CN104606464B - A kind of pharmaceutical composition for treating Alzheimer disease - Google Patents
A kind of pharmaceutical composition for treating Alzheimer disease Download PDFInfo
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Abstract
The invention discloses a kind of pharmaceutical composition for treating Alzheimer disease, it is made from the following raw materials in parts by weight：15 parts of RADIX POLYGONI MULTIFLORI PREPARATA, 15 parts of ganoderma lucidum, 12 parts of leech, 12 parts of the wind-weed.The present invention combines tcm clinical practice feature and the pharmaceutical research method plan side of traditional traditional Chinese medical theory and Alzheimer disease (AD), show through pharmaceutical research, medicine composite for curing AD of the present invention has the action character of multicomponent, Mutiple Targets, multipath.The pharmaceutical composition of the present invention can significantly improve a variety of AD rats, the learning and remembering ability of mouse model；There is obvious anti-inflammatory to AD models；Mitigate radical damage；Improve intracerebral energy metabolism impairment；It is obvious to suppress AD apoptosis of hippocampal neurons in rats rates, play cholinergic nerve of centrum protection and anti-dementia effect.
Description
Technical field
The invention belongs to pharmaceutical technology field, and in particular to a kind of Traditional Chinese medicine composition, and its treatment Alzheimer
The purposes of disease.
Background technology
Alzheimer disease (Alzheimer ' s Disease, AD) is a kind of with progressive amnesia and dull-witted to face
The central nervous system degenerative disease of bed feature.With the extension of human longevity, the AD incidences of disease steeply rise.Have due to lacking
The treatment method of effect, AD turn into No. 4 killer of the mankind after the heart, cerebrovascular disease, cancer.Therefore find, exploitation preventing and treating AD
Medicine be the task of top priority, turned into the primary brainstorm subject of global medical field to AD study on prevention.
AD pathological characters are mainly shown as senile plaque expelling (senile plaques, SP), neurofibrillary tangles
(neurofibrillary tangles, NFT) and neuron loss (neuron loss).At present, domestic and foreign scholars are from gene
The multiple fields such as, biochemistry, neuropathology are carried out widely studied, it is believed that and AD pathogenesis is sufficiently complex, including：①
The deposition and its neurotoxic effect of amyloid-beta (β-amyloid peptide, A β).2. cholinergic nerve functional defect
Theory.3. inflammation：Thinking AD pathogenesis includes the inflammatory processes such as inflammatory cell, inflammatory cytokine.4. free radical
Damage theory：Free radical can promote A beta peptide aggregations, make it cause ofneurodegenerative to become in intracerebral overacfivity, neuron loss,
Generation AD (Butterfield DA, Boy-Kimball D.Amyloid beta-peptide (1-42) contributes to
the oxidative stress and neurodegeneration found in Alzheimer disease
brain.Brain Pathol,2004,14(4):426~32.Kadowaki H,Nishitoh H,Urano F,et
al.Amyloid beta induces neuronal cell death through ROS-mediated ASKl
activation.Cell Death Differ,2005,12(1):19~24).A β can cause the increase of response to oxidative stress, make
Cholinergic reduces in AD brains；And A β depositions are aggravated after cholinergic system damage, promote the development of the AD state of an illness.5. can
Measure (Hoyer S, Oesterreich K, the Wagner O.Glucose metabolism as the such as dysbolism theory
site of the primary abnormality in early-onset dementia of Alzheimer’s dis-
ease.J Neurol,1988,235(3):143~148) AD generation and development can be promoted.
Study of pathogenesis based on AD, clinical treatment AD approach mainly have：1. cholinergic strengthens approach, for changing
Medicine (huperzine (huperzine A, donepezil (donepezil), the galanthamine of kind cholinergic nerve function
(glalantamine).2. prevent synthesis and the deposition approach of amyloid.3. anti-inflammatory treatment approach.4. nerve growth factor
Approach.5. antioxidant radical medicine such as epiphysin (melatomin) etc..These medicines largely belong to single targeted drug, although
There is the effect of certain to relief of symptoms, but be helpless to prevent or reverse the development of lesion, and there is certain adverse reaction.In recent years
Come, filter out aβ protein false folding inhibitor such as hypertensin conversion enzyme (Angiotensin-Converting
Enzyme, ACE), it can prevent amyloid deposition from accumulating (Hu J.Igarashi A, Kamata M.Angiotensin in intracerebral
converting enzyme degrades Alzheimer anyloid βpeptide(Aβ)；Retards Aβ
aggregation deposition,Fibril Formation；and inhibits cytoxicity.Biol
Chem.2001；(276):47863-4786713)；Ginkgo biloba extract EGb761, the formation of A betas can be suppressed, reduce nerve
Toxicity, but unclear (Luo Y, Smith J V, the Paramasivam V.Inhibition of amyloid of its mechanism of action
βaggregation and caspase 3 activation by the Ginkgo biloba extract
EGb761.Proc.Natl.Acad.ci.USA,2002；(99):12197-201).Because AD is multifactor complex disease, no
Few scholar thinks, attempts to be less likely with single factors treatment AD, it is necessary to carry out multifactor complex treatment.Clinical research table
It is bright, it is only capable of obtaining certain effect in finite time by certain a kind of medicine merely.Therefore, not generally acknowledging still so far,
Effective preventing and treating AD medicine comes out.
For many years, AD is the focus of TCM investigation, and motherland's traditional Chinese medicine theory has original understanding to AD, it is believed that AD belongs to
In traditional Chinese medicine " forgetful ", " dementia ", " dementia " category.With the proposition of Wang Yong inflammation academician " turbid poison ", summarized with reference to modern study
It is to cause aging and the AD Etiological interpretation of the cause, onset and process of an illness to go out turbid, malicious, and its sick position is in brain.Hemostasis, phlegm is turbid, malicious heresy is both knot that visceral-qi void declines
Fruit, is main pathogenic factor again, hemostasis, it is turbid, malicious influence each other, interaction turns into the complicated interpretation of the cause, onset and process of an illness of AD to suffer from, it is seen that turbid poison is
One of main pathogenic in AD morbidities.The present invention comes therefrom.
The content of the invention
The technical problems to be solved by the invention are the TCMs with reference to traditional traditional Chinese medical theory and Alzheimer disease
Bed feature, according to the pathogenesis that Alzheimer disease is complicated, for its multiple morbidity link, there is provided one kind has the more targets of medicine
Position effect, the Traditional Chinese medicine composition of Alzheimer disease can be effectively treated.
Technical scheme provided by the invention is：A kind of pharmaceutical composition for treating Alzheimer disease, its feature be, its
It is made from the following raw materials in parts by weight：RADIX POLYGONI MULTIFLORI PREPARATA 1-5 parts, ganoderma lucidum 1-5 parts, leech 1-2 parts, wind-weed 1-2 parts.
In the optimal technical scheme of the present invention, described pharmaceutical composition is made from the following raw materials in parts by weight：RADIX POLYGONI MULTIFLORI PREPARATA
1-3 parts, ganoderma lucidum 1-3 parts, leech 1-2 parts, wind-weed 1-2 parts.
In the optimal technical scheme of the present invention, described pharmaceutical composition is made from the following raw materials in parts by weight：RADIX POLYGONI MULTIFLORI PREPARATA
1.5 parts, 1.5 parts of ganoderma lucidum, 1 part of leech, 1 part of the wind-weed.
In the optimal technical scheme of the present invention, described pharmaceutical composition is pulvis, capsule, tablet, granule.
The second aspect of the present invention provides a kind of preparation method for the pharmaceutical composition for treating Alzheimer disease, in proportion
RADIX POLYGONI MULTIFLORI PREPARATA, ganoderma lucidum, leech, the wind-weed are taken respectively, adds water to cook secondary, is staticly settled after collecting decoction, are filtered, by filtrate at 80 DEG C
Thick paste is concentrated under reduced pressure into below, and dry, pulverize below 80 DEG C, is sieved, obtains extract powder, and packing is standby.
In the optimal technical scheme of the present invention, add water to cook secondary, 2 hours first times, second 1.5 hours.
In the optimal technical scheme of the present invention, per g extract powder 2-5g containing crude drug, preferably 2.5g.
The third aspect of the present invention be to provide a kind of foregoing pharmaceutical composition be used for prepare treatment Alzheimer disease medicine
The purposes of thing.
The present invention medicine can add various customary adjuvants required when preparing different dosage forms, as adhesive, lubricant,
Disintegrant etc. is prepared into any peroral dosage form, including pulvis, tablet, capsule, granule, pill, mouth in conventional manner
Take the formulations such as liquid.
RADIX POLYGONI MULTIFLORI PREPARATA is filling liver kidney simply, the good medicine of benefiting essence-blood, there is filling liver kidney benefiting essence-blood, anti-aging, has antioxygen
Change, raise brain tissue superoxide dismutase (SOD), reduce the effect of MDA (MDA) content.RADIX POLYGONI MULTIFLORI PREPARATA can be passed through by Radix Polygoni Multiflori
Cross deep processing to be prepared, mainly using Radix Polygoni Multiflori and black soya bean Powder Made by Steamed Method or cooking method, dry to obtain.
Ganoderma lucidum mental-tranquilization, increase intelligence strengthening the essence, have and improve SOD activity, remove free radical and cooperate with RADIX POLYGONI MULTIFLORI PREPARATA more preferably to send out
The effect of waving beneficial brain.
Leech contains heparin, antithromboxin and abundant protein, has anticoagulation, anti thrombotic action, and can suppress god
Through Apoptosis, blood-breaking eliminating the phlegm, promoting blood circulation are turbid.
The wind-weed can remove interior life " turbid poison " effect in AD morbidities, improve M choline receptor quantity, strengthen intracerebral choline acetyl
Transferase (ChAT) activity.
The dosage scope of pharmaceutical composition of the present invention is 0.24g-0.96g/kg/ days, preferably 0.48g/kg/ days.
The present invention combines tcm clinical practice feature and the pharmaceutical research method plan side of traditional traditional Chinese medical theory and AD, through medicine
Pharmacological research shows that pharmaceutical composition of the present invention has the action character of multicomponent, Mutiple Targets, multipath, the advantage is that：
1. the pharmaceutical composition of the present invention can improve Iibotenicacid (Ibotenic acid, IBO) damage bilateral
Meynert basal nucleis (nucleus basalis of Mynert, NBM) establish the learning and remembering ability of AD rat models.This
The pharmaceutical composition of invention is by reducing brain tissue acetylcholinesterase (acetylcholinesterase, AChE) and butyryl courage
The activity of alkali esterase (butyrocholinesterase, BuChE), suppress acetylcholine (acetylcholine, Ach) and decompose,
Increase intracerebral ACh contents, so as to improve the study of AD rats, memory disorders；Have to AD central nervous system of rats cholinergic system preferable
Protective effect.
2. the present invention pharmaceutical composition can improve bilateral hippocampus injection amyloid-beta (β-amyloid protein,
A β) establish the learning and remembering abilities of AD rat models.The pharmaceutical composition anti-inflammatory of the present invention：Reduce inflammatory factor leucocyte
The β of interleukin -1 (interlukin-1 β, IL-1 β), tumor necrosis factor-alpha (tumor necrosis factor- α, TNF-α)
Level, mitigate intracerebral inflammatory reaction；Mitigate the pathological change of AD rat cerebral tissues；It is obvious to suppress AD apoptosis of hippocampal neurons in rats
Rate, play neuroprotection and anti-dementia effect.
3. the pharmaceutical composition of the present invention can significantly improve D- galactolipins (D-galactose, D-Gal) and natrium nitrosum
(NaNO2) learning and remembering ability for establishing AD mouse models is injected intraperitoneally.The pharmaceutical composition antioxidant radical damage of the present invention
Wound：Raise brain tissue superoxide dismutase (SOD), suppress MDA (MDA) generation, mitigate radical damage；Raise Na+-K+- ATP enzyme and Ca2+-Mg2+- atpase activity；Play neuroprotection and anti-dementia effect.
Brief description of the drawings
Fig. 1 shows that the pharmaceutical composition of the present invention acts on more morbidity links of AD, has the more target position effects of medicine.
Fig. 2 is the change (Nissl's staining × 400) of each group CA 1 of Hippocampus, wherein：Fig. 2A is sham-operation group
(Sham), Fig. 2 B are model control group (A β), and Fig. 2 C are piracetam control group (A β+NFK), and Fig. 2 D are that the turbid powder of strengthening the essenceization is low dose of
Group (A β+YJHZF-L), Fig. 2 E are the turbid powder middle dose group of strengthening the essenceization (A β+YJHZF-M), and Fig. 2 F are the turbid powder heavy dose group of strengthening the essenceization
(Aβ+YJHZF-H)。
Fig. 3 is each group rat flow cytometry quantitative analysis figure and its data statistics figure, sham-operation group (Sham), model pair
According to group (A β), piracetam control group (A β+NFK), the turbid powder small dose group of strengthening the essenceization (A β+YJHZF-L), the turbid powder middle dosage of strengthening the essenceization
Group (A β+YJHZF-M), the wherein turbid powder heavy dose group of strengthening the essenceization (A β+YJHZF-H), lower-left in flow cytometry quantitative analysis figure
Quadrant is normal live cells (LL), and left upper quadrant is non-viable non-apoptotic cell (UL), and right lower quadrant is viable apoptotic cell bottom right (LR), right
Upper quadrant is non-viable apoptotic cell (UR)；Apoptotic cell sum is UR+LR sums.Compared with Sham groups, * P<0.05, * * P<
0.01；Compared with A β groups, #P<0.05.
Embodiment
Such scheme is described further below in conjunction with specific embodiment.It should be understood that these embodiments are to be used to illustrate this
Invent and be not limited to limit the scope of the present invention.The implementation condition used in embodiment can be done according to specific experiment condition into one
Successive step, unreceipted implementation condition are usually the condition in normal experiment.
First, pharmaceutical composition is prepared
The preparation of the pulvis of embodiment 1
RADIX POLYGONI MULTIFLORI PREPARATA 300g, ganoderma lucidum 300g, leech 200g, wind-weed 200g are weighed, is added water to cook secondary, 2 hours first times, the
Secondary 1.5 hours, staticly settle, filter after collecting decoction, filtrate is concentrated under reduced pressure into thick paste below 80 DEG C, and 80 DEG C with
Under dry, pulverize, sieve, obtain extract powder, add dextrin to dry in right amount, per g extract powder 2.5g containing crude drug, pulvis is obtained after crushing,
" the turbid powder of strengthening the essenceization " below.
The preparation of the capsule of embodiment 2
RADIX POLYGONI MULTIFLORI PREPARATA 300g, ganoderma lucidum 200g, leech 200g, wind-weed 150g are weighed, is added water to cook secondary, 2 hours first times, the
Secondary 1.5 hours, staticly settle, filter after collecting decoction, filtrate is concentrated under reduced pressure into thick paste below 80 DEG C, and 80 DEG C with
Under dry, pulverize, sieve, obtain extract powder, add right amount of auxiliary materials to be fitted into after dry, pulverize in gelatine capsule.
The preparation of the tablet of embodiment 3
RADIX POLYGONI MULTIFLORI PREPARATA 100g, ganoderma lucidum 100g, leech 100g, wind-weed 150g are weighed, is added water to cook secondary, 2 hours first times, the
Secondary 1.5 hours, staticly settle, filter after collecting decoction, filtrate is concentrated under reduced pressure into thick paste below 80 DEG C, and 80 DEG C with
Under dry, pulverize, sieve, obtain extract powder, add right amount of auxiliary materials, it is tabletted.
The preparation of the granule of embodiment 4
RADIX POLYGONI MULTIFLORI PREPARATA 150g, ganoderma lucidum 100g, leech 100g, wind-weed 100g are weighed, is added water to cook secondary, 2 hours first times, the
Secondary 1.5 hours, staticly settle, filter after collecting decoction, filtrate is concentrated under reduced pressure into thick paste below 80 DEG C, and 80 DEG C with
Under dry, pulverize into smalls, add ethanol and make binder, it is appropriate to add starch supplementary material, is pressed into granule.
Pharmaceutical composition of the present invention can effectively treat AD by Mutiple Targets pai n nursing, and central cholinergic system can be protected refreshing
Through；Play Na in anti-inflammatory, anti-oxidant, raising brain tissue+-K+- ATP enzyme and Ca2+-Mg2+- atpase activity, and anti-neuron wither
The effect died；Mitigate the pathological change of brain tissue, neuron is protected, so as to significantly improve the ability of learning and memory of AD animals.This
Study and provide experiment and theoretical foundation for clinical treatment AD, this international headache provides new Research Thinking for preventing and treating AD.
2nd, the beneficial effect experiment of pharmaceutical composition (following " the turbid powder of strengthening the essenceization " prepared from embodiment 1) of the present invention
Test example 1 uses stereotaxic apparatus from bilateral Meynert basal nucleis (nucleus basalis of
Mynert, NBM) injection Iibotenicacid (Ibotenic acid, IBO) establish AD central cholinergic neurons of rat system injuries
Model.
1.1 experiment purpose：Pharmaceutical composition of the present invention is observed, experiment applies the turbid powder of strengthening the essenceization being prepared in example 1 to AD
The protective effect of central nervous system of rats cholinergic system
The neuron of Meynert basal nucleis (nucleus basalis of Mynert, NBM) is mainly cholinergic nerve
There is serious missing or denaturation in member, the NBM neurons of AD patient's intracerebral, basal forebrain is projected cerebral cortex and hippocampus
Cholinergic nerve path is damaged, and causes the cholinergic neuron of cerebral cortex and hippocampus to significantly reduce.Now, intracerebral cholinergic god
Significantly lost through first, many researchs confirm the neuropathologic change of the impaired degree of cholinergic neuron and AD and dull-witted serious journey
Spend closely related.Excitatory amino acid Iibotenicacid (Ibotenic acid, IBO) is a kind of NMDA (one D of N- methyl mono- day
L-aminobutanedioic acid) receptor stimulating agent, there is strong neural information encoding to act on, can lasting and stably damaged animal study note
Recall.Rat NBM is damaged with IBO, NBM cholinergic neuron degenerations can be caused, nerve group area diminishes, and cell process quantity is obvious
Reduce, be comparatively ideal AD animal models.
This research is established using stereotaxic apparatus from bilateral Meynert basal nucleis (NBM) injection Iibotenicacid (IBO)
AD rat models, protective effect of the observation turbid powder of strengthening the essenceization to AD central nervous system of rats cholinergic systems.
1.2 experiment material：
1.2.1 experimental animal：Healthy adult male Sprague-Dawley (SD) rat, cleaning grade, body weight 240~
260g, Nantong University's Experimental Animal Center provide, credit number：SYXK (Soviet Union) 2007-0021.
1.2.2 Experimental agents：The turbid powder of strengthening the essenceization：It is made up of RADIX POLYGONI MULTIFLORI PREPARATA, ganoderma lucidum, leech and the wind-weed, by institute of traditional Chinese medicine of Nantong City
Pulvis is made in Drug Manufacturing Room, and the turbid powder 2.5g containing crude drug of every gram of strengthening the essenceization, specific method is as described in Example 1.Piracetam (Piracetam
Piece) (Nanjing Bai Jingyu pharmaceutical factories produce, lot number：20100812).
1.2.3 reagent and instrument：Iibotenicacid (U.S. Sigma-Aldrich, numbering：I2765)；Sterile saline
(Nanjing Zhengda Tianqing Pharmaceutical Co., Ltd, lot number：1012091)；Sodium carboxymethylcellulose (analyzes pure, Solution on Chemical Reagents in Shanghai station
Dispense factory).AChE determines kit (lot number：20101108), BuChE determines kit (lot number：20101108) it is purchased from south
Bioengineering Research Institute is built up in capital.Rat stereotaxic apparatus ((Lab StandardTM Stereotaxic Instrument,
Stoelting companies of the U.S.)；Syringe pump and micro syringe (Stoelting companies of the U.S.)；Y-Shaped (MG-B types) labyrinth stimulates
Device (emerging teaching machinery plant of Zhangjagang City three)；BS110S types electronic balance (Beijing Sai Duolisi balances company)；Microfuge
22R tabletop refrigerated centrifuges (Beckman companies of the U.S.)；The miniature vortex mixers of WH-3 (Shanghai Hu Xi analytical instrument factory)；-70
DEG C ultra low temperature freezer (SANYO, Japan)；MLS-3750 autoclave sterilizers (SANYO, Japan).
1.3 experimental method：
1.3.1 animal packet：The normal 60 SD rats of learning and remembering ability are filtered out with Y-Shaped labyrinth stimulator, with
Machine is divided into 6 groups, every group 10 (referring to table 1)：
The each group experimental animal situation of table 1
1.3.2 prepared by model：After SD rats adaptability is fed 2 days, 10% chloraldurate (4ml/kg) intraperitoneal injection of anesthesia,
It is fixed on stereotaxic apparatus.Standby skin of head, sterile surgical area skin.It is sterile lower to make 2.0cm stringers along head calvarium center line
Otch, hemostasis, blunt separation periosteum.With reference to rat brain stereotaxic atlas (George Paxions and Charles
Watson.The rat brain in stereotaxic coordinates.5th ed.USA:Elsevier Academic
Press,2005:36-55), in AP-1.14, ML ± 3.1, DV7.7 (away from skull surface 8.7mm), divide in bilateral skull corresponding points
The hole of a diameter 1mm is not bored not vertically.Injection Iibotenicacid (IBO) 1 μ l, injection time per side Meynert basal nucleis (NBM)
Continue 5min, let the acupuncture needle remain at a certain point 5min.The surface of a wound is sterilized after slowly pulling out pin, bone wax envelope skull hole, sutures scalp.Sham-operation group takes identical seat
Mark, Bilateral injections normal saline (normal saline, NS) after inserting needle.Whole surgical procedure follows strictly sterile working
Principle.Conventinal breeding in cage is put back to after rat is clear-headed.
1.3.3 dosage regimen：
The turbid powder of strengthening the essenceization and piracetam are prepared with 0.5% sodium carboxymethylcellulose.Positive controls give 0.4g/kg brains
Multiple health.The drug concentration ratio of the basic, normal, high dosage group of the turbid powder of strengthening the essenceization is followed successively by 1:2:4 (1.5,3.0,6.0g/kg), in postoperative
2d, start gastric infusion after the completion of Behavior test, 1 time/d, administration capacity is 10ml/kg.Sham-operation group, model comparison
Group such as gives at capacity physiological saline (NS) gavage, 1 time a day, Behavior test again after successive administration 30d.
Ingested the ordinary circumstances such as amount of drinking water, fecaluria color and amount, hair gloss in addition, observing activities in rats, 24h daily.
Rat body weight is weighed every 7d, and dosage is adjusted according to body weight.
1.3.4 learning and memory in rats aptitude tests (Y-Shaped labyrinth stimulator test)
Test the learning and remembering ability of rat respectively before modeling, after modeling and after medication.Rat is put into labyrinth stimulates
One arm of device, electro photoluminescence (60V, continuing 2s) is given after adapting to environment 3min.Any arm in remaining two-arm gives light signal,
It is shown as place of safety.Rat enters place of safety, is correct response, is otherwise wrong reaction.2nd time training using this place of safety as
Area is walked, and rat is rested on place of safety 1min with consolidating memory.Electro photoluminescence is given again, by that analogy, by direction (I → II
→ III → I) safe zone position is changed successively.
Learning test：Reach the number of shocks needed in continuous 10 times having before 9 (9/10) correct responses with test to taste
Examination frequency table shows learning ability (Ya Li, Hai-Qiang Qin, Qi-Sheng Chen, et al.Behavioral and
neurochemical effects of the intrahippocampal co-injection of β-amyloid
protein 1-40 and Ibotenic acid in rats.Int J Neurosci,2004,114(12):1521-
1531).Memory reproduces test：Learning test is in kind carried out after completing 24h, to reach the number of attempt before 9/10 standard
Represent memory capability (Ma Yiming, the influence Pharmacology and Clinics of Chinese Materia Medicas for He Qing tuber of multiflower knotweed god's Granules on Mouse ability of learning and memory of shutting out
.2001,17(4):35-37.)。
1.3.5 brain tissue AChE, BuChE determination of activity
Rat broken end takes brain, takes out Rat hippocampus immediately on 0 DEG C of ice bag, weighs 100mg, puts 10ml plastics with cover
In centrifuge tube, precooling NS 1.0ml are added into pipe rapidly, are fully homogenized with the 500r/min of XHF-1 high speed dispersers 1；4 DEG C from
Supernatant is taken after the heart 3 000r/min, 10min.Total protein content in Coomassie Brilliant Blue measure hippocampal tissue, azanol colorimetric method
Determine the activity of acetylcholinesterase (AChE) and butyrylcholine esterase (BuChE).
1.3.6 statistical method：
Experimental data mean ± standard deviationRepresent, variance is carried out with the statistical softwares of GraphPad Prism 5
Analyze and compare two-by-two, statistical analysis is carried out to data, with P<0.05 is statistically significant.
1.4 experimental result
1.4.1 rat ordinary circumstance
In rat drug treatment, the basic, normal, high dosage group rats eating of the turbid powder of strengthening the essenceization is multiple compared with sham-operation group, brain
The increase of health control group.Rats in sham-operated group activity freely, be quick on the draw, hair is glossy.Model group rats activity is reduced, reaction
Blunt, hair curling, gloss is not good enough.The middle and high dosage group rat state of mind of the turbid powder of strengthening the essenceization is good, it is movable freely, reactivity it is good,
Body weight increase is very fast, hair is glossy, hence it is evident that better than other each group rats.
1.4.2 influence of the turbid particle of strengthening the essenceization to AD rat learning abilities
The Y-Shaped maze method that this research is selected is the test more reliable method of rat behavior, extensively should be obtained at home
With (Zhao Chongkan, Cheng Guang, Chen Qi contain a kind of intelligentized Y-Shaped labyrinth [J] China applied physiology magazines of, 1997,13 (4)：
363-364.).Experimental selection has 9 times or more correct responses (correct response rate >=90%) big as judging using in continuous 10 times
The standard of mouse association, with the learning and remembering ability of overall merit rat.Rat study reach before association's standard needed for trial time
Number, represent study, the Memory result of the explanation reaction of its space.Required number more save your breath that bright pace of learning is faster or learning ability more
By force；Required number increases, and prompts learning and remembering ability to decline.
Before modeling：In the experiment of Y-Shaped labyrinth stimulator, each group rat reach before 9/10 correct response needed for trial time
Number no significant difference (P ＞ 0.05).
After modeling：Compared with sham-operation group, the number of attempt needed for model group rats and each treatment group rat significantly increases
Add (P ＜ 0.01)；Compared with model group, the number of attempt no significant difference (P ＞ 0.05) of each treatment group rat.
After treatment：Compared with sham-operation group, the piracetam and turbid powder of strengthening the essenceization is small, the number of attempt needed for middle dose group rat
There were significant differences (P ＜ 0.05 or P ＜ 0.01)；Compared with model group, piracetam and the middle and high dosage group rat institute of the turbid powder of strengthening the essenceization
The number of attempt needed substantially reduces (P ＜ 0.01).(being shown in Table 2)
The turbid powder of the strengthening the essenceization of table 2. to AD rat learning abilities influence (N=10)
Compared with sham-operation group,*p<0.05,**p<0.01；Compared with model group,ΔΔp<0.01
1.4.3 influence of the turbid powder of strengthening the essenceization to AD Rats With Memory abilities
Before modeling：In the experiment of Y-Shaped labyrinth stimulator, each group rat reach before 9/10 correct response needed for trial time
Number no significant difference (P ＞ 0.05).
After modeling：Compared with sham-operation group, the number of attempt needed for model group rats and each treatment group rat significantly increases
Add (P ＜ 0.01)；Compared with model group, the number of attempt no significant difference (P ＞ 0.05) of each treatment group rat.
After treatment：Compared with sham-operation group, the number of attempt needed for model group rats dramatically increases (P ＜ 0.05)；With mould
Type group is compared, and the required number of attempt of piracetam group and the middle and high dosage group rat of the turbid powder of strengthening the essenceization substantially reduces (P ＜
0.05).(being shown in Table 3)
The turbid particle of the strengthening the essenceization of table 3 to AD Rats With Memory abilities influence (N=10)
Compared with sham-operation group,*p<0.05,**p<0.01；Compared with model group,Δp<0.05
Study, memory disorders are AD important clinical performances, and in animal, learning and memory can only pass through exercisable time
Process (behavior for triggering conventional acquistion by appropriate stimulation) is recalled to detect.This experiment is detected using Y-Shaped labyrinth stimulator
Rat learning and remembering ability.Reach the number of shocks needed in continuous 10 times having before 9 (9/10) correct responses with test to taste
Examination frequency table shows learning ability；Tested again after learning ability tests completion 24h, to reach the number of attempt before 9/10 standard
Represent memory capability.
As a result it is visible, study, memory disorders are shown after IBO injury rats bilaterals NBM, required number of attempt is obvious
Increase (P ＜ 0.01), show that IBO causes rat study, memory disorders.Model group rats learning and remembering ability is worst, in Y-
In the stimulator experiment of type labyrinth, required number of attempt substantially increases (P ＜ 0.01).And significantly reduced after giving the turbid powder of strengthening the essenceization
Number of attempt needed for AD rats, compared with model group, strengthening the essence Hua Zhuofenge treatment groups statistically significant (P ＜
0.01)。
As a result prompt, the turbid particle of strengthening the essenceization can significantly improve the learning and remembering ability of AD rats.
1.4.4 the turbid powder of strengthening the essenceization is to AD Rat hippocampus acetylcholinesterase (AChE), butyrylcholine esterase (BuChE)
The influence of activity
(1) influence of the turbid powder of strengthening the essenceization to AD Rat hippocampus AChE activity
Model group rats hippocampal tissue AChE activity significantly raises (P ＜ 0.01) compared with sham-operation group；Small dose of the turbid powder of strengthening the essenceization
Amount group AChE activity is higher than sham-operation group (P ＜ 0.05)；Piracetam group and each dosage group Rat hippocampus of the turbid powder of strengthening the essenceization
AChE activity significantly reduces (P ＜ 0.01) compared with model group；The turbid powder small dose group AChE activity of strengthening the essenceization is higher than piracetam group (P
＜ 0.05).The middle and high dosage group of the turbid powder of strengthening the essenceization no significant difference (P ＞ 0.05) compared with sham-operation group.(being shown in Table 4).
The turbid powder of the strengthening the essenceization of table 4 to AD Rat hippocampus AChE activity influence (N=10)
Compared with sham-operation group,*p<0.05,**p<0.01；Compared with model group,ΔΔp<0.01
Compared with piracetam group,#p<0.05
As a result show, after model group rats cholinergic neuron is impaired, cerebral hippocampal tissue AChE activity shows compared with sham-operation group
Write rise (P ＜ 0.01)；But each dosage group Rat hippocampus AChE activity of the turbid powder of strengthening the essenceization significantly reduces (P ＜ compared with model group
0.01), the middle and high dosage group of the turbid powder of strengthening the essenceization no significant difference (P ＞ 0.05) compared with sham-operation group.
As a result prompt, the middle and high turbid powder of dosage strengthening the essenceization has preferable AChE inhibitory action.The turbid powder of strengthening the essenceization passes through suppression
AChE, ACh decomposition is reduced, so as to improve the study of AD rats, memory disorders.
(2) influence of the turbid powder of strengthening the essenceization to AD Rat hippocampus BuChE activity
Compared with sham-operation group, the significantly rise (P ＜ 0.05) of model group BuChE activity；Compared with model group, piracetam group
Dosage group BuChE activity middle and high with the turbid powder of strengthening the essenceization is obvious to reduce (P ＜ 0.05).(being shown in Table 5)
The turbid particle of the strengthening the essenceization of table 5 to AD Rat hippocampus BuChE activity influence (N=10)
Compared with sham-operation group,*p<0.05；Compared with model group,Δp<0.05
As a result show, the significantly rise (P ＜ 0.05) of model group rats hippocampal tissue BuChE activity, and piracetam and strengthening the essence
Change the middle and high dosage group Rat hippocampus BuChE activity of turbid powder substantially reduces (P ＜ 0.05) compared with model group.
As a result prompt, the turbid powder of strengthening the essenceization increases intracerebral ACh contents by suppressing hippocampal tissue AChE and BuChE activity
Add, so as to improve the learning and remembering ability of AD rats.
1.5 conclusion
1. the turbid powder of strengthening the essenceization can improve the learning and remembering ability that IBO damages bilateral NBM establishes AD rat models, big to AD
Mouse central cholinergic system has preferable protective effect.
2. the turbid powder of strengthening the essenceization suppresses ACh and decomposed by reducing brain tissue AChE and BuChE activity；Increase intracerebral ACh contains
Amount, so as to improve the study of AD rats, memory disorders.
Test example 2
2.1 experiment purpose：The turbid powder of strengthening the essenceization is observed to AD learning and memory in rats ability, hippocampal tissue morphosis, cell
The influence of apoptosis and the protective effect to brain tissue inflammation's damage
The basic pathology histologic characteristics of Alzheimer disease is to be formed in big intracerebral with amyloid-beta (beta-
Amyloid peptide, A β) be core neuritic plaque.A large amount of zooperies show：It can be led in the brain areas such as hippocampus injection A β fragments
The learning and remembering ability similar to AD is caused to decline, simulation inflammation-related factor change, nerve cell apoptosis, and there are A β depositions
Deng, can successfully prepare AD rat models (Liu Hui, Chen Jun throw, Tian Shiyu, wait hippocampal injections amyloid beta to rat learn
Memory and damaging action [J] Chinese Journal of Neurology of local neuron, 2000,33 (3):150-152.).Can be more complete
The behaviouristics that face simulation goes out mankind AD changes and part typical pathologic features.
Therefore the A β that this research plays the role of a nucleus from AD pathogenesis start with, and enter rat hippocampus using A β micro-injections
The AD rat models close to clinical pathology change are established, learning and memory in rats ability is detected by electric maze stimulator；Nissl contaminates
Color observes rat hippocampus Morphology；Enzyme linked immunosorbent assay (ELISA) detects hippocampal tissue tumor necrosis factor-alpha
(TNF-α) and interleukin-1 ' beta ' (IL-1 β) are horizontal.Flow cytomery Apoptosis of Hippocampal Neurons rate.Observe strengthening the essence
Therapeutic action of the turbid powder to AD rats.
2.2 experiment material
2.2.1 experimental animal：With test example 1
2.2.2 Experimental agents：With test example 1
2.2.3 main agents and instrument
Amyloid-beta dry powder (A β 1-40) (Sigma Co., USA).Nissl's staining agents useful for same：Dimethylbenzene, ethanol,
Cresyl viollet solution, optics natural gum etc. are that import or domestic analysis are pure.Leica CM1900 cryostats freezing microtome (Germany
Leica companies)；Light microscope, Japanese OLYMPUS companies；BD FACS Calibur flow cytometers (USA).Remaining is the same as real
Apply example 1.
2.3 method
2.3.1 A β 1-40 incubation
A β 1-40 are diluted to 2 μ g/ μ l by sterile saline, are incubated 1 week at 37 DEG C, so that it becomes the A β of coherent condition.
2.3.2 animal screening and packet
Filtered out with Y-Shaped labyrinth stimulator be swift in response, active 60 SD rats, be randomly divided into 6 groups, every group 10
(referring to table 6).
The each group experimental animal situation of table 6
2.3.3 prepared by model
Each group rats by intraperitoneal injection Chlorpent compound anesthetics 2ml/kg is anaesthetized, and is fixed on Naoliqing capsule after anesthesia
Instrument, the cropping of calvarium portion, after the tincture of iodine and alcohol disinfecting, skin is cut under aseptic condition, the outer periosteum of separation skull, appears bregma, remembers
Record bregma A (sagittal axis), L (frontal axis), V (vertical axis) coordinate, reference《Rat brain stereotaxic atlas》Determine bilateral sea
Horse CA1 zone positions, the elements of a fix：3.0mm after bregma, center line left and right side 2.0mm, Subdural space 2.8mm, bored at skull positioning
One aperture, slowly vertical inserting needle to target spot, gives bilateral hippocampus each disposable μ l of injection condensed state A β 1-40 5, and injection speed is
0.5 μ l/min, let the acupuncture needle remain at a certain point 5min, to ensure the abundant disperse of solution, then slowly remove pin, without skin suture after intracranialing hemorrhage, prepare AD
Model (George Paxions and Charles Watson.The rat brain in stereotaxic
coordinates.5th ed.USA:Elsevier Academic Press,2005:36-55).Sham-operation group injection equivalent life
Salt solution is managed, remaining operation is the same.All operations are aseptically carried out, the equal sub-cage rearing of Post operation rat.
2.3.4 experimental drug
The turbid powder of strengthening the essenceization and piracetam are prepared with 0.5% sodium carboxymethylcellulose.Positive controls give 0.4g/kg brains
Multiple health.The drug concentration ratio of the basic, normal, high dosage group of the turbid powder of strengthening the essenceization is followed successively by 1:2:4 (1.5,3.0,6.0g/kg), in postoperative
2d, start gastric infusion after the completion of Behavior test, administration capacity is 10ml/kg.Sham-operation group, model control group are given
The capacity physiological saline gavage such as give, 1 time a day, Behavior test again after successive administration 30d.
2.3.5 learning and memory in rats aptitude tests (Y-Shaped labyrinth stimulator test)
After 30d is administered, every group of SD rat is using the learning and remembering ability of MG-B types labyrinth stimulator detection rat.Tool
Body examination method for testing is the same as test example 1.
2.3.6 Nissl's staining (Nissle ' s staining) observation Hippocampal CA 1 morphological change
Behaviouristics determines the SD rats after terminating, and each group takes 2 rats perfusions to fix, takes out brain and carry out Coronal at random
Frozen section, the thickness of hippocampal tissue position section is 20 μm.Section is attached on the slide of poly-D-lysine coating, OK
Nissle is dyed, the micro- Microscopic observation Hippocampal CA 1 morphological changes of Leica.
2.3.7ELISA method detection Rat hippocampus Tumor necrosis factor-α (TNF-α) and leucocyte are situated between
The change of -1 β of element (IL-1 β) expression
Each group rat hippocampus are separated, take 100mg hippocampal tissues, is homogenized, takes supernatant to be grasped by ELISA kit method
Make, determine TNF-α, IL-1 β level in each group Rat hippocampus.
2.3.8 flow cytomery Apoptosis of Hippocampal Neurons rate
After each group rat medication terminates, yellow Jackets (40mg/kg) intraperitoneal anesthesia, after broken end takes brain, with cleaning for PBS
Blood in brain tissue, hippocampal tissue is taken out, coating and blood vessel are removed under surgical operation microscope.System matches somebody with somebody single cell suspension, through solid
After fixed, filtering and PI dyeing, the double mark dyeing detection apoptosis rates of-PI streamings of Annexin V.Experiment is repeated 3 times.
2.3.9 data processing and statistical analysis:With test example 1.
4 experimental results：
2.4.1 rat ordinary circumstance
Freely, reaction is quick, and hair is glossy for rats in sham-operated group activity, and diet, urine excrement are normal, body mass stable increase.
Activity is increasingly less freely after model control group rat modeling, and reaction owes sensitive, phenomena such as occurring being slow in action and be One's spirits are drooping,
Hair is crimped, and gloss is not good enough, and food-intake is slightly reduced, and increased weight speed is slow.Each dosage group of the turbid powder of piracetam group, strengthening the essenceization
Activities in rats and general body state are close to rats in sham-operated group, the change unobvious compared with before modeling.
2.4.2 influence of the turbid powder of strengthening the essenceization to each group rat learning and remembering ability
Study, impaired memory function are one of initial and most important behaviouristics changes of AD.A β are injected and are deposited on hippocampus
Neurotoxic effect can be directly produced in hippocampus, causes the pathological changes such as Neuron Apoptosis, its neurotoxic effect can also
By damaging cholinergic neuron, cause the functional lesion of hippocampal neuron indirectly, in turn result in serious study, memory function
Missing (Bornemann KD, Wiederhold KH, Pauli C.A beta induced inflammatory
processes in microglia cells of APP23 transgenic mice[J].Am J Pathol,2001,158
(1):63-73.)。
(1) influence of the turbid powder of strengthening the essenceization to each group rat learning ability：
As a result visible, compared with sham-operation group, model control group, piracetam control group, each dosage group of the turbid powder of strengthening the essenceization is big
The number of attempt that mouse reaches needed for 9/10 correct response dramatically increases (P<0.01 or P<0.05)；The turbid powder of strengthening the essenceization is low dose of
Number of attempt needed for group rat is apparently higher than piracetam group (P<0.01).Compared with model control group, piracetam control group and
Number of attempt needed for strengthening the essence Hua Zhuofenge treatment groups rat substantially reduces (P<0.01).(being shown in Table 7).
(2) influence of the turbid powder of strengthening the essenceization to each group Rats With Memory ability：
As a result it is visible, model control group rat reach before 9/10 correct response needed for number of attempt it is more notable than sham-operation group
Increase (P<0.01)；Compared with model group, the number of attempt needed for piracetam control group and strengthening the essence Hua Zhuofenge treatment groups rat
Substantially reduce (P<0.01).(being shown in Table 7)
The turbid powder of the strengthening the essenceization of table 7 to AD learning and memory in rats abilities influence (N=10)
Compared with sham-operation group,*p<0.05,**p<0.01；Compared with model group,ΔΔp<0.01；
Compared with piracetam group,##p<0.01
As a result prompt, the turbid powder of strengthening the essenceization can be obviously improved A β1-40The study of AD rat models caused by hippocampus micro-injection,
Memory capability.
2.4.3 the turbid powder of strengthening the essenceization is on the pathological influence in each group CA 1 of Hippocampus
Nissl's staining result is visible：Rats in sham-operated group brain tissue structure is normal, and hippocampus neuron is in hyacinthine, cell
The light dye in core area, karyon is big and justifies, and has no that neure damage and tigroid body are lost.Hippocampus cone neurone arranges in fence sample,
Neat rule, endochylema dyeing is clear, and tigroid body enriches.(see Fig. 2-A)
There is neuronal degeneration necrosis in model control group rat cerebral tissue, and hippocampus neuron still arranges in paliform,
But level and cell number significantly reduce, and normal configuration obscures, cell space swelling, arrangement is at random, and large area in endochylema
Tigroid body is lost, and dyeing is light.(see Fig. 2-B)
The turbid powder middle dosage of piracetam control group, strengthening the essenceization, high dose group rat hippocampus area pathological change are lighter, neuronal layers
Secondary complete, arrangement is still neat, and endochylema dyeing is deeper, and cellular morphology is significantly better than model control group, and the turbid powder low dosage of strengthening the essenceization
Group rat cerebral tissue lesion is heavier, close to model control group.(see Fig. 2-C, D, E, F).
As a result prompt, the turbid powder of strengthening the essenceization can mitigate A β1-40The neuron damage of AD rat models caused by hippocampus micro-injection
Wound, protect hippocampal neurons.
2.4.4 the turbid powder of strengthening the essenceization is to each group Rat hippocampus TNF-α, the influence of IL-1 β expression
It is now recognized that inflammatory reaction caused by amyloid-beta (A β) deposition is Alzheimer disease (AD) central pathological machine
One of system.
As a result visible, the turbid powder of model control group, strengthening the essenceization is small, the expression of middle dose group Rat hippocampus TNF-α
Obviously higher than sham-operation group (P ＜ 0.05 or P ＜ 0.01).The heavy dose of group of the turbid powder of strengthening the essenceization is compared with sham-operation group without obvious poor
Different (P ＞ 0.05).The expression and model comparison of piracetam group and each dosage group Rat hippocampus TNF-α of the turbid powder of strengthening the essenceization
Group more substantially reduces (P ＜ 0.01).It the results are shown in Table 8.
As a result it is visible, compared with sham-operation group, model group, piracetam group, the turbid powder of strengthening the essenceization are small, middle dose group rat hippocampus
Organize IL-1 β expressions increase (P ＜ 0.05 or P ＜ 0.01)；Compared with model group, each dose of the turbid powder of piracetam group, strengthening the essenceization
Amount group Rat hippocampus IL-1 β expressions significantly reduce (P ＜ 0.01)；The turbid powder small dose group IL-1 β expression of strengthening the essenceization
Level is higher than piracetam group (P ＜ 0.01), the middle and high dosage group of the turbid powder of strengthening the essenceization no significant difference (P ＞ compared with piracetam group
0.05).It is shown in Table 8.
The expression of table 8. each group Rat hippocampus TNF-α, IL-1 β
Compared with sham-operation group,*p<0.05,**p<0.01；Compared with model groupΔΔp<0.01；Compared with piracetam group,##p
<0.01
As a result prompt, the turbid powder of strengthening the essenceization can effectively be pressed down by reducing the content of IL-1 β of AD rat cerebral tissues, TNF-α
Hippocampal neuron inflammatory reaction processed.
2.4.5 flow cytomery apoptosis rate
Neuron loss in AD brains is mainly occurred in the form of apoptosis.Early apoptosis and late apoptic ratio sum are made
For total apoptosis rate.Flow cytometer showed result shows that model group (A β groups) apoptosis of hippocampus neurons percentage is notable compared with sham-operation group
Raise (P<0.01)；Piracetam control group and each medication group apoptosis of hippocampus neurons percentage of the turbid powder of strengthening the essenceization are higher than sham-operation group
(P<0.05)；But piracetam control group and each medication group apoptosis of hippocampus neurons percentage of the turbid powder of strengthening the essenceization significantly drop compared with model group
Low (P<0.05) (see Fig. 3).Although the turbid powder of strengthening the essenceization is small, middle and high dosage group relatively without significantly significant difference, one
Determine in dosage range with the increase of the turbid pulvis amount of strengthening the essenceization, the effect for reducing apoptosis rate is in increase tendency.
As a result prompt：The beta induced apoptosis of hippocampus neurons of A, the turbid powder of strengthening the essenceization can suppress the beta induced rat hippocampus nerves of A
First apoptosis.
2.5 conclusion
(1) the turbid powder of strengthening the essenceization can improve the learning and remembering ability of the beta induced AD rat models of A.
(2) the turbid powder of strengthening the essenceization has protective effect to the brain tissue pathology change of AD rat models.
(3) the turbid powder of strengthening the essenceization can significantly reduce the beta induced AD rat inflammation factors interleukin-1 ' beta 's (IL-1 β) of A, swell
Tumor necrosis factor-α (TNF-α) level, mitigate intracerebral inflammatory reaction.
(4) the turbid powder of strengthening the essenceization can substantially suppress the beta induced AD apoptosis of hippocampal neurons in rats of A, play neuroprotection and resist
Dementia effect.
Test example 3
3.1 experiment purpose：Therapeutic action of the turbid powder of strengthening the essenceization to AD is studied, inquires into the damage of its Green Tea Extract, protection nerve
The mechanism of member.
AD radical damage theory thinks：Free radical can promote A beta peptide aggregations, it is caused nerve in intracerebral overacfivity
First retrogression, neuron loss, AD occurs.A β can cause the increase of response to oxidative stress, make cholinergic system work(in AD brains
It can reduce；And A β depositions are aggravated after cholinergic system damage, promote the development of the AD state of an illness.
(a kind of new senile dementia animal model [J] Aged in China of the bright of Luo Huanmin, Chen Zi is miscellaneous for the report such as Luo Huanmin
Will, 2003,23 (3)：179~82), D-gal and NaNO2The standby Senlie dementia model of combination system simulates AD characteristics of incidence,
It is a kind of preferable model for studying AD and its medicine.Mouse long term injections D-gal, its metabolite galactitol can not be by
Further it is metabolized and is stacked on intracellular, increase osmotic pressure, causes cellular swelling, the rise of activity in vivo oxygen level, make cell
Membrane lipid is damaged；The oxidized enzymes of D-gal or dehydrogenase catalyzed oxidation are formed during xylulose with a large amount of free radicals such as
And H2O2Generation, cause the biological damage effect of Mediated by Free Radicals.Meanwhile galactolipin can also make cranial nerve cell produce one
There is class senile plaque expelling pathological change, hippocampus and Cerebral Cortex Neuronal Cells degeneration necrosis, make nerve in serial degeneration change, intracerebral
Cell number is reduced, and SOD vigor reduces in brain tissue, the rise of MDA contents, shows as ability of learning and memory decline.Mouse is longer
Period continuously injects NaNO2, NaNO2With hemoglobin combine make its denaturation, cause brain and body tissue repeatedly long-term ischemic, lack
Oxygen, Memory Process is damaged, destroy memory consolidation, be decreased obviously ability of learning and memory in mice.Therefore, this experiment uses D-gal
And NaNO2Establish AD mouse models, therapeutic action of the observation turbid powder of strengthening the essenceization to AD.
3.2 experiment material
3.2.1 experimental animal：ICR mouse, male and female half and half, cleaning grade, 20 ± 2g of body weight, by Nantong University experimental animal
The heart provides, credit number：Credit number：SYXK (Soviet Union) 2007-0021.
3.2.2 Experimental agents：With test example 1
3.2.3 main agents and instrument：
D- galactolipins：Shanghai Heng Xin chemical reagent Co., Ltd, (lot number：20100828).Natrium nitrosum：Shantou, Guangdong city
Western Gansu Province chemical plant, (lot number：20100813).Coomassie Brilliant Blue surveys protein reagent box, (lot number：20091225)；MDA
(malondialdehyde, MDA) kit, (lot number：20100831)；Superoxide dismutase (superoxide
Dismutase, SOD) kit, (lot number：20090914)；ATP enzyme kit (lot number：20091208)；Nanjing is purchased to build
Into biomedical engineering research institute.MG-B types labyrinth stimulator (emerging teaching machinery plant of Zhangjagang City three).The program control water mazes of SMG-2
(institute of Materia Medica,Chinese Academy of Medical Sciences development).Remaining is the same as test example 1.
3.3 method
3.3.1 animal packet, model preparation and experimental drug
Normal 90 mouse of ability of learning and memory are filtered out with labyrinth stimulator.6 groups are randomly divided into, Normal group
(Normal), model control group (Model), positive drug piracetam (0.4gkg-1·d-1) control group (NFK), the turbid powder of strengthening the essenceization
Small (YJHZF-L, 2.4g/kg)), in (YJHZF-M, 4.8g/kg), big (YJHZF-H, 9.6g/kg) dosage group, every group 15.
Normal group mouse peritoneal injection physiological saline, model control group and each administration group intraperitoneal injection 1%D- galas
Sugared 100mg/kg and 1% natrium nitrosum 90mg/kg, 1 time/d, continuous 60d.The turbid powder of strengthening the essenceization and piracetam are with 0.5% carboxylic first
Base sodium cellulosate is prepared, and starts gastric infusion while each group animal model, 1 time/d, continuous 60d, capacity, which is administered, is
10ml/kg.Normal group and model control group with etc. capacity physiological saline gavage, continuous 60d.
3.3.2 ability of learning and memory is tested
After administration terminates, each group takes 10 mouse to carry out ability of learning and memory test using the program control water mazes of SMG-2 at random.
Starting point, terminal (step) and many places cecum are set in the program control water mazes of SMG-2.In 22 DEG C of depth of water 9cm, 25 ± 2 DEG C of water temperature, room temperature bars
Under part, record mouse is put into water to the time for finding step, i.e. escape latency (escape latency) from starting point, if exceeding
180s is then recorded by 180s.1~3d after last dose, swimming instruction is carried out to each group mouse, and distance of swimming daily extends one
Individual cecum, 4d are tested, and record the escape latency of mouse.
3.3.3 brain tissue biochemical indicator detects
After Behaviors survey terminates, by each group mouse sacrificed by decapitation, cerebral cortex and sea are taken out immediately on 0 DEG C of ice bag
Horse, 100mg is weighed, by the use of the physiological saline of precooling as homogenate, 10% brain tissue homogenate is made, with refrigerated centrifuge 4
3000r/min under the conditions of DEG C, 10min is centrifuged, take supernatant to survey total protein content, Coomassie Brilliant Blue determines total in brain tissue
The content of albumen, thiobarbituricacidα- method determine the content of MDA in brain tissue, and xanthine oxidase determines SOD in brain tissue
Vigor, zymetology aerodynamic method measure brain tissue in Na+-K+-ATP、Ca2+-Mg2+The vigor of-ATP enzyme.
3.3.4 data processing and statistical analysis：With test example 1
3.4 result
3.4.1 influence of the turbid powder of strengthening the essenceization to each group ability of learning and memory in mice
Water maze laboratory result is visible：Model control group mouse escape latency is obviously prolonged (P ＜ than sham-operation group
0.01).Compared with model control group, piracetam group and each dosage group mouse escape latency of the turbid powder of strengthening the essenceization significantly reduce (P
＜ 0.05 or P ＜ 0.01).It the results are shown in Table 9.
As a result prompt, the turbid powder of strengthening the essenceization can significantly improve D- galactolipins (D-Gal) and natrium nitrosum (NaNO2) intraperitoneal injection
Establish the learning and remembering ability of AD mouse models.
The each group Mice water maze of table 9 tests the change of escape latency
Compared with sham-operation group,**p<0.01；Compared with model group,Δp<0.05,ΔΔp<0.01
3.4.2 influence of the turbid powder of strengthening the essenceization to AD rat model brain tissue MDA and SOD contents
As a result visible, AD model group MDA contents are apparently higher than sham-operation group (P ＜ 0.01).Piracetam control group and strengthening the essence
Change each dosage group MDA contents of turbid powder and be substantially less than model group (P ＜ 0.05 or P ＜ 0.01)；Each dosage group MDA of the turbid powder of strengthening the essenceization
Content is less than piracetam group, but not statistically significant (P ＞ 0.05).
AD model groups SOD activity be substantially less than sham-operation group (P ＜ 0.01), piracetam control group and the turbid powder of strengthening the essenceization it is small,
Middle dose group SOD activity is still significantly lower than sham-operation group (P ＜ 0.05 or P ＜ 0.01)；The middle and high dosage group SOD of the turbid powder of strengthening the essenceization
Content is higher than model group (P ＜ 0.05)；The middle and high dosage group SOD contents of the turbid powder of strengthening the essenceization are higher than piracetam group (P ＜ 0.05).Knot
Fruit is shown in Table 10.
Table 10 each group brain tissue MDA and SOD change (N=8)
Compared with sham-operation group,*p<0.05,**p<0.01；Compared with model group,Δp<0.05,ΔΔp<0.01
Compared with piracetam group,#p<0.05
As a result prompt, the turbid powder of strengthening the essenceization can improve the Scavenging activity of free radical, suppress intracerebral by increasing SOD activity
MDA is generated, and reduces lipid peroxidation injury degree, suppresses oxidative stress, protects brain tissue, mitigates damage of the free radical to brain tissue
Evil, so as to improve the ability of learning and memory of AD mouse, plays the therapeutic action to AD.
3.4.2 the turbid powder of strengthening the essenceization is to AD model mice brain tissues Na+-K+- ATP enzyme and Ca2+-Mg2+The shadow of-ATP enzyme vigor
Ring
Studies have shown that model control group brain tissue Na+-K+- ATP enzyme vigor is less than sham-operation group (P ＜ 0.05).Piracetam
Control group and the middle and high dosage group brain tissue Na of the turbid powder of strengthening the essenceization+-K+- ATP enzyme vigor is apparently higher than model control group (P ＜ 0.05
Or P ＜ 0.01).
Model control group brain tissue Ca2+-Mg2+- ATP enzyme vigor is significantly lower than sham-operation group (P ＜ 0.05).Piracetam pair
According to group and the middle and high dosage group brain tissue Na of the turbid powder of strengthening the essenceization+-K+- ATP enzyme vigor is apparently higher than model control group (P ＜ 0.05 or P
＜ 0.01).It the results are shown in Table 11.
The each group Mice brain tissues ATP enzyme vigor of table 11 change (N=8)
Note：Compared with sham-operation group,*p<0.05；Compared with model group,Δp<0.05,ΔΔp<0.01
As a result prompt, the turbid powder of strengthening the essenceization is by raising Na in brain tissue+-K+-ATP、Ca2+-Mg2+- ATP enzyme vigor, regulation
Cell traffic function, prevents nerve cell death.This is probably one of this medicine preventing and treating AD mechanism of action.
4 conclusions：
(1) the turbid powder of strengthening the essenceization can significantly improve D-gal and NaNO2 intraperitoneal injections and establish the study of AD mouse models, memory
Ability.
(2) the turbid powder antioxidant radical damage of strengthening the essenceization：Raise brain tissue superoxide dismutase (SOD) activity, suppress third
Dialdehyde (MDA) generates, and mitigates radical damage.
(3) the turbid powder rise Na+-K+-ATP enzymes of strengthening the essenceization and Ca2+-Mg2+- atpase activity, improve the intracerebral energy of AD mouse
Dysbolism is measured, so as to play neuroprotection and anti-dementia effect.
The foregoing examples are merely illustrative of the technical concept and features of the invention, its object is to allow person skilled in the art's energy
Solution present disclosure much of that is simultaneously implemented according to this, and it is not intended to limit the scope of the present invention.It is all spiritual according to the present invention
The equivalent transformation or modification that essence is done, should all be included within the scope of the present invention.
Claims (6)
1. a kind of pharmaceutical composition for treating Alzheimer disease, it is characterised in that it is made from the following raw materials in parts by weight：System
1.5 parts of the tuber of multiflower knotweed, 1.5 parts of ganoderma lucidum, 1 part of leech, 1 part of the wind-weed.
2. pharmaceutical composition according to claim 1, it is characterised in that described pharmaceutical composition is pulvis, powder, piece
Agent, capsule.
A kind of 3. method of the pharmaceutical composition of the treatment Alzheimer disease prepared described in claim 1, it is characterised in that press
Ratio takes RADIX POLYGONI MULTIFLORI PREPARATA, ganoderma lucidum, leech, the wind-weed respectively, adds water to cook secondary, is staticly settled after collecting decoction, filters, filtrate is existed
Less than 80 DEG C are concentrated under reduced pressure into thick paste, and dry, pulverize below 80 DEG C, sieving, obtain extract powder, and packing is standby.
4. preparation method according to claim 3, it is characterised in that add water to cook secondary, 2 hours first times, second
1.5 hour.
5. preparation method according to claim 3, it is characterised in that per g extract powders 2-5g containing crude drug.
What 6. a kind of any one of pharmaceutical composition or claim 3-5 described in any one of claim 1-2 preparation method obtained
Pharmaceutical composition is used for the purposes for preparing treatment Alzheimer disease drugs.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1651067A (en) * | 2004-12-29 | 2005-08-10 | 通化衛京药业股份有限公司 | Medicinal composition for treating and/or preventing heart brnin blood vessel disease and its preparation method |
| CN1679884A (en) * | 2005-02-04 | 2005-10-12 | 张海峰 | Injection of Naoxinkang for brain and heart and its making method |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1651067A (en) * | 2004-12-29 | 2005-08-10 | 通化衛京药业股份有限公司 | Medicinal composition for treating and/or preventing heart brnin blood vessel disease and its preparation method |
| CN1679884A (en) * | 2005-02-04 | 2005-10-12 | 张海峰 | Injection of Naoxinkang for brain and heart and its making method |
Non-Patent Citations (2)
| Title |
|---|
| 脑益康对D_半乳糖损伤的海马神经元的保护作用;朱燕;《中国病理生理杂志》;20090115;第25卷(第1期);第93页右栏第1段 * |
| 醒脑益智方对SAM_P_10老化痴呆鼠海马超微结构的影响;王平;《湖北中医学院学报》;20010630;第3卷(第2期);第18页摘要、右栏最后1段 * |
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Inventor after: Jiang Wei Inventor after: Zhou Ailing Inventor after: Zhang Xian Inventor after: Zhu Yan Inventor after: Mao Jiahui Inventor after: Hu Yae Inventor after: Geng Jinsong Inventor before: Zhou Ailing Inventor before: Jiang Wei Inventor before: Zhang Xian Inventor before: Zhu Yan Inventor before: Mao Jiahui Inventor before: Hu Yae Inventor before: Geng Jinsong |
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Effective date of registration: 20201016 Address after: 226019 No.205, building 6, Nantong University, No.9, Siyuan Road, Nantong City, Jiangsu Province Patentee after: Center for technology transfer, Nantong University Address before: 226019 Jiangsu city of Nantong province sik Road No. 9 Patentee before: NANTONG University |