CN102772418B - Application of pachysamine/pachysandrine G - Google Patents
Application of pachysamine/pachysandrine G Download PDFInfo
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Abstract
The invention provides an application of pachysamine/pachysandrine G. Specifically, the pachysamine/pachysandrine G can be applied as an acetylcholinesterase inhibitor, also can be used for preventing or treating senile dementia, and has obvious effects and development and utilization values.
Description
Technical field
The present invention relates to the purposes of pachysamine alkali G, be specifically related to pachysamine alkali G as acetylcholinesteraseinhibitors inhibitors and the application in the medicine of preparation prevention or treatment alzheimer disease.
Background technology
Alzheimer (Alzheimer ' s disease, AD) claim again alzheimer disease, be a kind of carrying out property neurodegenerative diseases, to hide onset, memory is faded, and cognitive disorder, dystropy and social obstacle etc. are main clinical manifestation.With extracellular, there is beta amyloid peptide (β-Amyloid protein, A β) assemble senile plaque (the senile plaque forming, SP), in cell, Protein tau Abnormal Phosphorylation forms neurofibrillary tangles (neurofibrillary tangles, NFTs) and neurocyte and the minimizing of synapse quantity etc. are main pathological characters.According to statistics, global old dementia patients surpasses 1,700 ten thousand, and China has more than 500 ten thousand, account for global 1/4th, over-65s elderly dementia prevalence is up to 10.1%, and mortality rate is only second to cardiovascular and cerebrovascular disease and cancer, and sickness rate increases with the growth at age.In recent years, along with the continuous increase of aging population, the research of alzheimer disease is also more and more received to more concern.Therefore, the further investigation tool of AD medicine be of great significance and become a study hotspot both domestic and external.Studies confirm that, in the generation of alzheimer disease and brain in patients, neurotransmitter---the disappearance of acetylcholine is closely related, the lytic response of acetylcholine esterase meeting catalysis acetylcholine, the disappearance that causes acetylcholine, nerve signal transmission failure is mainly to improve the levels of acetylcholine in patient body by suppressing acetylcholine esterase to the Drug therapy of senile dementia at present.
1907, after the clinical manifestation and pathological change of German scholar Alois Alzheimer reported first AD, the research and discovery deepening continuously through countless scientific research personnel, does not still find a kind of pathogenesis can explain definitely AD, does not find a kind of medicine of specially good effect to treat AD yet.Three liang of silver of Miao Ethnomedicine are dry root or the Herb of the wild fan flower of Buxaceae Sarcococca plant Sarcococca ruacifolia Stapf., Miao Ethnomedicine name: bub jid ngongx, have another name called delicate fragrance osmanthus, Radix Sophorae Tonkinensis etc., being distributed in the ground such as Southwestern China and Guangxi, Hunan, is that < < Guizhou Province Chinese crude drug, national quality of medicinal material standard > > record kind.Be mainly used in treating that gastropathy, traumatic injury impairment caused by overstrain, dizzy cardiopalmus, throat swell and ache, cooling blood and removing stasis, removing toxic substances sore.Because the distribution of this medicine is wide, output is larger, and therefore the effective ingredient in three liang of silver is carried out to deep pharmacological research has special realistic meaning.Applicant is through a large amount of experiments, and success is isolated pachysamine alkali G from three liang of silver of Miao Ethnomedicine, and confirms that through pharmacological research it has therapeutical effect to alzheimer disease, and is expected to become new acetylcholinesteraseinhibitors inhibitors.
Summary of the invention
Technical problem to be solved by this invention has been to provide the application of pachysamine alkali G as acetylcholinesteraseinhibitors inhibitors; Another technical problem to be solved by this invention is that the application of pachysamine alkali G in the medicine of preparation prevention or treatment alzheimer disease is also provided.
For solving the problems of the technologies described above, the present invention adopts following technical scheme to realize:
Pachysamine alkali G is as the application of acetylcholinesteraseinhibitors inhibitors.
The application of pachysamine alkali G in the medicine of preparation prevention or treatment alzheimer disease.
An anticholinesterase drug, described medicine comprises pachysamine alkali G.
Specifically, aforesaid anticholinesterase drug is mainly prepared from by pachysamine alkali G.
A medicine for prevention or treatment alzheimer disease, described medicine comprises pachysamine alkali G.
Specifically, the drug main of aforementioned prevention or treatment alzheimer disease will be prepared from by pachysamine alkali G.
The route of administration of said medicine is oral, subcutaneous, vein or intramuscular injection.
The molecular formula of described pachysamine alkali G is: C
28h
48n
2o, is colourless crystallization, and structural formula is:
Described pachysamine alkali G can be prepared like this: get three liang of silver, be ground into coarse powder, the alcohol reflux with 95% three times, each 3 hours, merge extractive liquid,, concentrated to obtain extractum, extractum adding distil water dissolves, and uses chloroform extraction 5 times, and chloroform extraction liquid concentrating under reduced pressure obtains extractum, extractum carries out column chromatography for separation with silica gel mixed sample, with petroleum ether-acetone-diethylamino 100:1:1,100:5:1,100:10:1,100:20:1,100:50:1,100:100:1 gradient elution, obtains 6 parts; Get part 2, upper silicagel column, with petroleum ether-diethylamino 100:1,100:3,100:5,100:10 gradient elution, wherein 100:3 stream part petroleum ether: diethylamino 100:1 eluting, eluent recrystallizing methanol, obtains.
Alzheimer (Alzheimer ' s disease, AD) (being alzheimer disease), is a kind of mostly occurring in old age, take the carrying out property neurodegenerative disease that brain β sample starch denaturalization, tangle be main pathological change.1907; clinical manifestation and the pathological change of German scholar Alois Alzheimer reported first AD; its pathological characteristics is to occur a large amount of senile plaque (SPs) and neurofibrillary tangles (NFTs), and on biochemistry, main manifestations is minimizing and the synthetic deficiency of CAT (ChAT) and acetylcholine (Ach) content.The pathogenesis of alzheimer disease is very complicated, and the factors such as its generation reason and cholinergic damage, β sample starch albumen (A β) abnormal deposition, Protein tau Abnormal Phosphorylation, inflammation mechanism are closely related.
Wherein, cholinergic damage is because cholinergic neurotransmitter is the important chemical substance in cerebral tissue, while suffering from AD, the cholinergic neuron of basal forebrain areas reduces, cause acetylcholine (ACh) synthetic, store and discharge and reduce, and then cause to remember and the series of clinical manifestations such as recognition function obstacle, can also optionally damage acetylcholine nerve, thereby affect people's the functions such as understanding learning and memory, so in the generation of AD and brain in patients, neurotransmitter---the disappearance of acetylcholine is closely related, to the Drug therapy of senile dementia, be mainly to improve the levels of acetylcholine in patient body by suppressing acetylcholine esterase at present.A β is by amyloid precursor protein (APP) cracking, and its abnormal deposition can cause Neuron Apoptosis, lesion wire mitochondria function, induces Oxidative Damage In Astrocytes, cause toxic action in cell, is the Important cause of disease of senile dementia.In AD patient's brain, one of significant pathological change is neurofibrillary tangles (neurofibrillary tangles in neurocyte, NFTs), the main component of NFFs is the Tau albumen of Hyperphosphorylationof, when Protein tau is by Hyperphosphorylationof, and in cell, formed after accumulation, can lose the function of himself, cause the infringement of microtubule function.Inflammation mechanism tool in the pathogenic process of AD has certain effect, wherein, the activation of microglia and astrocyte is two important steps of inflammatory reaction, research is found, at AD patient's brain endoplasm phospholipase A2 (cytosolic phosphor lipase A2, cPLA2) immunocompetent astrocyte is than the obvious increase of matched group, its domain of the existence is consistent with A β deposition region, point out in its brain and exist and worry property inflammatory reaction, meanwhile, also confirm that cPLA2 plays an important role to inflammatory mediator in brain and second message,second messenger's production process.
Given this, applicant to the anticholinesterasic activity of pachysamine alkali G, the mechanism of action of intervening A β and express, reduce Tau albumen peroxophosphoric acid, improve the anti-AD such as focal inflammation reaction carried out experimentation.And further set forth the present invention by following enforcement.
Experimental example 1: the inhibitory action to rat blood serum acetylcholine esterase
1. materials and methods
1.1 medicines and reagent
Pachysamine alkali G, derives from department of pharmacy of Guiyang College of Traditional Chinese Medicine; Acetylcholine esterase is measured test kit (Shanghai Rongsheng Bioisystech Co., Ltd, credit number 20030955); Dimethyl sulfoxide (DMSO); PH7.4 phosphate buffer.
1.2 instrument
Automatic clinical chemistry analyzer (glamour 2000), desk centrifuge
The preparation of acetylcholine esterase in 1.3 serum
1 of SD rat, male, 300g(is purchased from Guiyang Medical College Experimental Animal Center, credit number: SCXK(Guizhou Province) 2002-0001), brachycephaly is got blood 8ml, standing 6h is later with the centrifugal 15min of 3000r/min at normal temperatures, get supernatant 3ml, with phosphate buffer, press 1: 9 diluted for use.
The mensuration of 1.4 rat blood serum cholinesterase activities
Be divided into 4 groups, 10 parts every group, be respectively matched group, the large and small dosage group of pachysamine alkali G.The large and small dosage group of medicine to be measured is pressed crude drug amount 1g/1ml, 0.5g/1ml and is converted and to dissolve with equivalent DMSO, detects operation and is undertaken by test kit explanation, and the cholinesterase activity (U/L) of take is subject to the anticholinesterase activity of reagent as index evaluation.
1.5 statistical procedures
Data are analyzed with SPSS11.0 software, with mean+SD (
) represent, group difference adopts variance analysis, usings the standard of P<0.05 as significant difference.
2. result
Table 1 on the impact of acetylcholine esterase (
n=10)
| Group | Cholinesterase activity (U/L) |
| Matched group | 253.55±37.65 |
| Pachysamine alkali G is heavy dose of | 119.58±19.65 ★ |
| Pachysamine alkali G is low dose of | 83.65±14.68 ★ |
| DMSO | 221.32±29.51 |
Note: with matched group comparison,
★p < 0.05
3. conclusion
From this experimental result, can find out, pachysamine alkali G has obvious inhibitory action to rat blood serum acetylcholine esterase in vitro, illustrate that it has anticholinesterasic activity, and solvent DMSO has no significant effect to experimental result.In experimental example 2, continue to investigate the impact of pachysamine alkali G on acetylcholine esterase in brain, and the impact on amyloid beta and precursor protein (APP) for medicine, the aspects such as impact of Protein tau degeneration are studied.
Experimental example 2: the anti-AD rat of pachysamine alkali G study on mechanism
1 experiment material
1.1 laboratory animal
Healthy SPF level SD male rat, body weight (200 ± 10) g, is provided credit number by PLA Academy of Military Science Experimental Animal Center: 2007-004 SCXK-(army).Feedstuff, bedding and padding provide by animal center, freely ingest and drink water, natural circadian rhythm illumination, and indoor temperature is controlled at 25 ℃ of left and right, and relative humidity is 60-70%.
1.2 Experimental agents
Pachysamine alkali G(94.62%), derive from department of pharmacy of Guiyang College of Traditional Chinese Medicine.
Donepezil hydrochloride sheet (trade name: aricept): 5mg/ sheet, defend material (Beijing) pharmaceutcal corporation, Ltd, lot number: 091223A.
1.3 experiment reagent
D-galactose (D-gal, lot number: 100508), Bo Aosen bio tech ltd, Shanghai; Sodium nitrite (NaNO
2, lot number: 100901), Xilong Chemical Co., Ltd; Picric acid (lot number: 20081201), Taishan City Chemical Co., Ltd..
1.4 key instrument
Ordinary optical microscope ((CK-2 type), Japanese OLYMPUS company produces; Photomicroscope ((CH type), Japanese OLYMPUS company produces; Precise electronic balance (Libror AEL-200 type), Japanese Shimadzu produces; TGL-16G desk centrifuge, Anting Scientific Instrument Factory, Shanghai produces; Microplate reader (biorad680), U.S. biorad Bole produces.
2 methods and result
2.1 experimental technique
2.1.1 medicine preparation
2.1.1.1 the preparation of pachysamine alkali G
Pachysamine alkali G is dissolved in distilled water and (adds a certain amount of emulsifier tween-80), be mixed with respectively high, medium and low dosage, 4 ℃ of preservations, standby.
2.1.1.2 positive drug preparation
Get donepezil hydrochloride sheet, put into mortar pulverize, dissolve in distilled water, 4 ℃ of preservations, standby.
2.1.1.3 other drug
Get D-gal, NaNO
2dissolve in normal saline, D-gal (D-galactose) presses 85mgkg
-1lumbar injection, NaNO
2(sodium nitrite) presses 45mgkg
-1lumbar injection.
2.1.2 animal grouping, modeling and administration
2.1.2.1 animal grouping
At the spacious field analysis case of the self-control (1m * 1m * 40cm forming with plank, bottom is divided into 25 grid 20cm * 20cm) in, by observe every rat at 5min interior span lattice number of times, lift pawl number of times, defecation grain number, filter out qualified rat, adaptability is fed one week laggard line ordering, then adopt table of random number method to be divided into Normal group (10), model group (10), positive controls (10), the high, medium and low dosage group of pachysamine alkali G(, 10 every group).
2.1.2.2 animal model
Except Normal group gives with metering normal saline lumbar injection, all the other rat lumbar injection every day D-gal and NaNO
2, continue 60 days.
2.1.2.3 administering mode and dosage
The method of learning according to animal experiment method is according to the dosage of people's clinical medicine dose conversion rat, and reduction formula is as follows: the dosage of every rat=(people uses the conversion coefficient of dosage * people and Mus)/rat weight.In specific implementation process, ABW standard body weight ± all adopt the method to carry out administration in 20% scope.
From modeling the 21st day, carry out gastric infusion intervention simultaneously.Blank group, model group be all with same dosage normal saline gavage, to get rid of the diversity of stimulability with irrigation and damage, and continuous 40 days.
2.1.3 behavioristics is detected
Adopt Morris water maze to detect the orientation navigation of rat and space exploration ability of learning and memory, and the impact of pachysamine alkali G on learning and memory in rats ability inquired in subordinate act aspect.
2.1.3.1 orientation navigation learning and memory experiment
Morris water maze is mainly comprised of round pool, automatic camera and analytical system, the automatic acquisition and processing system of image is mainly comprised of video camera, computer, image monitor, animal starts monitoring device after entering water, record path of animal movement, test complete automatic analysis report relevant parameter.Round pool is made by rustless steel, diameter 120cm, and high 50cm, has a circular platform in pond, diameter 10cm, high 28cm.Along water maze center origin, be divided into four quadrants, platform is arranged in first quartile, i.e. target quadrant, wherein the distance of center circle pool wall 20cm of platform injects tap water in pond, and water temperature is 23-25 ℃, and the water surface exceeds platform 2cm.Overhead, pond is connected with computer by a video camera, when the training time of setting to or rat oneself climb to platform, computer stops following the tracks of and records rats'swimming track, time etc.It is relatively quiet that training period environment keeps, and leaves after rat is put into pond.Orientation navigation learning and memory experiment: from same pool wall place of entry, experimental rat is put into pond towards pool wall, automatically record the time that rat is found platform, as rat escape latency.Rat is climbed up after platform, allows the rat 10s that stands on platform; If rat is not found the platform in pond or fails to climb up platform in 120s, rat is dragged gently, cause the 10s that stands on platform.Rat is taken off from platform, and follow-on test 4d, as trained rat ability of learning and memory.5d records rat escape latency, detects rat orientation navigation memory ability.
2.1.3.2 space exploration learning and memory experiment
After orientation navigation experiment, platform is shifted out to pond, and by the method for orientation navigation experiment, computer tracking also records in rat 120s and swims and explore the track of platform, and record rat and after entering water, in 120s, swim across the number of times in region, former platform position, as wearing platform number of times.
2.1.4 the preparation of serum and cerebral tissue
Behavioristics carries out the preparation of serum and cerebral tissue after detecting, and before experiment, water is can't help in the fasting of 12h rat.
2.1.4.1 serum preparation
Take experimental rat body weight, with the fixing Mus of left hand, clutch skin of head as far as possible, make head fixing, and gently to lower compression cervical region both sides, cause head vein blood backflow difficulty, make the abundant evagination of eyeball (showing that eye socket rear vein beard is congested), the right hand is held curved forceps, inserts rotation get blood, the blood 2500rmin of taking-up along endocanthion eye socket rear wall to larynx direction
-1centrifugal 10min, gets serum, puts into-20 ℃ of refrigerators, standby.
2.1.4.2 brain tissue homogenate's preparation
Get after blood, broken end, opens cranial cavity fast, takes out rapidly full brain on ice platform, with cold saline, rinses blood, and filter paper blots after flushing liquor, puts into homogenizer, adds the normal saline that approximately 9 times of amount concentration are 0.9%, grinds to form brain tissue homogenate, 2500rmin
-1centrifugal 10min, gets supernatant, puts in EP pipe, puts into-20 ℃ of refrigerators, standby.
2.1.4.3 cerebral tissue perfusion is fixing
By body weight scale, extract 3.5% chloral hydrate intraperitoneal injection of anesthesia of equivalent, lie on the back and be fixed on Mus dissecting table, with shears, cut off thoracic cavity, and with curved forceps, clamp thoracic cavity bone and turn up, fully expose thoracic cavity.Being full of normal saline in transfusion device, and make the syringe needle of the 20ml syringe of special blunt ooze normal saline, with medium size Smooth forceps sub-folder, live heart, syringe needle is pierced into rapidly from the apex of the heart, go forward side by side and become owner of in arterial vessel, with anodontia pliers, fix, cut off the right auricle of rat, open fast the valve lock of transfusion device, speed first quick and back slow, until red liver becomes when pale, change fast 4% paraformaldehyde solution perfusion into.The rat of having filled with is placed on broken end frame and is breaked end immediately, with shears, cut off scalp, then disconnect skull with bone crushing forceps, expose cerebral tissue, with small size tweezers, press from both sides out cerebral tissue, drop into 4% paraformaldehyde solution, standby.
2.1.5 the detection of biochemical indicator
The detection of biochemical indicator all adopts in ELISA Fa Dui rat cerebral tissue the content of iNOS, IL-1 in selected index A β, Tau albumen, AchE, ChAT, GSK-3 β and serum to carry out biochemistry detection, by quantitative method, obtain the variation of each biochemical indicator, thereby for we provide Data support.
2.1.5.1Aβ
1-42
2.1.5.1.1 principle
Adopt rat A β in biotin DASELISA immunoabsorption (ELISA) working sample
1-42level.To being coated with in advance A β
1-42in the enzyme mark hole of monoclonal antibody, add A β
1-42, after incubation, add biotin labeled anti-A β
1-42antibody, then be combined with Streptavidin-HRP, form immune complex, then pass through incubation and washing, remove unconjugated enzyme, then add substrate A, B, produce blueness, and under sour effect, change into final yellow.A β in the depth of color and sample
1-42concentration be proportionate.
2.1.5.1.2 operating procedure
Dilution standard product: it is 1200pgmL that standard substance diluent is added to doubling dilution in former times of standard substance
-1, 600pgmL
-1, 300pgmL
-1, 150pgmL
-1, 75pgmL
-1;
Application of sample: blank well: blank hole does not add sample, biotin labeled anti-A β 1-42 antibody, Streptavidin-HRP, only adds developer A & B and stop buffer, and all the other each step operations are identical;
Standard substance hole: add standard substance 50 μ L, streptomycin-HRP50 μ L;
Testing sample hole: add sample 40 μ L, then respectively add anti-A β 1-42 antibody 10 μ L, streptavidin-HRP50 μ L, cover shrouding film, vibration mixes gently, 37 ℃ of incubations 60 minutes;
Dosing: by standby after 20 times of dilutions of 20 times of concentrated cleaning solutions use distilled water;
Washing: carefully take shrouding film off, discard liquid, dry, cleaning mixture is filled it up with in every hole, discards after standing 30 seconds, so repeats 5 times, pats dry;
Colour developing: every hole first adds developer A50 μ L, then adds developer B50 μ L, and light shaking mixes, 37 ℃ of lucifuge colour developing 10min;
Stop: every hole adds stop buffer 50 μ L cessation reaction (the now blue vertical yellow that turns);
Measure: with blank well zeroing, 450nm wavelength is sequentially measured the absorbance in each hole;
Calculate: according to the OD value of the concentration of standard substance and correspondence, calculate the linear regression equation of standard curve, then OD value per sample calculates corresponding sample concentration on regression equation.
2.1.5.2Tau albumen
2.1.5.2.1 principle: with 2.1.5.1.1 item.
2.1.5.2.2 operating procedure
Dilution standard product: it is 640pgmL that standard substance diluent is added to doubling dilution in former times of standard substance
-1, 320pgmL
-1, 160pgmL
-1, 80pgmL
-1, 40pgmL
-1.By method under 2.1.5.1.2 item, operate and get final product.
2.1.5.3AChE
2.1.5.3.1 principle: with 2.1.5.1.1 item.
2.1.5.3.2 operating procedure
Dilution standard product: it is 80UL that standard substance diluent is added to doubling dilution in former times of standard substance
-1, 40UL
-1, 20UL
-1, 10UL
-1, 5UL
-1.By method under 2.1.5.1.2 item, operate and get final product.
2.1.5.4IL-1
2.1.5.4.1 principle: with 2.1.5.1.1 item.
2.1.5.4.2 operating procedure
Dilution standard product: it is 400ngL that standard substance diluent is added to doubling dilution in former times of standard substance
-1, 200ngL
-1, 100ngL
-1, 50ngL
-1, 25ngL
-1.By method under 2.1.5.1.2 item, operate and get final product.
2.1.5.5GSK-3β
2.1.5.5.1 principle: with 2.1.5.1.1 item.
2.1.5.5.2 operating procedure
Dilution standard product: it is 150pmolL that standard substance diluent is added to doubling dilution in former times of standard substance
-1, 100pmolL
-1, 50pmolL
-1, 25pmolL
-1, 12.5pmolL
-1.By method under 2.1.5.1.2 item, operate and get final product.
2.1.5.6iNOS
2.1.5.6.1 principle: with 2.1.5.1.1 item.
2.1.5.6.2 operating procedure
Dilution standard product: it is 50nmolmL that standard substance diluent is added to doubling dilution in former times of standard substance
-1, 25nmolmL
-1, 12.5nmolmL
-1, 6.25nmolmL
-1, 3.12nmolmL
-1.By method under 2.1.5.1.2 item, operate and get final product.
2.1.5 morphology is measured
Morphologic detection adopts HE dyeing, two kinds of methods of immunohistochemical staining, adopt pathology picture and text analytical system to gather image, respectively A β (senile plaque), Tau albumen (neurofibrillary tangles) in cerebral tissue are carried out to qualitative analysis, and utilize pathology rgb image acquisition system to carry out relative quantitative assay to A β, Tau protein expression average optical density value in image.
2.1.5.1 specimen preparation
Get conventional fixing left cerebral tissue, dehydration of alcohol, waxdip, paraffin embedding, section, 15 of each continuous tissue sections, get 6 continuously, carry out HE dyeing, immunohistochemical staining.
2.1.5.2HE dyeing
Choose Hippocampus paraffin section; Dimethylbenzene dewaxing 10min, 2 times; With gradient alcoholic solution, dewater: 100% ethanol 2 times, each 3min, 95%, 85%, 75% ethanol each 1 time, each 1min; Distillation washing 1min; Hematoxylin dye liquor 6min transfect cell core; Distillation washing 10s; Enter 1% ethanol solution hydrochloride 5s, slough the painted of kytoplasm; Distillation washing 10s; Enter ammonia spirit 5s; Distillation washing 10s; Enter eosin stain 30s transfect cell slurry; With gradient alcoholic solution, dewater successively: 75%, 85%, 95%, 100% ethanol each once, each 10s; The transparent 3min of dimethylbenzene; Dry up section, use neutral gum mounting.
2.1.5.3 immunohistochemical staining
Choose Hippocampus paraffin section; 60 ℃ of roasting sheet 2h; Dewaxing: dimethylbenzene dewaxing 15min, three times; Aquation: pass through successively dehydrated alcohol, 95%, 90%, 80%, 70%, each 2min, distilled water (or tap water) 5min; PBS washes 5min, three times; 3% hydrogen peroxide sealing 20min, PBS washes 5min, three times; Antigen retrieval: citrate buffer boils repairs 15min, naturally cool to room temperature.PBS washes 5min, three times; The sealing of normal goats serum, 37 ℃, 15-20min; Primary antibodie is hatched, and 4 ℃ are spent the night.Rewarming 20min, PBS washes 5min, three times; Two anti-hatching, 37 ℃ of 20min (two resist for biotin labeled goat anti-rabbit igg), PBS washes 5min, three times; Three anti-hatching, 37 ℃ of 20min (three resist the streptavidin for Radix Cochleariae officinalis enzyme labelling), PBS washes 5min, three times; DAB colour developing, Microscopic observation result stops in good time; Haematoxylin is redyed 5min, and tap water rinses a moment, 75% hydrochloride alcohol solution differentiation 30s, and tap water rinses 5min oil blackeite; Dehydration: successively through 70%, 80%, 90%, 95%, dehydrated alcohol, each 2min; Dimethylbenzene 15min; Mounting: neutral gum mounting.
2.1.6.4 image processing method
Immunohistochemical staining result is utilized microscope, every group of every rat chosen 6 sections of same area at random, adopt pathology picture and text analytical system to gather image, and utilize pathology rgb image acquisition system to analyze protein expression average optical density value in image, as the experimental data of section.
2.1.7 statistical method
Experimental data adopts SPSS17.0 statistical software to process, mean ± standard deviation for data acquisition (
) represent, when the discontented sufficient variance analysis condition of each sample does not meet normality, do not adopt Friedman rank test, when each sample standard deviation Normal Distribution, adopt One-Way ANOVA(one factor analysis of variance) check, and analyze with Dunnett T3.
2.2 result
2.2.1 behavioristics's testing result
As can be seen from Table 2: with the comparison of blank group, model group rat escape latency extends, and difference has statistical significance (P<0.05); With model group comparison, tri-group rat escape latency of pachysamine alkali G shorten, and difference has statistical significance (P<0.05); With the comparison of positive group, each is for the reagent group equal no significant difference of rat escape latency (p>0.05).With the comparison of blank group, model group rat is worn platform number of times and reduces, and difference has statistical significance (P<0.05); With model group comparison, administration group and model group are relatively worn platform increased frequency, and difference has statistical significance (P<0.05); With the comparison of positive group, for reagent group rat, wear the equal no significant difference of platform number of times (p>0.05).
Table 2 respectively organize rat escape latency with wear the comparison of platform number of times (
n=6)
Note: with model group comparison,
★p<0.05.
2.2.2 biochemistry detection result
2.2.2.1A β 1-42 measures and Tau protein content
Result is as shown in Table 3: with the comparison of blank group, and A β in model group rat cerebral tissue
1-42amount increases, and difference has statistical significance (P<0.05); With model group comparison, A β in other group rat cerebral tissues
1-42amount reduces, and difference has statistical significance (P<0.05); With the comparison of positive group, the high, normal, basic group of equal not statistically significant of difference of pachysamine alkali G (p>0.05), point out the A β of its rat cerebral tissue
1-42content and positive group zero difference; No significant difference between tri-dosage groups of pachysamine alkali G.With the comparison of blank group, in model group rat cerebral tissue, Tau protein content increases, and difference has statistical significance (P<0.05); In model group rat cerebral tissue, Tau protein content and other are organized obvious increase, and difference has statistical significance (P<0.05); Each medicine group compares with positive group, the amount there was no significant difference (p>0.05) of Tau albumen; Pachysamine alkali G respectively organizes 3 dose comparisons, the amount difference nonsignificance (p>0.05) of Tau albumen.
Table 3 is respectively organized A β
1-42amount and the comparison of Tau protein content (
n=10)
Note: with model group comparison,
★p<0.05.
2.2.2.2AChE amount and GSK-3 β amount
Result is as shown in Table 4: with the comparison of blank group, in model group rat cerebral tissue, AChE content increases, and difference has statistical significance (P<0.05); With model group comparison, other groups AChE of rat cerebral tissue content obviously reduces, and difference has statistical significance (P<0.05); With the comparison of positive group, tri-equal no significant differences of dosage group of pachysamine alkali G; With the comparison of blank group, in model group rat cerebral tissue, GSK-3 β amount increases, difference not statistically significant (p>0.05); With model group comparison, other groups GSK-3 β of rat cerebral tissue quantitative change is without significant difference (p>0.05); With the comparison of positive group, the GSK-3 β of administration group rat cerebral tissue measures difference not statistically significant (p>0.05).
Table 4 respectively organize AChE amount and GSK-3 β amount relatively (
n=10)
Note: with model group comparison,
★p<0.05.
2.2.2.3IL-1 amount is measured with iNOS
Result is as shown in Table 5: with the comparison of blank group, in model group rat blood serum, IL-1 amount increases, and difference has statistical significance (P<0.05); With model group comparison, in other group rat blood serums, IL-1 amount reduces, and difference has statistical significance (P<0.05); With the comparison of positive group, IL-1 amount difference not obvious (p>0.05) in serum; Pachysamine alkali G respectively organizes 3 dose comparisons, and IL-1 measures difference nonsignificance (p>0.05).With the comparison of blank group, in model group rat blood serum, iNOS amount increases, difference not statistically significant (p>0.05); With model group comparison, in other group rat blood serums, iNOS quantitative change is without significant difference (p>0.05); With the comparison of positive group, administration group rat iNOS amount difference not statistically significant (p>0.05).
Table 5 respectively organize IL-1 amount, iNOS amount relatively (
n=10)
Note: with model group comparison,
★p<0.05.
2.2.3HE coloration result
Adopted specimen is carried out after HE dyeing, through microscopic examination, image acquisition visible (accompanying drawing 1).
Blank group: hippocampus region is larger, and neuronal structure is normal, clear, and number is more and cell arrangement is neat, has minority neurogliocyte; Nucleus is rounded, and cell space endochylema is light blue, projection endochylema pale red, accompanying drawing 1.
Model group: hippocampus region dwindles, neural cell injury distortion, decreased number, arrangement disorder are unordered, occur cell granulations vacuolar degeneration, and glial cells hyperplasia is obvious, and cell volume increases, and karyon dyeing increases the weight of, and kytoplasm increases; And the lumps material that occurs eosinophilic staining is the formation of class senile plaque, visible neurofibril increases thick twisted phenomena once in a while.
Positive drug group: with model group comparison, hippocampus region is larger, and neuron number is more, and cell arrangement is substantially neat, and glial cells hyperplasia is not obvious.
Pachysamine alkali G group: with model group comparison, neuron number is many and cell arrangement is substantially neat, structure is more clear.
2.2.4 immunohistochemical staining result
Adopted specimen is carried out after immunohistochemical staining to microscopic examination, image acquisition.Positive neuron is mainly distributed in the positions such as Hippocampus, temporal lobe, frontal cortex, basal forebrain, and neuron is fusiformis, ellipse, taper or polygon, and cell size is not etc.Positive staining position cell membrane is brown cell granulations, and the number of protein expression shows as the power of staining power, and staining power is optical density, dyes darker, and optical density value is larger, and positive staining position is more, and protein expression is more.Each is organized A β (accompanying drawing 2) and expresses average optical density value analysis in table 6 with Tau albumen (accompanying drawing 3).
Table 6 respectively organize A β and the comparison of Tau protein expression average optical density value (
n=10)
Note: with model group comparison,
★p<0.05; With the comparison of positive group,
◆p < 0.05.
As can be seen from the above table: with the comparison of blank group, in model group rat cerebral tissue, AChE content increases, and difference has statistical significance (P<0.05); With model group comparison, other groups A β of rat cerebral tissue expresses average optical density value and obviously reduces, and difference has statistical significance (P<0.05); His administration group compares protein expression with positive group and reduces, and difference has statistical significance (P<0.05); With the comparison of blank group, in model group rat cerebral tissue, AChE content increases, and difference has statistical significance (P<0.05); The Tau of model group rat cerebral tissue protein expression average optical density value and other groups relatively protein expression increase, and difference has statistical significance (P<0.05); Administration group compares no significant difference (p>0.05) with positive group.
3 conclusions and discussion
3.1 conclusion
3.1.1 adopt lumbar injection D-galactose and NaNO
2successfully set up AD animal model, experimental result illustrates this model stability, reliable, can be applicable to the pharmacological research of AD.
3.1.2 by Morris water maze laboratory, learning and memory in rats ability is detected, from escape latency shortening and the wearing of space exploration learning and memory of orientation navigation learning and memory, encircle increasing of number of times, known pachysamine alkali G can improve the learning memory disorder of AD rat.
3.1.3ELISA method, HE dyeing, immunohistochemical staining detect the content of rat A β, Tau albumen, confirm that pachysamine alkali G can improve the pathological change of AD rat cerebral tissue, delays the pathology process of alzheimer disease.
3.1.4ELISA the content results of method detection rat AChE shows that pachysamine alkali G is significantly improved to cholinergic nerve function, has the effect of neuroprotective unit.
3.1.5ELISA the content prompting pachysamine alkali G of method detection rat IL-1 makes moderate progress to focal inflammation reaction in AD patient's brain, has neuroprotective cytosis.
3.2 discuss
3.2.1 the foundation of animal model
At present, go back the animal model that neither one is simulated AD feature completely.Wherein, single model is just made for the AD cause of disease in a certain respect, can only simulate the Partial Feature of AD.And the cause and onset of disease of AD mechanism is complicated, the foundation of test model will directly affect basic research and the drug development of AD.Compound model is a kind of composite model that comprehensive two or more single model method obtains, and can comprehensively obtain various existing model features, meets the multifactor pathogenesis of AD, is the actual and effective method of of preparation AD model.
This experiment adopts D-gal to cause subacute aging model, and combines NaNO
2lumbar injection is prepared compound AD model.Within a certain period of time, continuously to animal injection D-gal, in cell, D-gal concentration raises, cause body to produce the deterioration of many organs, multisystem, finally cause the generation of body aging, existing more sure evidence shows that D-gal produces the old and feeble reaction of plan, has gained universal acceptance at present and has used at home and abroad.But the risk factor of old and feeble just AD morbidity, can not guarantee the aged animal AD that is just bound to develop into, and be normally not present the features such as the SP of AD and NFT.Therefore, NaNO in addition more simultaneously
2make rat cerebral anoxia repeatedly over a long time.Thereby press close to the pathological change process of AD complexity, for inquiring into the study model of Chinese medicine multiple target effect mechanism and active drug screening.
This compound AD model can be avoided the death that rat brain physical damnification is caused effectively, guaranteed carrying out smoothly of experimentation, and this experimental result also proves the stability of this model.Each index shows: with the comparison of blank group, model group behavioristics learning memory ability obviously declines, and in model group rat brain slice, also there is the pathological changes such as senile plaque and neurofibrillary tangles, the expression of the biochemical detection by quantitative of A β and Tau albumen also display model group A β and Tau albumen is significantly increased.
3.2.2 the utilization of control drug
The first-line treatment medicine of AD is acetylcholinesterase (AChE) inhibitor at present, donepezil hydrochloride (aricept) be unique a kind of while by U.S. FDA and Britain MCA approval listing for new drug light, moderate AD symptomatic treatment, it is second filial generation acetylcholine esterase (ChE) inhibitor, there is the effect that reversibly suppresses the acetyl gallbladder acyl hydrolysis that AChE causes and increase the acetyl choline content of acceptor site, may also comprise peptide, neurotransmitter receptor or Ca
2+the direct effect of passage, thus hypomnesis, unorientation, behavior and the personality change etc. that the carrying out property regression of AD cholinergic neuron causes improved.
Studies confirm that, aricept treatment is light, moderate AD, can significantly change AD patient's cognitive function, and safety is good.Therefore this experiment selects aricept to compare medicine.
3.2.3 the impact of pachysamine alkali G on AD learning and memory in rats ability
AD is a kind of brain degenerative disease, first shows as recent memory power obstacle in Clinical symptoms, then occurs persistence hypophrenia, judging and deducing Disability, the dyskinesia.Protection patient's AD neurocyte is avoided damage, improves memory, and the hypophrenia that stops patient is the important measures for the treatment of AD.Morris water maze laboratory is conventional learning and memory assay method, is divided into orientation navigation experiment and space search experiment.Orientation navigation experiment is that trained rat can utilize the spatial cues in environment to locate underwater platform after repetition training, can reach and measure the object that spatial reference memory obtains, in orientation navigation experiment, with rat escape latency, represent learning capacity, increase along with the training time, the rat escape latency time is shorter, shows that its learning capacity is stronger.And space search experiment is used for measuring the accurate memory of animal to platform space position, on the basis of orientation navigation test, by setting search time upper limit, rat is more at position of platform traversing times, shows that its memory ability is better.
Water maze orientation navigation experiment result of study shows: with the comparison of blank group, and the time significant prolongation (P<0.05) of model group escape latency; In space search experiment, model group is few compared with the blank group of cross-platform number of times of rat, and difference has statistical significance (P<0.05).Subordinate act detects, and confirms that model group's learning and memory ability weakens, and prompting is by D-gal+NaNO
2combine and successfully make AD model.Meanwhile, with model group comparison, tri-group rat escape latency of pachysamine alkali G significantly shorten (P<0.05), and tri-group rats of pachysamine alkali G are worn platform number of times and significantly increase (P<0.05).With the comparison of positive group, each is for the reagent group equal no significant difference of rat escape latency (p>0.05), and each wears the equal no significant difference of platform number of times (p>0.05) for reagent group rat.Comprehensive orientation navigation experiment and space search experimental result, each has obtained obvious improvement for reagent group and positive drug group rat ability of learning and memory after pharmaceutical intervention, and the cognitive competence of AD rat model is recovered.
3.2.4 the mechanism of action of the anti-AD rat of pachysamine alkali G
3.2.4.1 intervene A β and express, protection hippocampal tissue
A β is I type transmembrane protein amyloid precursor protein (amyloid precursor protein, APP) enzymatic hydrolysate, its deposition causes the formation of senile plaque, it is one of important channel causing AD generation, because neurocyte is abnormal, produce a large amount of A β, not only can produce senile plaque, and can cause the pathological changes such as cerebral nerve fibre matting and nerve cell death, thereby cause AD, medical circle thinks that A β causes the mankind to suffer from " arch-criminal " of Alzheimer's disease mostly at present.Experimentation has in the past confirmed that in hippocampus, amyloid beta deposition causes neurocyte significantly damage and loss.Given this, the present invention verifies modeling situation by detecting A β content, and the disturbed condition of pachysamine alkali G to rat brain aβ protein is also described simultaneously.
A β quantitative analysis results confirms: with the comparison of blank group, model group A β amount truly has increase, and both significant differences (P<0.05); HE dyeing picture also shows: the pathological changes such as corresponding senile plaque and cell death appear in model group; ImmunohistochemistryResults Results shows simultaneously: by the average optical density value to albumen, analyze, model group A β expresses and truly has remarkable increase (P<0.05); The raw variation of these 3 prompting A β volume productions is the result that modeling produces above, so modeling success has been described to a certain extent.ELISA method result shows; with positive controls comparison; pachysamine alkali G senior middle school low dose group reduces relatively remarkable; in addition; by average optical density value A β, measure, find that each administration group and positive group compare without significant difference, prompting pachysamine alkali G all has interference to rat brain aβ protein; and high, normal, basic group of interference of pachysamine alkali G is larger, thereby hippocampal tissue is protected.
3.2.4.2 reduce Tau albumen peroxophosphoric acid, protection microtubule function
The main Physiological Function of Tau albumen is to promote microtubule self aggregation and stable microtubule, and this transmission for neuron intracellular organic matter is most important.Studies have found that Tau albumen is mainly distributed in the aixs cylinder of maincenter and peripheral nervous system neurocyte, also can participate in cytocerastic adjusting.In AD patient's brain, one of significant pathological change is NFTs in neurocyte, and the main component of NFFs is the Tau albumen of Hyperphosphorylationof.When Protein tau is by Hyperphosphorylationof, and in cell, formed after accumulation, can lose the function of himself, caused the infringement of microtubule function.Have correlational study to show, the neurofibrillary tangles quantity in patient's brain can be used as an important indicator of Clinical detection AD degree.
This experimental result shows: with the comparison of blank group, model group Tau amount increases significantly, from pathological picture result, also can find out, produced neurofibrillary tangles, and the average optical density value of Tau protein expression is analyzed, show that the neurofibrillary tangles quantity in rat brain increases, so Tau albumen can be used as important indicator of Clinical detection AD degree and has obtained further confirmation.Simultaneously; with model group comparison; each average optical density value of organizing Tau protein expression is less; and significant difference (P<0.05); prompting administration group has relative minimizing with the neurofibrillary tangles quantity in positive group rat brain; suppressed to a certain extent Tau albumen peroxophosphoric acid, protected microtubule function, illustrated that thus pachysamine alkali G is inhibited to a certain extent to AD.
3.2.4.3 improve rat model cholinergic nerve function, neuroprotective unit
Cholinergic neurotransmitter is the important chemical substance in cerebral tissue, and while suffering from AD, the cholinergic neuron of basal forebrain areas reduces, cause Ach synthetic, store and discharge and reduce, and then cause to remember and the series of clinical manifestations such as recognition function obstacle.Can also optionally damage acetylcholine nerve, thereby affect people's the functions such as understanding learning and memory.Therefore, the mensuration to AChE in body, can learn the situation of body to acetylcholine function in the activity of AChE and body.
Experimental result shows: with the comparison of blank group, model group AChE content significantly increases (p<0.05), illustrates that the decomposition of ACh increases, and has reduced the content of ACh, thereby has caused memory dysfunction.With model group comparison, administration group rat AChE content significantly reduces, illustrate that ACh content increases relatively, reduced cerebral tissue lipid peroxide (LPO) content, thereby improve the anti-damage ability of Cholinergic, enhancing neurocyte, suppress the pyramidal cell degradation degeneration of brain and Hippocampus, thereby improve the cognitive disorder of AD rat.
3.2.4.4 improve the focal inflammation reaction of rat model, neuroprotective cell
There are some researches show in AD patient's brain and have strong focal inflammation reaction, near the microglia activating amyloid plaques can be expressed multiple CK and the complement molecules such as IL-1, IL-6 and TNF-α.It may be in brain due to immunity and the inappropriate activation of inflammatory reaction that the neural chronic degenerative that studies confirm that AD becomes, and superpower immunoreation can cause cell injury and death.IL-1, IL-6 in serum, TNF-alpha levels are lower under normal circumstances, and the microglia of activation can be secreted IL-1, IL-6, wherein most the visible IL-1 of microglia, IL-6 immunoreactivities around europathology infringement in AD brain.The distribution of the microglia of IL-1, the IL-6 positive is relevant to amyloid plaques.
This experimental result also shows, with model group comparison, blank group rat IL-1 amount reduces, difference has statistical significance (P<0.05), and with the comparison of positive group, pachysamine alkali G group IL-1 content reduces, and illustrates that administration group can improve the slow inflammatory pathological process that AD follows.
3.2.4.5 the impact on GSK-3 β, iNOS content
GSK-3 (comprising GSK-3 α, GSK-3 β), cyclin-dependent kinase-5 (cyclin dependent kinase-5, CDK-5) be important protein kinase, can catalysis Tau albumen generation phosphorylation reaction, it increases may cause Tau abnormal protein phosphorylation, make the forfeiture of Tau protein function, thereby cause AD.Result of study shows, in model group rat brain, GSK-3 β level, without significant change, illustrates this experiment institute modeling type this phosphokinase is produced to possible variation, or the Abnormal Phosphorylation of Tau albumen is that the impact of other phosphokinases causes.
Nitric oxide (NO) is that a kind of information of generally acknowledging is at present transmitted molecule, has not same-action of two kinds of neuroprotective and neurotoxicityes in cerebral ischemia.INOS is the carrier that a kind of gaseousness iuntercellular information is transmitted, once iNOS generates, its active duration is longer, can continue catalyzing N O in large quantities produces, and NO has potential neurotoxicity, be effective source of oxidative stress in AD, excessive NO can be damaged membranous structure, protein and DNA by number of ways, causes neuronal necrosis or apoptosis.Experimental result demonstration, model group rat iNOS level, without significant change, illustrates that this model does not produce certain impact to iNOS.
Compared with prior art, the present invention has following beneficial effect:
1, experimental example 1 confirms that pachysamine alkali G has obvious inhibitory action to rat blood serum acetylcholine esterase in vitro; experimental example 2 is tested the content that detects rat AChE by ELISA method in vivo; result shows that pachysamine alkali G is significantly improved to cholinergic nerve function, has the effect of neuroprotective unit.More than prove that pachysamine alkali G has anticholinesterasic activity, can become new acetylcholinesteraseinhibitors inhibitors.
2, confirmed that pachysamine alkali G has therapeutic effect to AD, in conjunction with many indexs result pachysamine alkali, G is effective, has value of exploiting and utilizing;
3, experimental result confirms that the effect of the anti-AD of pachysamine alkali G may be expressed with its intervention A β, reduces Tau albumen Hyperphosphorylationof, improves cholinergic nerve function, to improve focal inflammation reaction etc. relevant.
Accompanying drawing explanation
Fig. 1 is each group section HE colored graph (400 *); Wherein, A-aricept group, the blank group of K-, M-model group, dosage group, Y3-pachysamine alkali G high dose group in Y1-pachysamine alkali G low dose group, Y2-pachysamine alkali G;
Fig. 2 is each group section aβ protein immunohistochemical staining figure (100 *); Wherein, the blank group of K-, M-model group, Y-aricept group, dosage group, Y3-pachysamine alkali G high dose group in Y1-pachysamine alkali G low dose group, Y2-pachysamine alkali G;
Fig. 3 is each group section Tau protein immunization group colored graph (100 *); Wherein, the blank group of K-, M-model group, Y-aricept group, dosage group, Y3-pachysamine alkali G high dose group in Y1-pachysamine alkali G low dose group, Y2-pachysamine alkali G.
The specific embodiment
Embodiment 1.
Technique: get pachysamine alkali G 100g, mix with the starch of 40g, incapsulate, make 1000, obtain.
Pachysamine alkali G is prepared like this: get three liang of silver, be ground into coarse powder, the alcohol reflux with 95% three times, each 3 hours, merge extractive liquid,, concentrated to obtain extractum, extractum adding distil water dissolves, and uses chloroform extraction 5 times, and chloroform extraction liquid concentrating under reduced pressure obtains extractum, extractum carries out column chromatography for separation with silica gel mixed sample, with petroleum ether-acetone-diethylamino 100:1:1,100:5:1,100:10:1,100:20:1,100:50:1,100:100:1 gradient elution, obtains 6 parts; Get part 2, upper silicagel column, with petroleum ether-diethylamino 100:1,100:3,100:5,100:10 gradient elution, wherein 100:3 stream part petroleum ether: diethylamino 100:1 eluting, eluent recrystallizing methanol, obtains.
Usage and dosage: oral, 3 times on the one, each using dosage is counted 3mg with pachysamine alkali G.
Function with cure mainly: acetylcholinesteraseinhibitors inhibitors; Also can be used for treating alzheimer disease, improve memory, delay patient's hypophrenia etc.
Embodiment 2:
Technique: get pachysamine alkali G, be dissolved in water for injection and embedding in corresponding container, 115 ℃ of pressure sterilizings 30 minutes, cooling after, sucking filtration, filtrate injects dilute with water, incipient fusion filter bulb filters, embedding, 115 ℃ of hot repressing sterilizings 30 minutes, are prepared into injection.
Pachysamine alkali G is commercially available.
Usage and dosage: intramuscular injection, intravenous injection or intravenous drip, every day, use amount was counted 2mg with pachysamine alkali G.
Function with cure mainly: be used for the treatment of alzheimer disease, improve memory, stop patient's hypophrenia etc.
Embodiment 3:
Technique: get pachysamine alkali G, be dissolved in water for injection, add 2% mannitol, dry, be prepared into freeze-dried powder injection.
The preparation method of pachysamine alkali G is with embodiment 1.
Usage and dosage: intramuscular injection, intravenous injection or intravenous drip, every day, use amount was counted 1mg with pachysamine alkali G.
Function with cure mainly: acetylcholinesteraseinhibitors inhibitors.
Embodiment 4:
Technique: get pachysamine alkali G, add PEG400 to mix, then feed liquid is transferred in the hopper of pellet processing machine, dripping or be pressed into soft gelatin capsule and obtain soft capsule.
Pachysamine alkali G is commercially available.
Usage and dosage: oral, every day 3 times, each using dosage is counted 4mg with pachysamine alkali G.
Function with cure mainly: acetylcholinesteraseinhibitors inhibitors; Also can be used for treating alzheimer disease, improve memory, delay patient's hypophrenia etc.
Embodiment 5:
Technique: get pachysamine alkali G, mix with the starch of the medicine amount of making 7%, pill, dry, sugar coating, obtains pill.
Pachysamine alkali G is commercially available.
Usage and dosage: oral, every day 2 times, each using dosage is counted 2mg with pachysamine alkali G.
Function with cure mainly: acetylcholinesteraseinhibitors inhibitors; Also can be used for treating alzheimer disease, improve memory, delay patient's hypophrenia etc.
Embodiment 6:
Technique: get pachysamine alkali G, mix with the magnesium stearate of the medicine amount of making 1%, tabletting, film coating, obtains tablet.
Pachysamine alkali G is commercially available.
Usage and dosage: oral, every day 3 times, each using dosage is counted 5mg with pachysamine alkali G.
Function with cure mainly: acetylcholinesteraseinhibitors inhibitors; Also can be used for treating alzheimer disease, improve memory, delay patient's hypophrenia etc.
Embodiment 7:
Technique: get sucrose and add in the water boiling and make to be uniformly dissolved, filter, make simple syrup, pachysamine alkali G is added in simple syrup, add sodium benzoate, dissolve, add water to 1000ml, with sodium hydroxide, adjust PH to 5.00-5.25, stir evenly, filter, embedding, obtains oral liquid.
Pachysamine alkali G is commercially available.
Usage and dosage: oral, every day 2 times, each using dosage is counted 6mg with pachysamine alkali G.
Function with cure mainly: acetylcholinesteraseinhibitors inhibitors; Also can be used for treating alzheimer disease, improve memory, delay patient's hypophrenia etc.
Claims (6)
1. the application of pachysamine alkali G in the medicine of preparation prevention or treatment alzheimer disease.
2. application as claimed in claim 1, it is characterized in that: described pachysamine alkali G is prepared like this: get three liang of silver, be ground into coarse powder, alcohol reflux with 95% three times, each 3 hours, merge extractive liquid,, concentrate to obtain extractum, extractum adding distil water dissolves, and uses chloroform extraction 5 times, and chloroform extraction liquid concentrating under reduced pressure obtains extractum, extractum carries out column chromatography for separation with silica gel mixed sample, with petroleum ether-acetone-diethylamine 100:1:1,100:5:1,100:10:1,100:20:1,100:50:1,100:100:1 gradient elution, obtains 6 parts; Get part 2, upper silicagel column, with petroleum ether-diethylamine 100:1,100:3,100:5,100:10 gradient elution, wherein 100:3 stream part petroleum ether: diethylamine 100:1 eluting, eluent recrystallizing methanol, obtains.
3. an anticholinesterase drug, is characterized in that: described medicine comprises pachysamine alkali G.
4. anticholinesterase drug as claimed in claim 3, is characterized in that: described drug main will be prepared from by pachysamine alkali G.
5. a medicine for prevention or treatment alzheimer disease, is characterized in that: described medicine comprises pachysamine alkali G.
6. prevent or treat as claimed in claim 5 the medicine of alzheimer disease, it is characterized in that: described drug main will be prepared from by pachysamine alkali G.
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