CN101904838A - Application of ligustilide in preparing composition for preventing and treating senile dementia - Google Patents
Application of ligustilide in preparing composition for preventing and treating senile dementia Download PDFInfo
- Publication number
- CN101904838A CN101904838A CN2009100595054A CN200910059505A CN101904838A CN 101904838 A CN101904838 A CN 101904838A CN 2009100595054 A CN2009100595054 A CN 2009100595054A CN 200910059505 A CN200910059505 A CN 200910059505A CN 101904838 A CN101904838 A CN 101904838A
- Authority
- CN
- China
- Prior art keywords
- ligustilide
- application
- composition
- components
- senile dementia
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention relates to an application of ligustilide in preparing a composition for preventing and treating senile dementia, which comprises ligustilide as an effective component and other components acceptable pharmaceutically and/or acceptable in food or auxiliary components. As required, the effective components can comprise at least one of acetylcholinesterase inhibitor, excitatory amino acid and peptide transmitter components, Abeta fibration and sedimentation disturbance components, antioxidant, microcirculation improving components and calcium antagonist accounting for 10%-40% of the total weight. Shown by experimental results, through the protective actions of the ligustilide on damage caused by oxidation, apoptosis and inflammatory reaction induced by the Abeta amyloid peptide fragment and aging neuron degenerative change, the composition can obviously improve the descending of learning and memory caused by the Abeta amyloid peptide fragment and the aging neuron degenerative change, and can be used as a medicine and/or a health food composition.
Description
Technical field
The present invention relates to the novel medical use of a kind of ligustilide (Ligustilide), specifically is to be used for preparing the application that control senile dementia disease compositions comprises medicine and/or health edible composition.
Background technology
Senile dementia is serious threat senior health and fitness's a kind of commonly encountered diseases and a frequently-occurring disease, lasting comprehensively hypophrenia appears in the patient under the consciousness waking state, show as ability obstacles such as memory, understanding, calculating, judgement, orientation, language, abstract thinking, oneself's control, cause the patient to live on one's own life and the ability to work forfeiture.Senile dementia is one group of syndrome of the senior cerebral disorder that causes of the organic damage of chronic progressive external brain structure, and its cause of disease is not illustrated at present as yet fully.Studies show that: beta-amyloyd peptide (A β) is to constitute senile dementia patient cerebral nerve sexually transmitted disease (STD) to become speckle and cause the impaired main pathological characters of a series of function of nervous system in the amyloid deposition of brain memory and the formation of cognitive domain of dependence, and oxidative damage, inflammation and apoptotic response are the important pathogenesis that this disease generation develops.The main policies of treatment senile dementia is to improve cognitive dysfunction to improve patient's viability at present.The drug main that is used for the senile dementia treatment both at home and abroad will comprise that cholinergic preparation, excitatory amino acid and peptide class mediator (as Securan-11-one., Fructus Cnidii etc.), A beta form and sedimentary interfering material (as salvianolic acid B, emodin etc.), antioxidant (as pressure differential self, tea polyphenols, Semen Ginkgo extrac etc.), microcirculation improve material (as TANSHINONES and danshensu, ligustrazine and ferulic acid etc.), calcium antagonist (as Butylphthalide, berberine etc.) etc.But discover in a large number: existing medicine exists mostly that the long-term treatment effect is not good enough, untoward reaction is big, be difficult to see through blood brain barrier, be difficult for absorbing or the pathogenesis of senile dementia complexity is lacked the reasons such as intervention effect of specificity and integration, has a strong impact on the clinical prevention effect of these medicines to senile dementia.Reasons such as along with social population enters the process of aging, dementia patients increases year by year, and long, treatment of the prevalence of primary disease and disability rate height, the course of disease and nursing spending be big in addition all bring huge burden and influence for patient, family and society.Therefore, based on the latest developments of senile dementia morbidity Study on Molecular Mechanism in the world, the newtype drug that the many target spots of active research are intervened the senile dementia main pathogenesis is significant.
Summary of the invention
At above-mentioned situation, the present invention will provide a kind of compositions that the control of senile dementia disease is had remarkable result through experiment confirm.
The said compositions that the senile dementia disease is had preventive and therapeutic effect of the present invention, be as effective ingredient with ligustilide, forming jointly with other composition or the auxiliary element of one or more solids of acceptable or liquid form in pharmacy and/or the food, is as the medicine of control senile dementia disease and/or a kind of application in the auxiliary therapy compositions (comprising functional food) with known medicinal ingredient ligustilide.
Ligustilide (Ligustilide) is a kind of water-fast oily matter, and its structure is suc as formula shown in (I).
Ligustilide at present can be by the conventional Chinese medicine that contains this composition, obtain as extracting in the plants such as Radix Angelicae Sinensis, Rhizoma Chuanxiong, Cnidium officinale, also can obtain by the synthesis mode artificial preparation, as Chinese patent literatures such as publication number CN 1552702A, CN 1084851A and CN 1778799A, and other pertinent literature (as: Hu Changying, the extraction of ligustilide in the Radix Angelicae Sinensis such as fourth night continuous heavy rain, separation and structure are identified) at " Wuxi is through worker's college journal " 2003, report among the 9:69 all has synthesis mode to prepare the relevant report of ligustilide.Because ligustilide all exists in many natural plants/medicines, and mostly be the cis-structure form, therefore in the said compositions of the present invention, the ligustilide that uses as effective ingredient, more favourable with the originate ligustilide of the natural existence form of horn of plenty more of employing
Present research and application to Ligustilide, mostly be at its effect and effect at treating cardiac and cerebral vascular diseases, all related to the medicine of ligustilide as publication number CN 1732921A, Chinese patent literatures such as CN 1748676A, CN1857534A, reported among the CN1543859A ligustilide is used to prevent and treat atherosclerotic purposes etc. as cerebral ischemia diseases such as prevention and treatment ischemic brain infarctions.
The application mode of the said compositions of the present invention can be used as the suitable administration form of senile dementia disease medicine use and the pharmaceutical composition of dosage form.Because the ligustilide that just will be have at present used as ingredient that the present invention relates to is in the application mode of expanding aspect this frontier of control senile dementia disease, therefore as drug use the time, at present the various dosage forms and/or the application mode of the ligustilide that uses as ingredient equally all are applicable to the said compositions of the present invention.For example, ligustilide is oily liquids and instability, and according to above-mentioned using dosage needs, the technical staff of pharmaceutical field is by selecting suitable adjunct ingredient to make to meet the various oral formulations of medicine and health product stability requirement.For example, can be prepared into soft capsule preparation according to the mode of publication number CN1732922A report; Or after carrying out enclose according to the mode of CN1732923A report by cyclodextrin or cyclodextrin derivative, further be prepared into the multiple operational medicament forms of injection-type preparations such as comprising multiple oral type preparation and transfusion, liquid drugs injection, powder pin.
Except that as the medicine, above-mentioned composition of the present invention also can be as partner treatment or the health edible composition that uses as prevention senile dementia disease.
Above-mentioned composition as medicine and/or health edible composition, for further improving its prevention effect to the senile dementia disease, on the basis of above-mentioned said active drug composition ligustilide, can also be at present known Chinese medicine monomer and other effective ingredient that the senile dementia disease is had definite curative effect effect, comprise as acetylcholinesteraseinhibitors inhibitors (as: huperzine compounds etc.), excitatory amino acid and peptide class mediator composition (as: Securan-11-one., Fructus Cnidii etc.), the A beta forms and sedimentary interference component (as: salvianolic acid B, emodin etc.), antioxidant composition (as: pressure differential self, tea polyphenols, Semen Ginkgo extrac etc.), microcirculation improves composition (as: TANSHINONES and danshensu, ligustrazine and ferulic acid etc.), calcium antagonist (as: Butylphthalide, berberine etc.) etc. in the composition, as required, select wherein one or more to occupy the mode of imitating composition gross weight 10%～40%, as complementary effective ingredient, the effective ingredient of forming the compound recipe form uses.
Above-mentioned composition of the present invention is when being used for prevention and treatment senile dementia disease, to determining of the concrete consumption of effective ingredient ligustilide (dosage), need depend on multiple factor, the order of severity that comprises age of using object, body weight, individual variation, disease, and the individuation embodiment on the corresponding clinical treatment etc., therefore can adjust according to the treatment and the needs of reality.Test shows, suitable consumption (dosage) scope of every day can be in 0.1～500mg/kg weight range generally speaking, can adopt once a day property or is divided into and repeatedly waits the different modes use according to the embodiment of clinical treatment.Except that taking separately the above-mentioned compositions, also can merge and use, and in reasonable range, adjust consumption with the other treatment medicine.
Experiment in vitro confirms; adopt A β starch inducing peptide human neuroblastoma strain SH-SY5Y damage; oxidation, apoptosis and inflammatory reaction index of correlation have been measured; damage has protective effect to ligustilide to the human neuroblastoma strain SH-SY5Y of A β starch inducing peptide, and relevant with antiinflammatory action with the antioxidation of ligustilide, anti-apoptosis.
In testing in the body, at first cause dysmnesia model by setting up the three-dimensional locating injection A of Wistar rat bilateral tricorn brain β starch peptide, with of the influence of Morris determined with Morris water ligustilide to nearly memory of animal and space function, the result show ligustilide to dementia rats memory and spatial orientation power, closely remember and the locus functional impairment improves significantly; Simultaneously, the pathology result of this model shows that also the three-dimensional locating injection A of brain β starch peptide causes dull-witted rat prefrontal lobe district's floor and Hippocampus CA1 district neuronal cell disappearance obviously, and obvious karyopycnosis phenomenon, dense dying are arranged; The hippocampal neuron number reduces to some extent, and deficient phenomena is obvious.After the ligustilide treatment, obviously improve the damage of rat prefrontal lobe cortical neuron, make the neuronal cell form normal substantially; The Hippocampal Neuron Cells degree of impairment obviously improves, and cellular morphology is normal substantially, and marshalling does not have obvious karyopycnosis phenomenon, illustrates that ligustilide has preventive and therapeutic effect for the neuron degeneration that A β starch peptide causes.
Because the senile dementia disease is that a class is the brain degenerative disease of main feature with carrying out property hypomnesis, and the closest with the growth relation at age, thereby be one of common disease of aged crowd.In addition, also because the pathogenesis complexity of senile dementia disease, comprise that mainly inherited genetic factors is (as APP, apoE, Ps1, gene mutation such as Ps2), the cytoskeleton that the Protein tau of unusual excessive phosphorylation causes changes, neurotransmitter metabolism obstacles such as the interior acetylcholine of brain, starch precursor protein Metabolic disorder causes the formation of extracellular senile plaque, the intracellular Ca2+ metabolism disorder, the immune inflammation reaction, pathogenesis such as radical damage theory and neuronal apoptosis, therefore, only cause the senile dementia rat model still to be not enough to simulate fully and prove the process of clinical senile dementia disease with regard to above-mentioned A β starch peptide.
Cultivate success heritability quick aging mice (SAM) by the bamboo field professor of Kyoto Univ Japan, the R that comprises SAMP and anti-quick aging is on the basis of (SAMR), wherein the SAMP8 subbreed has typical dull-witted feature and brain pathological change, this animal maincenter learning and memory function fails fast with carrying out property of age growth, it is the heritability animal model of present research senile dementia of generally acknowledging both at home and abroad, SAMR1 then shows as usual aging, can be used as the normal control of SAMP8.Therefore proving that ligustilide has on the basis of improvement effect the dysmnesia model that A β starch peptide causes, the present invention further uses the dull-witted SAMP8 mouse model of quick aging again, adopts the experiment of Y type labyrinth and diving tower behavioristics to detect the influence of ligustilide to the animal learning memory function.These further go deep into result of the test and can show fully that ligustilide improves significantly to the dementia mice damage in learning and memory, have further confirmed the preventive and therapeutic effect that ligustilide has the senile dementia disease.
Below, foregoing of the present invention is described in further detail again by the specific embodiment by the accompanying drawing illustrated embodiment.But this should be interpreted as that the scope of the above-mentioned theme of the present invention only limits to following example.Do not breaking away under the above-mentioned technological thought situation of the present invention, various replacements or change according to ordinary skill knowledge and customary means are made all should comprise within the scope of the invention.
Description of drawings
Fig. 1 protects the figure as a result of the SH-SY5Y cell injury of A β starch inducing peptide for showing ligustilide.
Fig. 2 suppresses the figure as a result that ROS produces in the A β starch inducing peptide SH-SY5Y cell for showing ligustilide.
Fig. 3 is for showing the as a result figure of ligustilide to the influence of A β starch inducing peptide SH-SY5Y cell mitochondrial approach expression of apoptosis protein.
Fig. 4 is for showing the SH-SY5Y cellular expression inflammatory associated protein TNF-α of ligustilide to A β starch inducing peptide, the figure as a result of the influence of COX-2.
Fig. 5 is for showing the as a result figure A (n=10) of ligustilide to the influence of the three-dimensional locating injection A of brain β starch peptide rat water maze learning and memory.
Fig. 6 is for showing the as a result figure B (n=10) of ligustilide to the influence of the three-dimensional locating injection A of brain β starch peptide rat water maze learning and memory.
Fig. 7 is for showing the as a result figure C (n=10) of ligustilide to the influence of the three-dimensional locating injection A of brain β starch peptide rat water maze learning and memory.
Fig. 8 is for showing the as a result figure D (n=10) of ligustilide to the influence of the three-dimensional locating injection A of brain β starch peptide rat water maze learning and memory.
Fig. 9 is for showing the as a result figure of ligustilide to the neuronal cell influence in three-dimensional locating injection A β starch peptide rat brain prefrontal lobe cortical area of brain and Hippocampus CA1 district.
Figure 10 is for showing the as a result figure of ligustilide to the influence of the three-dimensional locating injection A of brain β starch peptide rat brain prefrontal lobe cortical area paraprotein APP, A β and Tau expression.
Figure 11 is for showing the as a result figure of ligustilide to the influence of the three-dimensional locating injection A of brain β starch peptide rat hippocampus CA1 district paraprotein APP, A β and Tau expression.
Figure 12 is for showing the as a result figure of ligustilide to the influence of the three-dimensional locating injection A β starch peptide rat brain prefrontal lobe cortical area of brain and Hippocampus CA1 district inflammatory protein TNF-α and NF-κ B expression.
Figure 13 is for showing the as a result figure A (n=12) of ligustilide to the handicapped influence of the quick aging learning and memory of little mouse of SAMP8.
Figure 14 is for showing the as a result figure C (n=12) of ligustilide to the handicapped influence of the quick aging learning and memory of little mouse of SAMP8.
Wherein, the relevant block diagram acceptance of the bid implication that is marked with * or * * is: * represents with model group significant difference P＜0.05 is arranged relatively; * represents with model group significant differences P＜0.01 is arranged relatively.
The specific embodiment
Embodiment 1
Ligustilide (Ligustilide, LIG) the SH-SY5Y cell injury of protection A β starch inducing peptide
Cell culture: human neuroblastoma cell SH-SY5Y cell strain is available from U.S. ATCC.The SH-SY5Y cell is with high sugared DMEM complete medium (containing 10% (V/V) FBS, 1% (V/V) NEAA, 100U/ml penicillin, 100 μ g/ml streptomycins), at 37 ℃, and 5%CO
2, cultivate in the cell culture incubator of saturated humidity.Changed a subculture in every 2-3 days, went down to posterity once in 4-6 days.Take the logarithm the trophophase cell inoculation in culture plate, be used for different experiments.
Reagent and medicine:
N-2supplement U.S. Gibco company
Non essential amino acid (NEAA) U.S. Sigma company
A β (25-35) U.S. Sigma company
TNF-α polyclonal antibody U.S. CST company
Cox2 polyclonal antibody U.S. CST company
All the other reagent are commercially available analytical pure.
Instrument:
ESCO two stage biological safety cabinet Singapore ESCO Science and Technology Ltd.
Leica DMIL HC fluorescence inverted microscope Germany Lycra Instr Ltd.
SHELLAB 2323-2CO
2Cell culture incubator U.S. Shellab company
ELGA PureLab Ultra/Classic pure water system Britain ELGA LabWater company
Grouping and administration:
1. blank group: only cultivate the SH-SY5Y cell, do not add A β (25-35) and LIG and intervene with the DMEM complete medium that contains 0.1%DMSO.
2. A β (25-35) damage group: act on the SH-SY5Y cell with A β (25-35).
3. after LIG intervention group: LIG is hatched the SH-SY5Y cell, give A β (25-35) again, according to Concentraton gradient, the LIG intervention group is divided into four concentration groups of 5.0,2.5,1.0,0.1 μ g/ml again.
The preparation of A β (25-35) solution:
With sterile distilled water preparation 1mM/l A β (25-35) solution, after PBS was diluted to variable concentrations, the reference literature method was standby after aging 14 days in 37 ℃.
Cell viability detects:
Detect cell survival rate with mtt assay.The take the logarithm SH-SY5Y cell of trophophase is made into unicellular solution with the DMEM complete medium, with 1.2 * 10
4Individual cell/cm
2The density of cell suspension is inoculated in the 96 porocyte culture plates, and every hole 100 μ l put into 37 ℃ with postvaccinal Tissue Culture Plate, 5%CO
2, cultivate in the cell culture incubator of saturated humidity.1. after inoculating 24h, replacing contains the serum-free medium of N-2, and simultaneously every hole adds the DMEM complete medium and wherein contains 0.1%DMSO, behind the 24h, add A β (25-35) the effect 24h of variable concentrations, observe the influence of variable concentrations A β (25-35) pair cell survival rate; 2. after inoculating 24h, change liquid, add 5.0,2.5,1.0,0.1 μ g/ml LIG, blank group and A β (25-35) damage group add the DMEM complete medium and wherein contain 0.1%DMSO, hatch 24h after, add A β (25-35) (50uM) act on 24h after, add MTT solution 10 μ l/ holes, 37 ℃ continue to hatch 4h after, inhale and remove supernatant, every hole adds DMSO 150 μ l dissolve purple crystallizations, measures each hole absorbance (OD with microplate reader under the 570nm wavelength
570nm), be calculated as follows cell survival rate.
Cell survival rate=experimental group (OD
570nm)/blank group (OD
570nm) * 100%
ROS detects:
DCFH-DA is a ROS probe in the most frequently used, the most sensitive up to now cell, itself does not have the DCFH-DA of fluorescence can pass cell membrane and enters cell, transforms in cell and generates DCFH.Under the condition that active oxygen exists, the oxidized generation fluorescent material of DCFH DCF, green fluorescence intensity is directly proportional with intracellular reactive oxygen level.This experiment is with reference to Hong andJoeph method and improve to some extent, behind the SH-SY5Y cell inoculation 24h, change liquid, add 5.0,2.5,1.0,0.1 μ g/ml LIG, blank group and A β (25-35) damage group add DMEM complete medium (the DMSO final concentration is 0.1%), hatch 24h after, add A β (25-35) (50uM) act on 3h after, add 10 μ M DCFH-DA, 37 ℃ of lucifuges are hatched 30min.Shift out the solution that contains DCFH-DA, the PBS washing, every hole fluorescence intensity detects (excitation wavelength 485nm, emission wavelength 520nm) with microplate reader.
Immunoblotting assay:
Get protein sample 20-40 μ g, with 4: 1 volume mixture of 5 * sample-loading buffer, 95 ℃ of heating 5min make albuminous degeneration.Protein sample after handling is slowly added in the well, and a swimming lane adds 5 μ l and dyes molecular weight of albumen Marker in advance therein.80V constant voltage electrophoresis when the protein sample swimming extremely concentrates glue and separation gel intersection, changes 100V constant voltage electrophoresis into.When treating the swimming of bromophenol blue line, stop electrophoresis extremely apart from glue edge.With reference to molecular weight Marker, downcut the required part of separation gel, it is standby to immerse commentaries on classics film liquid.Take off pvdf membrane, the one side of labelling carrier protein molecule is dipped among the TBST, washes film 3 times, each 5min.Pvdf membrane is dipped in 5% defatted milk powder room temperature sealing 2h.Pvdf membrane is dipped among the TBST, washes film 3 times, each 10min.Take out pvdf membrane, immerse an anti-(GAPDH antibody dilution in 1: 5000 with the TBST dilution; Bax, TNF-α, the dilution in 1: 1000 of Cox-2 antibody; The dilution in 1: 1000 of Bcl-2 antibody; Cytochrome c antibody 1: 500), incubated at room 1h.Pvdf membrane is dipped among the TBST, washes film 3 times, each 10min.Radix Cochleariae officinalis enzyme labelling two anti-that adds each antibody correspondence, Bax goat anti-rabbit igg, β-actin, Bcl-2, cytochrome c goat anti-mouse IgG.Two anti-TBST with 1: 2000 dilute.Incubated at room 2h.Pvdf membrane is dipped among the TBST, washes film 3 times, each 10min.Photographic fixing is developed in chemiluminescence.Strip analysis is used Image Pro-Plus software analysis purpose band gray value, and each sample is proofreaied and correct with the GAPDH confidential reference items, relatively each histone differential expression.
Statistical analysis:
Experimental data represents with Mean ± SD that all experimental result adopts SPSS11.0 software to carry out statistical analysis, relatively adopts one factor analysis of variance LSD method between group, is significant difference with P＜0.05.
Interpretation of result:
Cell survival rate adopts mtt assay to detect.SH-SY5Y cell and variable concentrations A β (25-35) (1～200uM) hatch 24h after, cell survival rate increases and reduces along with A β (25-35) concentration.1uM A β (25-35) causes 15.93% cell injury, along with A β (25-35) concentration increases, cell injury increases, when A β (25-35) concentration is 50uM, 100uM, 200uM, SH-SY5Y cell injury percentage rate is respectively 53.74%, 73.49% and 86.74%.According to damage percentage, selecting concentration is that 50uM A β (25-35) is used for subsequent experimental.After LIG (0.1,1.0,2.5, the 5.0 μ g/ml) treatment, the minimizing SH-SY5Y cell injury of energy dose dependent improves cell survival rate, sees Fig. 1.
Active oxygen has important function in the process of cell death of A β (25-35) mediation.Can estimate the effect of LIG in A β (25-35) inducing cell generation oxidative stress by ROS level in the DCF fluorescent probe mensuration born of the same parents, Fig. 2 result shows, the SH-SY5Y cellular exposure A β (25-35) (50uM) in behind the 3h, the ROS level obviously increases in the born of the same parents, after variable concentrations LIG (0.1,1.0,2.5,5.0 μ g/ml) intervenes, the increase of fluorescence intensity in the born of the same parents is alleviated on dose dependent ground, and wherein 2.5 and 5.0 μ g/ml LIG effects are remarkable.
A β (25-35) induces the SH-SY5Y apoptosis by activating mitochondrion Caspase approach.In apoptosis pathway, cytochrome c is released into cytoplasm from mitochondrion, in conjunction with starting the Caspase cascade reaction behind the apoptosis inducing factor Apaf-1.The Bcl-2 family protein is to be one group of apoptosis regulatory protein of representative with Bcl-2, and as mitochondrial embrane-associated protein, Bcl-2 activates to bring into play by expression, cytochrome C release, the procaspase3 that suppresses Bax and suppresses effect of apoptosis.The apoptosis cascade reaction is early stage, and cell survival depends on the poised state of pro apoptotic protein and anti-apoptotic proteins in the Bcl-2 family to a great extent.This experimental result shows that A β (25-35) obviously raises the expression of Bax and endochylema cytochrome c, reduces the Bcl-2 protein level simultaneously, promotes apoptosis.But concentration is the LIG of 0.1,1.0,2.5,5.0 μ g/ml can significantly suppress the inductive apoptosis process of A β (25-35), reduces the expression of Bax and endochylema cytochrome c, and raises the Bcl-2 protein level, sees that (a is that bax expresses sketch map to Fig. 3; B is that bcl-2 expresses sketch map; C is that cytochrome c is expressed sketch map).
The SH-SY5Y cell can produce tumor necrosis factor (TNF-α) and be discharged into cell culture fluid.TNF-α is one of main proinflammatory cytokine, is the important regulatory factor that body inflammatory is replied.TNF also can make the COX-2 up-regulated, free radical is generated increase, the exacerbate inflammation damage.This experimental result shows that after A β (25-35) effect, SH-SY5Y cellular expression TNF-α obviously increases, and LIG can obviously reduce TNF-α protein expression.The beta induced SH-SY5Y cellular expression of A COX-2, after A β (25-35) effect, SH-SY5Y cellular expression COX-2 obviously increases, and LIG can obviously reduce the COX-2 protein expression, sees that (figure a is a TNF-alpha expression sketch map to Fig. 4; Figure b is that COX-2 expresses sketch map).
Ligustilide is to the influence of rat memory dysfunction due to the three-dimensional locating injection A of the brain β starch peptide
Animal: male Wistar rat, body weight 300～350 grams, the SPF level is bought by Sichuan Province's medical college academy of science institute of lab animals.Room temperature remain on 25 ℃ left back, ad lib and drinking-water.
Reagent and medicine: used ligustilide chemical purity＞98%, 3% tween 80 preparation;
All the other reagent are commercially available analytical pure.
Instrument: Morris water maze, monitor etc.
The preparation of A β (25-35) solution:
With physiological saline solution preparation A β (25-35) solution (5mM/l), the reference literature method is standby after aging 7 days in 37 ℃.
The modelling of rat memory dysfunction due to the three-dimensional locating injection A of the brain β starch peptide:
After experimental rat is anaesthetized with 1% pentobarbital sodium (40mg/kg), stereotaxic instrument fixing head, calvarium district preserved skin, routine disinfection field of operation skin.Make about 2mm otch along the calvarium center line, separate periosteum and make the skull exposure, in bregma 0.8mm backward, other each 1.5mm that opens about center line, open skull with the dental burr brill, vertically advance microsyringe pin 3.8mm, 10ulA β (50nmol) solution is slowly injected, be 5 minutes inject time, let the acupuncture needle remain at a certain point 5 minutes, to guarantee the abundant disperse of solution, slowly removes pin then, afterwards with penicillin powder sterilization, the skin of sewing up the incision.The sham operated rats operation technique is identical, the normal saline of injection equal volume.
Grouping and administration:
Animal is divided into three groups at random, sham operated rats, model control group and LIG (40mg/kg) oral medication group, 10 every group.All animals all carry out the training experiment (5 days) of water maze before the operation, promptly carry out operation after training experiment finishes.The back test (7 days) that began to carry out water maze in seven days of performing the operation is divided into concealment platform (3 days), explores platform (1 day) and new platform (3 days) is tested.Promptly began administration from the same day of operation, and finished until water maze laboratory, and be total to two weeks, model control group gives the solvent (3% Tween 80) of same dose.In behavioristics's experiment, all in experiment administration in preceding 1 hour.
Water maze laboratory:
The modeling senile dementia clinically of rat memory dysfunction due to the present invention's memory of nearly memory of Morris determined with Morris water rat and locus, the three-dimensional locating injection A β starch peptide of requiring mental skill.The Morris water maze mainly is made up of a metal cylindricality pond (the high 60cm in pond, diameter 120cm) and safety island (high 20cm, the platform of diameter 10cm).In the pond, inject clear water in advance, add fresh milk amidin then (after milk powder earlier becomes white homogeneous solution with the small amount of thermal water dissolution, add in the pond, the limit edged stirs), make Chi Shui become opaque milky, the water surface exceeds platform 15cm, and animal can not arrive platform by listening, look with olfactory sensation like this, so that detect the acuteness of animal to the locus.Water temperature remains on 23 ± 1 ℃, and the pond is divided into 4 quadrants (east, south, west, north), and platform is put the center with Southwest Quadrant.Preoperative water maze training experiment carried out 5 days continuously, being the experiment of concealment platform (is that the water surface exceeds platform, platform is invisible) 2 training searchings of every rat acceptance in 1 day platform, twice head is towards the pool wall entry respectively from the mid point of Northeast Quadrant, Northwest Quadrant.Twice training 10 minutes at interval.The record rat is found the time (incubation period) of platform, and twice experimental result averaged.If rat was not found platform in 60 seconds, then calculated by 60 seconds incubation period.No matter whether rat found platform in 60 seconds, and rat all will stop on platform 10 seconds.Earlier rat is placed on the platform before the experiment beginning for the first time and adapts to 10 seconds.Operation back water maze laboratory carried out 7 days continuously, was the experiment of concealment platform in 1-3 days, and method is with preceding described identical, and the record rat is found the time (incubation period) of platform; The 4th day for exploring platform experiment (promptly removing platform), rat place of entry invariant position, and the record rat is in time of staying of place, original platform position quadrant and pass through number of times; 5-7 days is new platform experiment (be that the position of platform changes, be placed on the center of Northeast Quadrant), rat place of entry invariant position, and the record rat is found the time (incubation period) of platform.
Statistical analysis:
All experimental results adopt Mean ± SD to represent.Diversity ratio incubation period of each group adopts the two-way analysis of variance (Two-way AVONA) of replication in the water maze laboratory.The water maze platform is explored experiment and is adopted one factor analysis of variance.Histopathological analysis adopts monofactorial variance analysis (One-way AVONA) (SPSS11.0 software).Group difference adopts post hoc LSD check.P＜0.05 is for there being significant difference.
In water maze laboratory, study and reservation experiment are often used in the spatial memory ability of estimating dementia rats.In the preceding 5 days training experiment of operation, there is not significant difference between each group.Through 5 days training, the Swimming Achievements of each group all improved, and search strategy also progressively turns to trend formula and orthoscopic from marginal mode and random mode, on average descends about 60% incubation period.Postoperative water maze laboratory is divided into the experiment of concealment platform, explores platform experiment and new platform experiment from performing the operation back seven days.In the concealment platform experiment, compare greatly before the incubation period of model group and the operation and prolonged, extend to 27.5 second (postoperative three day) from 18.2 seconds (preoperative the 5th day) incubation period.Search strategy also transfers random mode and marginal mode to, shows the damage of memory ability.LIG group and sham operated rats search strategy still for after the trend formula and orthoscopic (being respectively the 3rd day incubation period 6.55 seconds and 7.4 seconds) compare the continuation shortening with perform the operation preceding (be respectively the 5th day incubation period 16.8 seconds with 16.2 seconds).And LIG and sham operated rats and model group are relatively, and obvious difference incubation period (P＜0.01) is seen Fig. 5.Explanation through the rat of LIG (40mg/kg) treatment near normal level.After the experiment of 3 days concealment platforms finishes, carry out platform and explore experiment, platform is removed with the test rat whether formed spatial memory to platform.Sham operated rats and LIG administration group are in time of staying of target quadrant and pass through number of times all greater than model control group, between group relatively except that sham operated rats on passing through number of times with the model group there was no significant difference, all the other more all have significant difference (P＜0.01), see Fig. 6 and Fig. 7.In new platform experiment, sham operated rats and LIG treatment group and model control group relatively shorten obviously (P＜0.01) incubation period, see Fig. 8.Illustrate animal by learning and mastering the strategy of certain searching platform.Confirm nearly memory and locus functional impairment the have clear improvement effect of LIG to A β starch fragments of peptides injury rats.
Ligustilide is to the influence of rat neuronal damage due to the three-dimensional locating injection A of the brain β starch peptide
Animal: male Wistar rat, body weight 300～350 grams, the SPF level is bought by Sichuan Academy of Medical Sciences institute of lab animals.Room temperature remain on 25 ℃ left back, ad lib and drinking-water.
Reagent and medicine: used ligustilide chemical purity＞98%, 3% tween 80 preparation;
All the other reagent are commercially available analytical pure.
The preparation of A β (25-35) solution
With physiological saline solution preparation A β (25-35) solution (5mM/l), the reference literature method is standby after aging 7 days in 37 ℃
The modelling of rat memory dysfunction due to the three-dimensional locating injection A of the brain β starch peptide
After experimental rat is anaesthetized with 1% pentobarbital sodium (40mg/kg), stereotaxic instrument fixing head, calvarium district preserved skin, routine disinfection field of operation skin.Make about 2mm otch along the calvarium center line, separate periosteum and make the skull exposure, in bregma 0.8mm backward, other each 1.5mm that opens about center line, open skull with the dental burr brill, vertically advance microsyringe pin 3.8mm, 10ulA β (50nmol) solution is slowly injected, be 5 minutes inject time, let the acupuncture needle remain at a certain point 5 minutes, to guarantee the abundant disperse of solution, slowly removes pin then, afterwards with penicillin powder sterilization, the skin of sewing up the incision.The sham operated rats operation technique is identical, the normal saline of injection equal volume.
Grouping and administration
Animal is divided into three groups at random, sham operated rats, model control group and LIG (40mg/kg) oral medication group, 10 every group.Promptly began administration from the same day of operation, and be total to two weeks, model control group gives the solvent (3% Tween 80) of same dose.
After the results are shown in Figure 9:HE dyeing, observe sham operated rats and LIG treatment group and ANOMALOUS VARIATIONS all do not occur Hippocampus and cortex, the cortex structural integrity, neurocyte is arranged in order, clear layer, cellular morphology is normal, and structural integrity, endochylema enrich, kernel is clear.But model control group is after injection two weeks of A β (25-35), neuron degenerative change has in various degree all appearred in Hippocampus and cortex, as seen neuronal cell obviously reduces, cell is arranged at random, tissue edema, painted shoaling, and cell volume slightly dwindles, after birth is obvious with boundary on every side, the many pyknosis of nucleus are triangle, and structure and kernel disappear, and the cavity phenomenon is serious.After being described, LIG administration group (40mg/kg) treatment can obviously improve the beta induced neuronal cell injury of A.
Embodiment 4
Ligustilide is to the influence of rat prefrontal lobe cortical area due to the three-dimensional locating injection A of the brain β starch peptide and Hippocampus CA1 district A β starch peptide relevant diseases protein A PP, A β and Tau and inflammatory protein TNF-α and NF-κ B expression
Animal: male Wistar rat, body weight 300～350 grams, the SPF level is bought by Sichuan Academy of Medical Sciences institute of lab animals.Room temperature remain on 25 ℃ left back, ad lib and drinking-water.
Reagent and medicine: used ligustilide chemical purity＞98%, 3% tween 80 preparation;
A β 1-42, β-APP rabbit antibody, Phospho-tau (pSer202) rabbit antibody, NF-κ Bp65 rabbit antibody, TNF-α rabbit antibody are provided by Wuhan doctor's moral biotechnology development corporation, Ltd.;
All the other reagent are commercially available analytical pure.
The preparation of A β (25-35) solution:
With physiological saline solution preparation A β (25-35) solution (5mM/l), the reference literature method is standby after aging 7 days in 37 ℃.
The modelling of rat memory dysfunction due to the three-dimensional locating injection A of the brain β starch peptide:
After experimental rat is anaesthetized with 1% pentobarbital sodium (40mg/kg), stereotaxic instrument fixing head, calvarium district preserved skin, routine disinfection field of operation skin.Make about 2mm otch along the calvarium center line, separate periosteum and make the skull exposure, in bregma 0.8mm backward, other each 1.5mm that opens about center line, open skull with the dental burr brill, vertically advance microsyringe pin 3.8mm, 10ulA β (50nmol) solution is slowly injected, be 5 minutes inject time, let the acupuncture needle remain at a certain point 5 minutes, to guarantee the abundant disperse of solution, slowly removes pin then, afterwards with penicillin powder sterilization, the skin of sewing up the incision.The sham operated rats operation technique is identical, the normal saline of injection equal volume.
Grouping and administration:
Animal is divided into three groups at random, sham operated rats, model control group and LIG (40mg/kg) oral medication group, 10 every group.Promptly began administration from the same day of operation, and be total to two weeks, model control group gives the solvent (3% Tween 80) of same dose.
The results are shown in Figure 10 and Figure 11: estimate APP, A β and the proteic expression of phosphorylation Tau of rat layer and hippocampus by the method for SABC, the result is presented at sham operated rats, these three kinds of proteic expression are all very faint, and the expression of model control group obviously strengthens, positive cell number quantity is many, color depth, and the distribution comparatively dense.LIG (40mg/kg) is the expression of Profilin obviously, positive cell quantity reduces, painted more shallow, the results are shown in Figure 12: the method by SABC is estimated rat layer and NF κ β, the TNF-α of hippocampus, proteic expression, the result is presented at sham operated rats, and these two kinds of proteic expression are all very faint, and (and the expression of model control group obviously strengthens, and positive cell number quantity is many, color depth, and the distribution comparatively dense.LIG (40mg/kg) is the expression of Profilin obviously, and positive cell quantity reduces, and is painted more shallow.
Ligustilide is to the neuroprotective of the dull-witted SAMP8 mice of quick aging
Animal: 48 of male SAMP8 quick aging of 10 monthly ages of SPF level mices, about body weight 30g; 12 of 10 monthly age SAMR1 aging resistance mices provide by The First Affiliated Hospital of Tianjin University of Traditional Chinese Medic.Room temperature remain on 25 ℃ left back, ad lib and drinking-water.
Reagent and medicine:
Used ligustilide chemical purity＞99% faces with preceding 3% tween 80 preparation;
All the other reagent are commercially available analytical pure.
Instrument and reagent:
The diving tower instrument is provided by Chinese Academy of Sciences institute of materia medica.
Y type labyrinth stimulator, factory provides by Zhangjagang City, Jiangsu Province Lignum Pini Nodi Biomedical Instruments.
Grouping and administration:
It is five groups that animal is divided into, and the SAMP8 mice is divided into dosage group (20mg/kg), LIG high dose group (40mg/kg) among model group, LIG low dose group (10mg/kg), the LIG at random, and the SAMR1 mice is as the normal control group, 12 every group.Gastric infusion, 1 day 1 time, altogether 8 weeks of administration, model group and normal group are irritated stomach and are waited equal-volume blank solvent (3% Tween 80).
The test of mice behavioristics:
The diving tower experiment
During experiment mice is put into the diving tower experimental box, energized is jumped onto platform after mice is shocked by electricity, most mices are rebound copper grid once more, jump onto platform after being shocked by electricity again, and the mice biped contacts the number of times that the copper grid are promptly shocked by electricity simultaneously in the record 5min, be designated as errors number, as training achievement.Repeated experiments behind the 24h is for memory keeps test.The errors number of mice in record 5min during test is as the diving tower test result.
Y type maze experiment
Experimental provision is 1 lost case of being made by the opaque plastics plate of Y type, and it is linked to each other with adjustable transformer, and voltage is 36V.By the electric shock controller, can make the zone that surrounds mutually by the terminal long 18cm section of 3 support arms, alternately as starting area or place of safety.During training mice being put into the starting area, handle the electric shock controller, after mice is shocked by electricity, is correct response as if directly escaping to the place of safety, otherwise is wrong reaction.Training mice 10 times carries out same test behind 24h, write down correct response number of times in 10 times, as the labyrinth test result.
Statistical analysis:
All experimental results adopt Mean ± SD to represent.Analyze and adopt monofactorial variance analysis (One-way AVONA) (SPSS11.0 software).Group difference adopts post hoc LSD check.P＜0.05 is for there being significant difference.
Mice diving tower experimental result is seen Figure 13, SAMR1 group errors number is starkly lower than model group (P＜0.01), LIG high dose group and model group significant difference (P＜0.05) is relatively arranged and among the LIG, low dose group and model group relatively have the trend of minimizing but no difference of science of statistics.
Mice Y type maze experiment the results are shown in Figure 14, compares with model group, and SAMR1 group and LIG height, the correct time number average of middle dosage group have significant difference (P＜0.01), and low dose group mice achievement also increases, but there was no significant difference.
Above-mentioned cell in vitro experiment, whole animal level, histopathologic result of study show that ligustilide can very significantly improve the SH-SY5Y cell injury of A β starch inducing peptide; Obviously improve the neuron degeneration that the nearly memory of rat due to the A β starch peptide and locus dysfunction and three-dimensional locating injection A β starch peptide cause, the dementia that three-dimensional locating injection A β starch peptide is caused has therapeutical effect; Significantly improve the damage in learning and memory of SAMP8 quick aging mice, have the effect of anti-senile dementia treatment of conditions.Thereby, can be prepared into and be used to prevent and treat the medicine of senile dementia disease and/or the edible combination of health.
Claims (7)
1. the application of ligustilide in preparation control senile dementia disease compositions is characterized in that with ligustilide forming jointly with acceptable auxiliary element in the pharmacy as effective ingredient.
2. application as claimed in claim 1 is characterized in that said ligustilide is a cis form ligustilide.
3. application as claimed in claim 1 is characterized in that also can including in the said effective ingredient and occupies that acetylcholinesteraseinhibitors inhibitors, excitatory amino acid and peptide class mediator composition, the A beta of imitating composition gross weight 10%～40% form and deposition interference component, antioxidant, microcirculation improve at least a in composition, the calcium antagonist.
4. as the described application of one of claim 1 to 3, it is characterized in that said compositions is pharmaceutical preparation.
5. application as claimed in claim 4 is characterized in that said compositions is an oral drug preparation.
6. application as claimed in claim 4, it is characterized in that said pharmaceutical preparations composition for suitable every day consumption be the dosage form of 0.1～500mg/kg body weight.
7. as the described application of one of claim 1 to 3, it is characterized in that said compositions is the health food compositions.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009100595054A CN101904838A (en) | 2009-06-04 | 2009-06-04 | Application of ligustilide in preparing composition for preventing and treating senile dementia |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009100595054A CN101904838A (en) | 2009-06-04 | 2009-06-04 | Application of ligustilide in preparing composition for preventing and treating senile dementia |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101904838A true CN101904838A (en) | 2010-12-08 |
Family
ID=43260527
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2009100595054A Pending CN101904838A (en) | 2009-06-04 | 2009-06-04 | Application of ligustilide in preparing composition for preventing and treating senile dementia |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101904838A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103479743A (en) * | 2013-01-28 | 2014-01-01 | 钱昌美 | Pharmaceutical composition for treating senile dementia and preparation method thereof |
| CN103830116A (en) * | 2014-03-26 | 2014-06-04 | 清华大学 | Application and use method of ligustilide in preparing skin-care product for preventing skin photoaging |
| US9889117B2 (en) | 2014-07-02 | 2018-02-13 | Everfront Biotech Inc. | Method for treating and/or delaying the degeneration of Purkinje cells |
| CN108315350A (en) * | 2018-03-01 | 2018-07-24 | 昆明医科大学 | It is overexpressed COX5A/ low expression BDNF transgenic mouse models and its construction method and application |
| CN110772510A (en) * | 2019-12-13 | 2020-02-11 | 浙江省淡水水产研究所 | Novel application of ligustilide |
| CN114377045A (en) * | 2022-01-17 | 2022-04-22 | 江西中医药大学 | Extraction method of hemlock parsley extract, capsule and application |
-
2009
- 2009-06-04 CN CN2009100595054A patent/CN101904838A/en active Pending
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103479743A (en) * | 2013-01-28 | 2014-01-01 | 钱昌美 | Pharmaceutical composition for treating senile dementia and preparation method thereof |
| CN103479743B (en) * | 2013-01-28 | 2015-07-08 | 钱昌美 | Pharmaceutical composition for treating senile dementia and preparation method thereof |
| CN103830116A (en) * | 2014-03-26 | 2014-06-04 | 清华大学 | Application and use method of ligustilide in preparing skin-care product for preventing skin photoaging |
| CN103830116B (en) * | 2014-03-26 | 2017-01-04 | 清华大学 | Ligustilide prevents purposes and the using method of skin photoage skin care item in preparation |
| US9889117B2 (en) | 2014-07-02 | 2018-02-13 | Everfront Biotech Inc. | Method for treating and/or delaying the degeneration of Purkinje cells |
| CN108315350A (en) * | 2018-03-01 | 2018-07-24 | 昆明医科大学 | It is overexpressed COX5A/ low expression BDNF transgenic mouse models and its construction method and application |
| CN108315350B (en) * | 2018-03-01 | 2021-08-27 | 昆明医科大学 | COX5A overexpression/BDNF low expression transgenic mouse model and construction method and application thereof |
| CN110772510A (en) * | 2019-12-13 | 2020-02-11 | 浙江省淡水水产研究所 | Novel application of ligustilide |
| CN110772510B (en) * | 2019-12-13 | 2022-07-19 | 浙江省淡水水产研究所 | Application of ligustilide |
| CN114377045A (en) * | 2022-01-17 | 2022-04-22 | 江西中医药大学 | Extraction method of hemlock parsley extract, capsule and application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| ES2732559T3 (en) | Compositions comprising acid extracts of mastic | |
| CN101904838A (en) | Application of ligustilide in preparing composition for preventing and treating senile dementia | |
| Hu et al. | Danggui-Shaoyao-San and its active fraction JD-30 improve Aβ-induced spatial recognition deficits in mice | |
| Li et al. | Tibetan medicine Duoxuekang ameliorates hypobaric hypoxia-induced brain injury in mice by restoration of cerebrovascular function | |
| CN101869600A (en) | Medicinal composition for treating degenerative change in central nervous system, preparation method and application thereof | |
| Inanaga | Amycenone, a nootropic found in Hericium erinaceum | |
| Li et al. | Coeloglossum viride var. bracteatum extract attenuates Aβ-induced toxicity by inhibiting RIP1–driven inflammation and necroptosis | |
| Zhang et al. | α‑lipoic acid attenuates spatial learning and memory impairment induced by hepatectomy | |
| CN103751228A (en) | Medicine for treating Alzheimer disease and preparation method thereof | |
| CN100496482C (en) | Application of ligustilide in preparation of medicine for preventing and treating cerebral ischemia diseases | |
| CN104042797A (en) | Aiweixin oral solution for treating cardiovascular diseases and preparation method thereof | |
| CN102697757B (en) | Application of p-hydroxy benzylidene acetone in preparation of drugs for preventing and/or treating encephalopathy | |
| CN104473519A (en) | Medicine pillow capable of improving and maintaining memory ability | |
| CN104095938B (en) | A kind of Mongolian medicinal preparation of Cure of depression | |
| CN102813914A (en) | Medicinal composition used for treating or preventing cerebrovascular disease and related diseases | |
| CN102813659A (en) | Use of 20alpha-dimethylamino-2alpha-hydroxy-3beta-tiropramide-5alpha-pregnane | |
| KR101470241B1 (en) | Pharmaceutical composition for the prevention or treatment of depression | |
| CN108210879A (en) | A kind of pharmaceutical composition for treating acute Cerebral bleeding and its application | |
| JPH07509494A (en) | Novel anti-anxiety drug | |
| CN106344609A (en) | Application of ozonized oil in preventing and controlling nervous system diseases | |
| CN103006633A (en) | Application of hydroxysafflor yellow A in preparation of medicament for resisting Alzheimer disease | |
| CN102028681B (en) | Application of peperphentonamine or salt thereof in preparing drug for preventing/treating encephalopathy | |
| CN102836152B (en) | Application of physalin B in preparation of medicine for curing and/or preventing schistosomiasis | |
| CN101361831B (en) | Application of geniposide in preparation of medicine for treating vascular dementia | |
| CN101642465B (en) | Application of Kadsura heteroclite (Roxb.) Craib polysaccharide of Guangdong province for preparing medicaments for preventing and/or treating senile dementia |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20101208 |
|
| WD01 | Invention patent application deemed withdrawn after publication |