawing explanation

Fig. 1: AT promotes adult mice hippocampus neural stem cells propagation and neuranagenesis.(A) the experimental design diagram of detection cell proliferation of nerve cord.(B, C) mice is administered (B) control solvent (Ctrl) or (C) AT, scheme, as shown in figure (A), carries out immunofluorescence dyeing, detection BrdU (red) and DAPI (blue) to its Hippocampus DG district.Scale, 100um.(D) quantitative statistics of BrdU+ cell.Often organize N=5-6.(E, F) is administered by method detection (E) control solvent (Ctrl) of immunofluorescence or the GFAP (red) in mice DG district, Nestin (green) and the BrdU (Lycoperdon polymorphum Vitt) of (F) AT administration.Scale, 25um.(G) quantitative statistics GFAP+Nestin+BrdU+ cell number.Often organize N=5-6.The mice DG district that (H) control solvent (Ctrl) is administered by (H, I) or (I) AT is administered carries out Tbr2 (green), BrdU (red) and DAPI (blue) dye altogether.Scale, 100um.(J) quantitative statistics Tbr2+BrdU+ cell number.Often organize N=5-6.(K, L) is administered by method detection (K) control solvent (vehicle) of immunofluorescence or the Ki67 (red) in mice DG district, Dcx (green) and the DAPI (blue) of (L) AT administration.Scale, 100um.(M) quantitative statistics Dcx+Ki67-cell number.Often organize N=5-6.(N) neural stem cell differentiating experimental design diagram is detected.(O, P) mice is administered (O) control solvent (vehicle) or (P) AT, scheme, as shown in figure (N), carries out immunofluorescence dyeing, detection BrdU (red) and NeuN (green) to its Hippocampus DG district.Scale, 100um.(Q) quantitative statistics of BrdU+NeuN+ cell.Often organize N=7.(R) BrdU+NeuN+ cell accounts for the ratio of BrdU+ cell.Often organize N=7.The little figure in lower right is to shoot under high power lens, and scale is 10um.Quantitative result is expressed as meansigma methods ± standard error；* P ＜ 0.05, * * P ＜ 0.01, double tail t check.

Fig. 2: the AT impact that adult hippocampal neural is regenerated.(A) mice is administered solvent control (Ctrl) or AT 28 days, detects the volume of its cerebral granule cells layer.Often organize N=5-6.(B) quantitative statistics Ki67+ cell number.Often organize N=5-6.(C-E) with the Sox2 (green) and DAPI (blue) in the mice DG district that method detection (D) control solvent (Ctrl) is administered or (E) AT is administered of immunofluorescence；And carry out quantitative statistics (C).Scale, 100um.Often organize N=3.(F-G) with method (F) the detection Ki67 (red) in mice DG district, the Dcx (green) and DAPI (blue) of immunofluorescence；And quantitative statistics Dcx+Ki67+ cell number.Scale, 10um.(H) the double positive cell of quantitative statistics Dcx and TUNEL.Often organize N=3.(I) in the hippocampus of mice DG district section that AT is administered, c-Fos (blue), BrdU (red) and NeuN (green) are contaminated altogether.Scale, 10um.(J) experimental program is shown in Fig. 1 N.The section of mice DG district contaminates GFAP, NeuN and BrdU, and quantitative statistics GFAP+BrdU+ cell number altogether.N=4-5 often group.Quantitative result is expressed as meansigma methods ± standard error；* P ＜ 0.05, * * P ＜ 0.01, double tail t check.

Fig. 3: the AT hippocampal neural regeneration promoting Aged Mice and transgenic AD mice.(A, B) 18-23 month big Aged Mice oral administration (A) solvent control (Ctrl) and (B) AT 28 days, and injected BrdU at initial 7 days simultaneously.Experimental program is shown in Fig. 1 N.DG section carries out BrdU (red) and the dyeing of NeuN (green).(C) quantitative statistics BrdU+NeuN+ cell number.Often organize N=7.(D) BrdU+NeuN+ ratio in BrdU+ cell.Often organize N=7.(E-H) wild type (WT) and APP/PS1 Mouse oral are administered solvent control (Ctrl) and AT 17 days, and at last 7 days injection BrdU.DG section to mice carries out BrdU (red) and the dyeing of DAPI (blue).(I) quantitative statistics BrdU+ cell number.Often organize N=6-8.(J-M) in the DG brain sheet of solvent control (Ctrl) and the wild type of AT administration and APP/PS1 mice, Ki67 (red) and the dyeing of DAPI (blue) are carried out.(N) quantitative statistics Ki67+ cell number.Often organize N=6-8.(O-R) in the DG section of solvent control (Ctrl) and the wild type of AT administration and APP/PS1 mice, BrdU (red) and the dyeing of NeuN (green) are carried out.Experimental program is shown in Fig. 1 N.(S) quantitative statistics BrdU+ cell number.Often organize N=5.(T) quantitative statistics BrdU+NeuN+ cell number.Often organize N=5.(U) BrdU+NeuN+ ratio in BrdU+ cell.Quantitative result is expressed as meansigma methods ± standard error；* P ＜ 0.05, * * P ＜ 0.01, one-way ANOVA analyze.Scale, 100um.The little figure in lower right is to shoot under high power lens, and scale is 10um.

Fig. 4: external AT promotes cell proliferation of nerve cord.(A-C) adhere-wall culture adult hippocampus neural stem cells in the training liquid subtracting somatomedin (1ng/ml EGF and 1ng/ml bFGF), and process cell 24 hours with the AT of variable concentrations.Cell is fixed first 2 hours and is added EdU.The cell that DMSO (A, Ctrl) or 0.1mg/ml AT (B) processes carries out EdU (red) and DAPI (blue) dyeing.(C) ratio of quantitative statistics EdU+ cell.(D-F) adhere-wall culture adult hippocampus neural stem cells in without the training liquid of somatomedin, and process cell 14 hours with the AT of variable concentrations.Cell is fixed first 2 hours and is added EdU.The cell that DMSO (D, Ctrl) or 0.1mg/ml AT (E) processes carries out EdU (red) and DAPI (blue) dyeing.(F) ratio of quantitative statistics EdU+ cell.(G-I) adhere-wall culture embryo neural stem cells in the training liquid subtract somatomedin, and process cell 24 hours with the AT of variable concentrations.Cell is fixed first 2 hours and is added EdU.The cell that DMSO (G, Ctrl) or 0.1mg/ml AT (H) processes carries out EdU (red) and DAPI (blue) dyeing.(I) ratio of quantitative statistics EdU+ cell.(J-L) adhere-wall culture embryo neural stem cells in without the training liquid of somatomedin, and process cell 14 hours with the AT of variable concentrations.Cell is fixed first 2 hours and is added EdU.The cell that DMSO (J, Ctrl) or 0.1mg/ml AT (K) processes carries out EdU (red) and DAPI (blue) dyeing.(L) ratio of quantitative statistics EdU+ cell.Quantitative result is expressed as the meansigma methods ± standard error of 8 experiments；* P ＜ 0.05, * * P ＜ 0.01, * * * P ＜ 0.001, one-way ANOVA analyze.Scale, 100um.

Fig. 5: external AT is on neural stem cell differentiating impact.(A) adult neural stem cell expresses Nestin (green) and (Sox2).(B) embryo neural stem cells expresses Nestin (green) and (Sox2).(C), after adult hippocampus neural stem cells breaks up 5 days in vitro, cell expresses Tuj 1 (green, neuronal marker) or GFAP (red, star spongiocyte mark).(D) cell that quantitative statistics AT the processes ratio (left figure) of Tuj 1+ cell and ratio (right figure) of GFAP+ cell after differentiation 5 days.Quantitative result is expressed as the meansigma methods ± standard error of 5 experiments；One-way ANOVA analyzes.Scale, 100um.

Fig. 6: asaricin is the main active that AT promotes cell proliferation of nerve cord.(A) extraction of Rhizoma Acori Graminei composition and fractionation.(B) under the condition of culture subtracting somatomedin (1ng/ml EGF and 1ng/ml bFGF), the ratio of the EdU+ cell in the embryonic neural that 3 components process respectively.(C) the EdU+ cell proportion in the embryo neural stem cells of α-asaricin, beta-Asarone and 2 analog process.(D) ratio of the EdU+ cell in the adult hippocampal neurons that asaricin processes.(E) process adult hippocampus neural stem cells with DMSO (Ctrl), 1uM α-asaricin and 1uM beta-Asarone, and EdU (red) and DAPI (blue) is dyeed.(F) under the condition of culture without somatomedin, the ratio of the EdU+ cell in the adult hippocampal neurons that asaricin processes.(G) under the condition of culture without somatomedin (1ng/ml EGF and 1ng/ml bFGF), the ratio of the EdU+ cell in the adult hippocampal neurons that asaricin processes.Quantitative result is expressed as the meansigma methods ± standard error of 8 experiments；* P ＜ 0.05, * * P ＜ 0.01, * * * P ＜ 0.001, one-way ANOVA analyze.Scale, 100um.

Fig. 7: efficient liquid phase chromatographic analysis AT, ATE, ATB and ATW.β-asarone, tR=25.0min；α-asarone, tR=27.3min.

Fig. 8: asaricin is on the neural stem cell differentiating and impact of neurite outgrowth.(A) adhere-wall culture adult hippocampus neural stem cells in without the training liquid of somatomedin, and process cell 14 hours by the 3 of variable concentrations each components.Cell is fixed first 2 hours and is added EdU.EdU is dyeed, and the ratio of quantitative statistics EdU+ cell.N=8 independent experiment.(B) the EdU+ cell proportion under the condition of culture without somatomedin, in the embryo neural stem cells of α-asaricin, beta-Asarone and 2 analog process.N=8 independent experiment.(C) the adult hippocampal neurons that quantitative statistics asaricin the processes ratio (left figure) of Tuj 1+ cell and ratio (right figure) of GFAP+ cell after differentiation 5 days.(D-F) neuron of original cuiture processes with AT or asaricin the 3-7 days cultivated, and MAP2 is carried out immunofluorescence dyeing, detects neurite outgrowth.(D) cell processed with DMSO (Ctrl), 10ug/ml AT, 0.1uM α-asaricin or 0.1uM beta-Asarone, dyes to MAP2 (red) and DAPI (blue).(E) the synapse length of quantitative statistics MAP2+ neuron.N=4 independent experiment.(F) the average number of synapses of each cell of quantitative statistics.N=4 independent experiment.Quantitative result is expressed as meansigma methods ± standard error；* P ＜ 0.05, * * P ＜ 0.01, * * * P ＜ 0.001, one-way ANOVA analyze.Scale, 100um.

Fig. 9: AT and asaricin impact that neuroblastoma cell line is bred.In the training liquid of serum-free, process SH-SY5Y cell 2 days with different pharmaceutical, analyze cell proliferation with mtt assay.Quantitative result is expressed as meansigma methods ± standard error；* * P ＜ 0.001, one-way ANOVA analyze.

Figure 10: asaricin promotes adult hippocampus neural stem cells propagation and neuranagenesis.(A) detection cell proliferation of nerve cord experimental design diagram.(B-D) 8 weeks big mices are administered (B) control solvent (Ctrl), (C) α-asaricin or (D) beta-Asarone, scheme is as shown in figure (A), its Hippocampus DG district is carried out immunofluorescence dyeing, detection BrdU (red) and DAPI (blue).(E) quantitative statistics of BrdU+ cell.Often organize N=5-6.(F) neural stem cell differentiating experimental design diagram is detected.(G-I) mice is administered (G) control solvent (Ctrl), (H) α-asaricin or (I) beta-Asarone, scheme is as shown in figure (F), its Hippocampus DG district is carried out immunofluorescence dyeing, detection BrdU (red) and NeuN (green).(E) quantitative statistics of BrdU+NeuN+ cell.Often organize N=4-7.(K) BrdU+NeuN+ cell ratio in BrdU+ cell.Often organize N=4-7.(L-N) 18-23 month big mice is administered (L) control solvent (Ctrl), (M) α-asaricin or (N) beta-Asarone, scheme is as shown in figure (F), its Hippocampus DG district is carried out immunofluorescence dyeing, detection BrdU (red) and NeuN (green).(O) quantitative statistics of BrdU+NeuN+ cell.Often organize N=5-7.(P) BrdU+NeuN+ cell ratio in BrdU+ cell.Often organize N=5-7.Quantitative result is expressed as meansigma methods ± standard error；* P ＜ 0.05, * * * P ＜ 0.001, one-way ANOVA analyze.Scale, 100um.

The impact that adult hippocampal neural is regenerated by Figure 11: asaricin.(A-C) experimental program is as shown in Figure 10 A.(A) the DG brain sheet of control solvent (Ctrl), (B) α-asaricin and (C) beta-Asarone administration mice carries out immunofluorescence dyeing, detection GFAP (red), Nestin (green) and BrdU (Lycoperdon polymorphum Vitt).Scale, 25um.(D) quantitative statistics GFAP+Nestin+BrdU+ cell number.Often organize N=5-6.(E-G) the DG brain sheet of (E) control solvent (Ctrl), (F) α-asaricin and (G) beta-Asarone administration mice carries out immunofluorescence dyeing, detection BrdU (red), Tbr2 (green) and DAPI (blue).Scale, 100um.(H) quantitative statistics Tbr2+BrdU+ cell number.Often organize N=5-6.(I-K) the DG brain sheet of (I) control solvent (Ctrl), (J) α-asaricin and (K) beta-Asarone administration mice carries out immunofluorescence dyeing, detection Ki67 (red), Dcx (green) and DAPI (blue).Scale, 100um.(L-N) quantitative statistics (L) Ki67, (M) Dcx+Ki67+ and (N) Dcx+Ki67-cell number.Often organize N=5-6.(O) quantitative statistics solvent control (Ctrl) and asaricin are administered the Sox2+ cell number in mice DG district.Often organize N=3.(P) quantitative statistics solvent control (Ctrl) and asaricin are administered the Dcx+TUNEL+ cell number in mice DG district.Often organize N=3.(Q) in the hippocampus of mice DG district section that asaricin is administered, c-Fos (blue), BrdU (red) and NeuN (green) are contaminated altogether.Experimental program is shown in Fig. 5 F.Scale, 10um.(R) experimental program is shown in Fig. 5 F.Mice DG district contaminates GFAP, NeuN and BrdU, and quantitative statistics GFAP+BrdU+ cell number altogether.N=4-5 often group.(S-T) Aged Mice new object identification analysis.(S) mice is at the preference function of training period.(T) cognitive index of mice test phase after 1 day.Often organize N=5-8.Quantitative result is expressed as meansigma methods ± standard error；* P ＜ 0.05, * * P ＜ 0.01, one-way ANOVA analyze；#P ＜ 0.05, ##P ＜ 0.01, the monocyte sample t check analysis compared with 50% probability level.

Figure 12: AT and asaricin activate ERK cascade reaction and promote cell proliferation of nerve cord, but do not affect Akt cascade reaction.(A) under the condition (1ng/ml EGF and 1ng/ml bFGF) subtracting somatomedin, cultivate embryo neural stem cells 24 hours, be subsequently adding 1mg/ml AT, 1uM a-asaricin or 1uM b-asaricin and hatch certain time.With the phosphorylated CREB 1/2 and Akt in immunoblotting detection protein sample.The amount of total ERK and Akt is as the comparison of applied sample amount.4 independent experiments obtain the result being similar to.(B) in embryo neural stem cells, mek inhibitor U0126 (U is added；0.1uM), ERK inhibitor FR180204 (F；10uM), PI3K inhibitor LY294002 (L；10uM) or Akt inhibitor MK-2206 (M；10uM).After 30 minutes, add AT or asaricin hatches 5 minutes.With the phosphorylated CREB 1/2 in immunoblotting detection protein sample, total ERK, phosphorylation Akt and total Akt.4 independent experiments obtain the result being similar to.(C-E) under conditions of subtracting somatomedin, cultivate embryo neural stem cells 24 hours, in cell, add mek inhibitor U0126 (U；0.1uM), ERK inhibitor FR180204 (F；10uM), PI3K inhibitor LY294002 (L；10uM) or Akt inhibitor MK-2206 (M；10uM).After 30 minutes, add (C) AT, (D) a-asaricin or (E) b-asaricin and hatch 24 hours.The EdU of last 2 hours are mixed and dyes and quantitative statistics.(C-E) all experimental grouies in compare with the only experimental group added with AT, α-asaricin or beta-Asarone in this figure respectively.N=5 independent experiment.(F-G) Mouse oral is administered solvent control (Ctrl), AT or asaricin 5 days.Dui Qi DG district carries out immunofluorescence dyeing, the ERK1/2 (F, red) and Sox2 (F, green) of detection phosphorylation；And p-ERK+Sox2+ cell quantification is added up (G).Often organize N=4-6.Scale, 20um.Quantitative result is expressed as meansigma methods ± standard error；* P ＜ 0.05, * * P ＜ 0.01, * * * P ＜ 0.001, one-way ANOVA analyze.

Figure 13: AT and asaricin short-term administration promote hippocampus neural stem cells propagation.11 weeks big Mouse oral are administered AT or asaricin 5 days, are injected the cell of labelling propagation by BrdU.With the BrdU (red) in the mice DG district of immuno-fluorescence assay (A) solvent control (Ctrl), (B) AT, (C) α-asaricin and (D) beta-Asarone administration；And quantitative statistics BrdU+ cell number (E).Often organize N=5-7.Quantitative result is expressed as meansigma methods ± standard error；* P ＜ 0.05, * * P ＜ 0.01, one-way ANOVA analyze.Scale, 100um.

Detailed description of the invention

Below by way of specific instantiation, embodiments of the present invention being described, those skilled in the art can be understood further advantage and effect of the present invention easily by the content disclosed by this specification.The present invention can also be carried out by the most different detailed description of the invention or apply, and the every details in this specification can also carry out various modification or change based on different viewpoints and application under the spirit without departing from the present invention.

Before further describing the specific embodiment of the invention, it should be appreciated that protection scope of the present invention is not limited to following specific specific embodiments；It is also understood that the term used in the embodiment of the present invention is to describe specific specific embodiments rather than in order to limit the scope of the invention；In description of the invention and claims, unless additionally explicitly pointed out in literary composition, singulative " ", " one " and " this " include plural form.

When embodiment provides numerical range, it should be appreciated that unless the present invention is otherwise noted, between two end points and two end points of each numerical range, any one numerical value all can be selected for.Unless otherwise defined, the same meaning that all technology used in the present invention and scientific terminology are generally understood that with those skilled in the art of the present technique.In addition to the concrete grammar used in embodiment, equipment, material, according to those skilled in the art's grasp to prior art and the record of the present invention, it is also possible to use any method, equipment and the material of the prior art similar or equivalent with the method described in the embodiment of the present invention, equipment, material to realize the present invention.

Unless otherwise indicated, the experimental technique that disclosed in this invention, detection method, preparation method all use conventional molecular biology, biochemistry, chromatin Structure and the analysis of the art, analytical chemistry, cell to cultivate, recombinant DNA technology and the routine techniques of association area.These technology have improved explanation in existing document, specifically can be found in the MOLECULAR CLONING:A LABORATORY MANUAL such as Sambrook, Second edition, Cold Spring Harbor Laboratory Press, 1989and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley&Sons, New York, 1987and periodic updates；The series METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..

The preparation of embodiment 1 Rhizoma Acori Graminei extract and the separation of component

The present embodiment prepares Rhizoma Acori Graminei extract (AT), and carries out Quality Control by the method for high performance liquid chromatography.

60% alcohol reflux of Rhizoma Acori Graminei dried roots 70L of 19.5kg 3 times, each 2h.After the extract merging obtained, evaporation and concentration obtains the Rhizoma Acori Graminei total extract (AT) of 2.2kg, and preparation yield is 11.3% (w/w).AT is dissolved again with 10L distilled water, successively with ethyl acetate (10L, in triplicate) and n-butyl alcohol (10L, in triplicate) separate each component, organic solvent is removed subsequently with vacuum evaporation, the each component obtained is the component (ATE) that (1) is dissolved in ethyl acetate, 346.8g, yield 1.8%, (2) are dissolved in the component (ATB) of n-butyl alcohol, 127.4g, yield 0.7%, (3) residue is dissolved in the component (ATW) of water, 1.25kg, yield 6.4%.Compound composition and content UV passage on Waters 2695 chromatograph in each component detect, use Cosmosil chromatographic column (C18,5 μm, 250X 4.6mm), 35 DEG C, flow velocity 1.0ml/ minute, flowing is methanol (A phase) and water (B phase) mutually, 0-10 minute (10-55%, A), linear gradient is: 10-20 minute (55-60%, A), 20-40 minute (60-75%, A), 40-50 minute (75-100%, A), 50-65 minute (100%, A).

Embodiment 2 experimental technique

(1) mice is administered and BrdU injects

α-asaricin, beta-Asarone and the AT water dissolution containing 0.8% tween 80.In the administration experiment of 8 weeks size young mice, every mouse stomach is administered 100 μ l, administration concentration is respectively α-asaricin 10mg/kg Mouse Weight, beta-Asarone 30mg/kg Mouse Weight and AT 20g/kg Mouse Weight, contain the water of the tween 80 of 0.8% to 100 μ l referring additionally to group, once a day, it is administered continuously 28 days, in short-term administration experiment in 5 days, is then administered 5 days.In the experiment of detection neural stem cell proliferation in vivo, last be administered after the BrdU of point 3 lumbar injection 150mg/kg Mouse Weights, every time between be spaced 3h, the last time 2h after BrdU injection, by PFA perfusion after mouse anesthesia, take full brain and carry out next step experiment.In the experiment of detection neural stem cell differentiation in vivo, starting to be administered the BrdU of pneumoretroperitoneum injection 50mg/kg Mouse Weight from the 1st day, once a day, injection 7 days, by PFA perfusion after mouse anesthesia after being administered 28 days, take full brain and carry out next step experiment altogether.In the administration of AD mice is tested, reference group is all identical with young mice with AT group administration concentration, administration time is 17 days, in the experiment of detection neural stem cell proliferation in vivo, within 10th day, start to be administered the BrdU of pneumoretroperitoneum injection 50mg/kg Mouse Weight, once a day, injection 7 days altogether, in the experiment of detection neural stem cell differentiation in vivo, started to be administered the BrdU of pneumoretroperitoneum injection 50mg/kg Mouse Weight from the 1st day, once a day, injection 7 days altogether, both of which after being administered 24h last day by mouse anesthesia PFA perfusion, take full brain and carry out next step experiment.The experimental procedure of aged mice administration experiment in 20 months and 8 weeks size young mice detection neural stem cell differentiation in vivo of detection is identical.

(2) cell is cultivated

The separation reference of adult mice hippocampus neural stem cells, and make suitably modified.First the chloral hydrate of 8 weeks size mouse peritoneal injection 10% (w/v) is given, dosage is 5ml/kg Mouse Weight, 70% ethanol whole body spray disinfectant after anesthesia, cut off skull, take out full brain, be placed in the PBS of pre-cooling, remove cerebellum, press center line again to be cut by remainder, strip out left and right sides Hippocampus.Hippocampus is placed in PDD (2.5U/ml, the papain of 500 μ l preheatings；1U/ml, dispase；250U/ml, DNase) in, shred to the fragment less than 1mm3 with eye scissors, every mice adds 2ml Digestive system, blow and spread out even rear addition in 60mm Tissue Culture Dish, put into and digest 20 minutes on 37 DEG C of cell culture incubator shaking tables, sucking-off is to 15ml centrifuge tube together with not digesting fragment, adding 2.5ml NeuroCult NSC basal and train liquid, blow and beat 10 times with the 1ml rifle head cutting head, natural subsidence is after 30 seconds, supernatant adds 40 μm strainer filterings, the most in triplicate, train liquid flushing filtering net with 5ml the most again, merge the cell of all filtrations.Take part cell, mix with Trypan Blue dye 1:1, add blood cell counting plate counting (living cells that only meter Trypan Blue is negative).Enter 24 orifice plates with the plating cells density kind of 1X105/ml again, and add somatomedin (20ng/ml bFGF, 20ng/ml EGF, 2 μ g/ml heparin).The fresh training liquid adding 1/10 volume in every 2 days, after 6 days, the neural ball of major part can grow to more than 50 μm, now cell can be passed on, Accutase 37 DEG C digests 5 minutes, pass on the plating cells density of 1X105/ml, owing to firm isolated cell there being neurocyte etc. pollute, therefore passed on for 3 generations and above cell just can be tested.Unnecessary cell carries out frozen, 10%DMSO (v/v), 30%Serum Replacement (v/v).

Separating from the C57BL/6 mice embryonic of E12.5 of embryo neural stem cells, after taking out embryo, strip off skull, take the cortex of mice embryonic, digestion is cultivated, concrete steps are with adult hippocampus neural stem cells, and being used training liquid is the DMEM/F12 adding B27, wherein supplements 10ng/ml bFGF and 20ng/ml EGF.

(3) external EdU mixes experiment

nullNeural ball is after digesting with Accutase，It is resuspended in the NeuroCult NSC propagation training liquid in DMEM/F12 training liquid (embryo neural stem cells) containing 1ng/ml bFGF and 1ng/ml EGF and B27 or containing 1ng/ml bFGF and 1ng/ml EGF，It is inoculated into the cell density of 4X104/cm2 in 96 orifice plates being coated in advance that (PDL is coated overnight，After washing one time with water，It is coated overnight with laminin again)，After cultivating 24 hours，Add AT，α-asaricin and beta-Asarone，In the experiment having inhibitor，Add AT，α-asaricin，Before beta-Asarone 30 minutes with ERK and Akt signal pathway inhibitor (U0126，0.1μM；LY294002,1 μM；MK-2206,10 μMs；FR180204,10 μMs) pretreatment, hatch altogether after dosing 24 hours, added the EdU of 10 μMs at the 22nd hour, after hatching 2 hours, receive cell dyeing.

(4) EdU dyeing and analysis

Cell 4%PFA fixes 15 minutes, after PBS washes one time, then processes 15 minutes with the PBS punching containing 0.1%TritonX-100, PBS washes one time, PBS containing 4%BSA closes 30 minutes, and Nestin (1:100, the PBS containing 4%BSA) resists 4 DEG C of overnight incubation, after PBS washes one time, two anti-(1:1000, the PBS containing 4%BSA) incubated at room 1 hour, after PBS washes one time, add the EdU dyeing liquor prepared to dye 30 minutes, shown in formula table 1 below.

Table 1

After EdU dyeing, PBS washes one time, and DAPI (1:5000, PBS) dyes 5 minutes, and PBS washes twice, is detected and analyzed on operetta.

(5) neural stem cell vitro differentiation

Neural ball is after digesting with Accutase, it is resuspended in the NeuroCult NSC propagation training liquid containing 10ng/ml bFGF and 20ng/ml EGF, it is inoculated into the cell density of 2.5X104/cm2 in 96 orifice plates being coated in advance that (PDL is coated overnight, after washing one time with water, it is coated overnight with laminin again), after cultivating 24 hours, training liquid is changed into NeuroCult NSC differentiation training liquid, it is simultaneously introduced AT, α-asaricin, beta-Asarone, the most every other day change training liquid, after training liquid changes NeuroCult NSC differentiation training liquid into 5 days, the differentiation marker molecule of each pedigree is carried out staining analysis.

(6) immunoblot experiment

Neural ball is after digesting with Accutase, it is resuspended in the DMEM/F12 containing 1ng/ml bFGF and 1ng/ml EGF and B27 and trains liquid, then be inoculated into the cell density of 3X106/ml in 12 orifice plates being coated in advance, after cultivating 24 hours, add AT, α-asaricin, beta-Asarone, in the experiment having inhibitor, add AT, α-asaricin, before beta-Asarone 30 minutes with ERK and Akt signal pathway inhibitor (U0126,0.1M；LY294002,1M；MK-2206,10M；FR180204,10M) pretreatment, cell is put on ice by after dosing 5 minutes, twice is washed with the PBS of pre-cooling, adding 1XLB, 95 DEG C process 5 minutes, run SDS/PAGE glue, go on cellulose acetate film, with phospho-ERK, ERK, phospho-Akt, Akt mono-is anti-and IRDye800CW labelling two is anti-hatches, and carries analysis on software with the detection of Odyssey far infrared picture system and at it.

(7) immunofluorescence experiment

The mouse brain that PFA perfusion is crossed continues to fix 24 hours in 4%PFA, then stands 72 hours in 30% sucrose fixative.After being disposed, removing cerebellum part, be the most uprightly placed on filter paper by brain olfactory bulb ,-80 DEG C frozen at least 24 hours.With 30 μ m thick vertically longitudinal frozen section, taking DG part by mouse brain collection of illustrative plates, cut 8X 9X=72 altogether and open, altogether 2.16mm, arrange by section order, every 8 take 1, and compiling after merging is one group of brain sheet, dyes.The brain sheet cut is placed in organization protection's liquid (30% sucrose, 30% ethylene glycol, 0.1M PB), can preserve half a year more than at-20 DEG C.Taking one group of brain sheet during dyeing, in confining liquid (10% donkey serum, 0.3%TritonX-100, PBS), room temperature is closed 45 minutes, and addition one afterwards resists 4 DEG C of overnight incubation, and (an anti-dilution ratio is: big mouse-anti BrdU, 1:2000；The anti-Ki67 of rabbit, 1:1000；Goat-anti Dcx, 1:200；Mouse-anti NeuN, 1:200；Goat-anti Sox2,1:60), then with the suitable anti-incubated at room of fluorescence two 1 hour.DAPI is to nuclear targeting.With Olympus FV100i or Leica SP-8, the brain sheet of dyeing is taken pictures afterwards.The quantity of positive or double dye cell is by Image Pro Plus software analysis.For contaminating altogether with BrdU, needing to carry out the brain sheet of antigen retrieval, before serum is closed, in antigen retrieval buffers (10mM sodium citrate, pH 6.5), 95 DEG C process 20 minutes.BrdU dyes, and brain sheet, before serum is closed, processes 30 minutes at 37 DEG C with the hydrochloric acid of 2M, then cleans with the borate buffer (pH 8.5) of 0.1M.

(8) MTT experiment

SH-SY5Y being digested to unicellular, adds 96 orifice plates with the density in 4000/hole, every pore volume is 200 μ l.Dosing after cell attachment, 5 secondary orifices of every kind for the treatment of conditions.37 DEG C of cell culture incubators are cultivated 20 hours, every hole adds the MTT solution (5mg/ml) of 20 μ l, continue to cultivate 4 hours, terminate cultivating, carefully sucking culture fluid in hole, every hole adds 150 μ l DMSO, is placed in low speed on shaking table and shakes 10 minutes, make crystal fully dissolve, at enzyme-linked immunosorbent assay instrument OD 490nm, measure the light absorption value in each hole.Experiment needs arrange zeroing hole (not having cell, only train liquid, MTT and DMSO) simultaneously, and control wells (there is no medicine, only cell, the medicine dissolution medium of same concentrations, train liquid, MTT and DMSO).

(9) data statistic analysis

All experimental datas are expressed as mean+/-standard error.T inspection is used to compare between different disposal group.One-way ANOVA is used to be analyzed between many group results, and carry out post-hoc tests with Fisher ' s protected least significant difference test or Bonferroni t test, or use two-way ANOVA to be analyzed, and carry out post-hoc tests with Tukey post hoc test.P < thinks there is significant difference between group when 0.05.

Embodiment 3 Rhizoma Acori Graminei promotes adult mice cell proliferation of nerve cord

We give 8 weeks big C57BL/6 mices according to the oral dose AT of 200mg crude drug/20g body weight；Control group mice is then administered orally respective volume solvent (vehicle, in water dissolve 0.8%Tween-80), gastric infusion, every day 1 time, totally 28 days.In last day, inject 3 bromodeoxyribouridines (bromodeoxyuridine, BrdU) and carry out the cell that labelling is bred.Immunohistochemical analysis result shows, major part BrdU positive cell is positioned at the subgranular zone (subgranular zone, SGZ) that neural stem cell is concentrated.Compared with control group mice, oral AT makes the quantity of BrdU positive cell increase by 31% (as shown in Figure 1B-D).Dyeing of another propagation marker molecule Ki67 is also obtained similar result (such as Fig. 1 K, shown in L, Fig. 2 B).And AT the most substantially changes the volume (as shown in Figure 2 A) of granular cell layer.Research before shows, there is two kinds of adult neural stem cell in SGZ district, is radial neuroglia like cell (radial glia-like cells) and non-radioactive shape neural stem cell (nonradial neural progenitor cells) respectively.We detect the change of both cells further, find that oral AT promotes the quantity (as shown in Fig. 1 H-J) of the non-radioactive shape neural stem cell of the double positive propagation of Tbr2 and BrdU, and GFAP, Nestin and BrdU tri-the radial neuroglia like cell quantity of propagation of the positive the most substantially change.Additionally, the Sox2 positive cell number in SGZ district does not has significant change yet, show that oral AT does not affect the size (as shown in Fig. 2 C-E) in neural stem cell storehouse.We are further by the dyeing of Dcx, detection hippocampal dentate (dentate gyrus, DG) district neuroblast (neuroblast) and non-mature neuron.After oral AT, the double positive neuroblast of Dcx and Ki67 and the non-mature neuron that Dcx is positive, Ki67 is negative all dramatically increase (as shown in Fig. 1 K-M, Fig. 2-F).TUNEL experiment shows, the survival rate of Dcx positive cell is not affected (as illustrated in figure 2h) by AT.These results indicate that oral AT promotes adult hippocampus neural stem cells propagation.

In order to detect the newborn neuron produced by neural stem cell further, we were at initial 7 days of mice administration, every day, 1 injection BrdU carried out the cell of labelling propagation, behind 28 days away from injection BrdU for the first time, mouse brain detected (as shown in Fig. 1 N).We have detected mature neuron mark NeuN in BrdU positive cell, finds compared with control group mice, there is the double positive neuron (as shown in Fig. 1 O-Q) of more BrdU and NeuN in the mouse brain of oral AT.A portion neuron the most also expresses c-Fos, shows that these neurons are active (as shown in figure 2i).But, after oral AT, in BrdU positive cell, the ratio of the double positive cell of BrdU and NeuN the most substantially changes (as shown in Fig. 1 R), shows that AT has no effect on neural stem cell as the lineage (lineage commitment) in neuron direction.Further, it has been found that after oral AT, the quantity of the double positive cell of GFAP and BrdU does not has significant change yet, shows that colloid regeneration (gliogenesis) is not affected.

Embodiment 4 Rhizoma Acori Graminei promotes mouse aging and the neuranagenesis of transgenic AD mice

First, we give 18-23 month big Aged Mice injection BrdU for continuous 7 days, and oral administration AT or control solvent 28 days.Compared with the mice big with 8 weeks, the quantity of the double positive cell of BrdU and NeuN in these Aged Mice brains significantly reduces (as shown in Figure 3A；To shown in such as Fig. 1 O).What is interesting is, it has been found that oral AT dramatically increases the quantity of the double positive cell of BrdU and NeuN, and (～90%, as shown in figures 2 a-c), and the ratio of the double positive cell of BrdU and NeuN in BrdU positive cell is not changed in (as shown in Figure 3 D).These results indicate that oral AT can promote the neuranagenesis of Aged Mice.

Then, AT or control solvent are administered orally to 8-12 month big middle aged APP/PS1 transgenic mice and wild type litter control mice thereof, then it are injected BrdU.Compared with the mice big with 8 weeks, in the wild-type mice brain of 8-12 month, BrdU positive cell number significantly reduces (such as Fig. 3 E, shown in G, I), shows AD transgenic mice cell proliferation of nerve cord existing defects.It was found that oral AT makes the cell proliferation of nerve cord in wild-type mice brain increase by 36% (such as Fig. 3 E, shown in F, I), and in APP/PS1 mouse brain, amplification has reached 98% (such as Fig. 3 G, shown in H, I) especially.Dyeing to Ki67 displays that similar result (as shown in Fig. 3 J-N).We also have detected the neuranagenesis of the APP/PS1 mice of oral AT.To this end, we give APP/PS1 mice and their wild type litter control mice injection BrdU, and oral administration AT 28 days.We have found that compared with wild-type mice, the ratio of the double positive cell of the quantity of the BrdU positive cell in APP/PS1 mice DG district and wherein BrdU and NeuN is all remarkably decreased (such as Fig. 3 O, shown in Q, S-U).After APP/PS1 Mouse oral AT, the ratio of the double positive cell of the quantity of BrdU positive cell and wherein BrdU and NeuN the most substantially rises (as shown in Fig. 3 O-U), shows that oral AT promotes survival and the quantity of increase newborn neuron of regenerative cell.These results indicate that oral AT decreases AD model mice cell proliferation of nerve cord and the defect of neuranagenesis.

In vitro, Rhizoma Acori Graminei promotes cell proliferation of nerve cord to embodiment 5

In order to probe into whether AT directly acts on neural stem cell, we are separation and Culture neural stem cell from adult mouse Hippocampus.In cell, about 90% is the neural stem cell (as shown in Figure 5A) that Nestin and Sox2 is positive.The neural stem cell of these adhere-wall culture expands rapidly in the training liquid containing 20ng/ml EGF and 20ng/ml bFGF.There are some researches show, in the brain of aging or neurological, the decline of growth factor levels and the rising of tranquillization regulatory factor level are probably the reason causing neuranagenesis to reduce.Therefore, we decrease EGF and the bFGF concentration in neural stem cell training liquid, simulate this physiology or pathological conditions, and in this cell culture system, are mixed the impact detecting AT to cell proliferation of nerve cord by EdU.As shown in Figure 4 A, in the training liquid subtracting somatomedin containing 1ng/ml EGF and 1ng/ml bFGF, the cell that there are about 20% presents the EdU positive, and training in liquid without somatomedin without EGF and bFGF, this ratio drops to 1% (such as Fig. 4 A, D).In both cell culture systems, AT makes EdU mix in dose dependent increases (such as Fig. 4 A-F) (increase～30% under conditions of reducing somatomedin, increase～65% under conditions of without somatomedin).Similarly, AT also can promote the propagation (such as Fig. 5 B, Fig. 4 G-L) of the neural stem cell separated from cerebral cortex.

On the other hand, under conditions of differentiation, AT does not change that Tuj 1 is positive or the ratio (such as Fig. 5 C, D) of GFAP positive cell, shows that AT in vitro does not affect the lineage of neural stem cell.This phenomenon that it was experimentally observed that in vivo with us consistent (such as Fig. 1 R).

Embodiment 6 asaricin is the main active regulating and controlling neural stem cell in Rhizoma Acori Graminei

In order to find the active component in Rhizoma Acori Graminei further, we are divided into 3 each components (Fig. 6 A) AT.We detect embryo neural stem cells and are subtracting somatomedin and mixing (Fig. 6 B and Fig. 7 A) without the EdU under the conditions of somatomedin two kinds.The concentration of every kind of component is converted to the concentration of initial crude drug by us according to yield.As shown in Fig. 6 B and Fig. 7 A, ethyl acetate molten component (ATE) dosing is similar with AT dosing, it is possible in the ratio dose-dependently improving EdU positive cell.Compared with matched group, EdU levels of incorporation is then had not significant impact by n-butyl alcohol and soluble component, i.e. ATB and ATW.Therefore, the key component of cell proliferation of nerve cord is promoted during ATE is AT.

In order to find the main active in ATE further, We conducted efficient liquid phase chromatographic analysis (Fig. 7 A-D).ATE mainly has 2 chemical composition enrichments, accounts for the 20% of ATE gross mass, be beta-Asarone and α-asaricin (Fig. 7 B) respectively.Asaricin (Fig. 7 A) in AT, containing 3%.In ATB and ATW, only detect the asaricin (Fig. 7 C, D) of trace.

We have detected the asaricin impact on cell proliferation of nerve cord.As shown in Fig. 6 C and Fig. 7 B, α-asaricin and beta-Asarone can be in dose-dependently increasing EdU levels of incorporation.Then cell proliferation of nerve cord is had not significant impact on the contrary, process cell with 2 analog cis-methylisoeugenol or asarylaldehyde of asaricin in ATE.Further, asaricin also has the effect (Fig. 6 D-G) promoting propagation in dose dependent to adult hippocampus neural stem cells.In order to detect whether asaricin also has the similar effect promoting propagation to other cell, especially cancerous cell further, we have cultivated neuroblastoma cells SH-SY5Y under conditions of serum-free, and have processed with asaricin.MTT experiment result shows, asaricin is different from the effect of EGF and bFGF, and the propagation of SH-SY5Y is had not significant impact (Fig. 9), shows that asaricin may selectively promote cell proliferation of nerve cord.Further, compared with matched group, the cell proportion that produced by the neural stem cell after asaricin process, Tuj 1 is positive and GFAP is positive is constant, shows similar with AT, and the lineage of neural stem cell is had not significant impact (Fig. 8 C) by asaricin.In the primary neuron that newborn mice cerebral cortex and Hippocampus are originated, AT and asaricin are in dose-dependently increasing the length of nerve synapse, but their quantity is had not significant impact (Fig. 8 D-F).It is interesting that the optium concentration that asaricin promotes neurite outgrowth is 0.1-0.3 μM, promoting that than them the optimal dose (0.3-3 μM) of adult neural stem cell propagation is low, hint there may be different mechanism of action.These results indicate that α-asaricin and beta-Asarone are the main active that AT promotes cell proliferation of nerve cord in vitro.

Embodiment 7 asaricin in vivo promotes hippocampus neural stem cells propagation and neuranagenesis

In order to detect whether asaricin can promote as AT that adult hippocampal neural regenerates in vivo further, we give 8 weeks big C57BL/6 Mouse oral α-asaricins and beta-Asarone 28 days, and inject the cell (Figure 10 A) that BrdU labelling is bred.Compared with control group mice, the neural stem cell increasing number (Figure 10 B-E) of the propagation positive for mouse brain SGZ district BrdU that α-asaricin and beta-Asarone are administered.The staining analysis of Ki67 also presents similar result (Figure 11 I-L).Being administered similar with AT, asaricin is administered the propagation (Figure 11 E-H) mainly promoting non-radioactive shape neural stem cell positive for Tbr2, and the propagation of positive radial neuroglia like cell double to GFAP and Nestin has not significant impact (Figure 11 A-D).The size in the neural stem cell storehouse of Sox2 positive cell composition the most substantially changes (Figure 11 O).Asaricin is administered and adds the double positive neuroblast of Dcx and Ki67 and the Dcx positive, the quantity (Figure 11 I-K, M, N) of non-mature neuron negative for Ki67, and the survival to these two groups of cells has not significant impact (Figure 11 P).In order to detect the newborn neuron produced by neural stem cell further, we asaricin be administered first 7 days give injected in mice BrdU, take brain analysis (Figure 10 F) after 28 days.Immunohistochemical analysis finds, compared with matched group, has the double positive newborn neuron (Figure 10 G-J) of more BrdU and NeuN in the mouse brain that asaricin is administered.Wherein part newborn neuron expresses c-Fos simultaneously, shows that it is in functional activation state (Figure 11 Q).But, after asaricin is administered, NeuN and BrdU double positive cell ratio in BrdU positive cell does not change (Figure 10 K), shows that asaricin is similar with AT, does not affect the lineage in Neural Stem Cells direction.What is interesting is, it has been found that the double positive cell number of GFAP and BrdU too increases, show that asaricin also can promote that colloid regenerates (Figure 11 R).These results show that asaricin and AT promote the cell proliferation of nerve cord in adult mice Hippocampus, and further enhancing neuranagenesis.

We also have detected the neuranagenesis of the Aged Mice that asaricin is administered.To this end, we carry out labelling proliferative cell to 7 days BrdU of 18-23 month big injected in mice, and it is administered asaricin or control solvent 28 days.Similar to the result of AT, asaricin makes the quantity of the double positive cell of BrdU and NeuN add about 70% (Figure 10 L-O), but Neuronal lineage differentiation is not changed in (Figure 10 P).These tables of data detail octyl ethers promote the neuranagenesis of Aged Mice.

Along with the increase at age, cognitive function and neuranagenesis decline.For probing into AT and asaricin are administered whether to improve behavioristics's performance further, we have used new object identification experiment to detect cognition and memory relevant to Hippocampus in Aged Mice.During mice is trained, the mice of matched group, AT administration group and asaricin administration group shows equal preference function (Figure 11 S) to two same object.After 24 hours, new object is explored the probability level without departing from 50% by control group mice, shows that it cannot be distinguished by the object (Figure 11 T) that makes new advances.On the contrary, new object table is revealed more more Preference than familiar objects by the mice that AT and asaricin are administered, show that they also maintain the memory to object (Figure 11 T).These results indicate that AT and asaricin decrease the cognition and memory defect of Aged Mice.

Embodiment 8 Rhizoma Acori Graminei and asaricin activate ERK cascade reaction, but do not affect Akt cascade reaction

Research shows, the activation of ERK and/or Akt cascade reaction is the most crucial for cell proliferation of nerve cord and self renewal.Therefore, we detect the activation of these cascade reactions by the method for immunoblotting.As illustrated in fig. 12, after AT and asaricin dosing, intracellular ERK phosphorylation level increases, and at 2-10 minute to peaking, is then slowly declined to foundation level in 30 minutes.But, Akt phosphorylation level does not then have significant change (Figure 11 A).Further, the activation of ERK can be blocked by mek inhibitor U0126 and ERK inhibitor FR180204 and can not be blocked (Figure 11 B) by PI3K inhibitor LY294002 or Akt inhibitor MK-2206 by AT and asaricin.Whether the facilitation of cell proliferation of nerve cord depends on the activation of ERK cascade reaction in order to detect AT and asaricin further, and we process neural stem cell with U0126, FR180204, LY294002 or MK-2206.As shown in Figure 11 C-E, process cell with U0126 and FR180204 and can block AT and the effect of asaricin promotion cell proliferation of nerve cord, but LY294002 or MK-2206 then can not, show MEK/ERK cascade reaction, rather than PI3K/Akt cascade reaction is necessary to AT and asaricin promotion cell proliferation of nerve cord.The effect played in cell proliferation of nerve cord to further determine that internal ERK signal path to promote at asaricin, we give 11 weeks big Mouse oral administration AT or asaricin 5 days.Compared with control group mice, the number of the proliferative cell positive for mice SGZ district BrdU that AT and asaricin are administered adds 25%, shows that AT and asaricin have direct and quick facilitation (Figure 13 A-E) to cell proliferation of nerve cord.Meanwhile, we have carried out common dye in SGZ district to phosphorylated CREB and Sox2, find that the neural stem cell quantity of the phosphorylated CREB positive of AT and asaricin administration mice the most substantially increases (Figure 12 F-G).

Embodiment 9 tests brief summary

We have found that AT promotes the effect of cell proliferation of nerve cord in AD and aged mice body, it is better than its effect in young mice, this is probably and is caused by many factors: first AD and aged mice IC somatomedin such as EGF, FGF and BDNF etc. are less than young mice, therefore the effect that AT is administered easily shows in lower Background proliferation background, and the promotion proliferation function that AT is produced by growth factor signal path simultaneously also can emerge from；Secondly there is A β in AD and aged mice brain, the chronic inflammatory disease environment that tau and injured neuron etc. cause, and this environment serves the effect of inhibition for cell proliferation of nerve cord, report AT has the effect of certain suppression inflammatory reaction before, the most this inflammation that presses down acts on AT itself and promotes that the effect of cell proliferation of nerve cord is worked in coordination with mutually, has given play to the effect preferably promoting neuranagenesis on AD and aged mice.

Our result indicate that, the function of AT and asaricin is multiplicity, and this characteristic has the advantage of its uniqueness in treatment includes many virulence factors disease of AD.

MEK/ERK and PI3K/Akt signal path functionally has the most crucial effect in regulation neural stem cell.Activating MEK/ERK signal path and can promote the propagation of neural stem cell, and PI3K/Akt signal path is incessantly critically important to the propagation of neural stem cell, it also can regulate differentiation and the survival of neural stem cell simultaneously.Our result indicate that, the effect of AT and asaricin is by activating MEK/ERK signal path rather than PI3K/Akt signal path.It coincide with this result, the activation of suppression MEK or ERK, can suppress AT and asaricin for the effect of cell proliferation of nerve cord.On the other hand and, AT and asaricin process the differentiation for neural stem cell not to be affected, also confirmed AT and asaricin has not activated the phenomenon of PI3K/Akt signal path.Illustrate that asaricin has cell-specific to the facilitation of propagation.

We have found that AT and asaricin process the propagation that under the vitro culture conditions that micro-growth factor exists, can promote neural stem cell.And in conventional addition excess growth culture environment, AT and asaricin the most do not promote the effect of propagation.This finds that hint AT and asaricin may play its function promoting neuranagenesis under the physiology lacking somatomedin and pathological conditions.Match with these in vitro results, it is observed that AT promotes that the effect of neuranagenesis is also superior to young mice (8 weeks big) in middle age mice (8 months big).

In sum, we are not only found that AT and can promote the propagation of neural stem cell with active component asaricin therein, we also establish the Culture of neural stem cells system adding micro-growth factor simultaneously, may be used for the physiology in analogue body and pathology environment, provide new method to the medicine of screening treatment neurodegenerative diseases.

Above embodiment illustrates that embodiment disclosed by the invention, can not be interpreted as limitation of the present invention.Additionally, various amendments listed herein and invention in method, the change of compositions, be apparent from for those skilled in the art without departing from the scope and spirit in the present invention.Although the multiple particular preferred embodiment having combined the present invention has carried out concrete description to the present invention, it is to be understood that, the present invention should not be limited only to these specific embodiments.It is true that various as above for those skilled in the art obvious amendment obtain invention and be intended to be included within the scope of this invention.

Claims (10)

1. asaricin or the purposes in preparation promotes cell proliferation of nerve cord medicine of the material containing asaricin.

Purposes the most according to claim 1, it is characterised in that described asaricin is selected from α-asaricin, beta-Asarone, γ-thin The combination of any one or more in octyl ether.

Purposes the most according to claim 1, it is characterised in that asaricin is that the drug effect promoting cell proliferation of nerve cord medicine becomes / mono-or unique active ingredient.

Purposes the most according to claim 1, it is characterised in that the described material containing asaricin is Rhizoma Acori Graminei or Rhizoma Acori Graminei has Effect component extract.

Purposes the most according to claim 1, it is characterised in that Rhizoma Acori Graminei or Rhizoma Acori Graminei effective component extracts are described promotion One of active ingredient of cell proliferation of nerve cord medicine or unique active ingredient.

Purposes the most according to claim 1, it is characterised in that described promotion cell proliferation of nerve cord medicine be to defying age and The medicine of neurodegenerative diseases.

7., for promoting a pharmaceutical preparation for cell proliferation of nerve cord, its main pharmacodynamics composition is asaricin, Rhizoma Acori Graminei or stone Chang Pu effective component extracts.

Pharmaceutical preparation the most according to claim 7, it is characterised in that described pharmaceutical preparation also includes one or more routines Pharmaceutically acceptable adjuvant.

Pharmaceutical preparation the most according to claim 8, it is characterised in that described principal agent active ingredient and pharmaceutically acceptable adjuvant Gross weight ratio can be 1: 0.1～10.

Pharmaceutical preparation the most according to claim 7, it is characterised in that described pharmaceutical preparation is oral drug preparation.