CN102397526A - Application of Jingzhaotoxin-V to preparation of medicine for resisting cognitive, learning and memory dysfunction - Google Patents

Abstract

The invention discloses application of Jingzhaotoxin-V to the preparation of a medicine for resisting cognitive, learning and memory dysfunction. In the invention, a chilobrachys jingzhao toxin extract, namely JZ-V prepared by a chemical method is used for preparing a medicine for preventing, alleviating and/or treating diseases which can cause the disorder of cognitive function and/or learning and memory function such as Alzheimer's disease and the like, particularly a medicine and/or a medicinal composition for improving the cognitive function and/or the learning and memory function. Through the experiment, the JZ-V can remarkably improve the hippocampal synaptic transmission effect of a rat, and ensure that the induction of long-term potentiation (LTP) of a synaptic transmission path of the rat is remarkably strengthened, so that the error latency of dementia animals can be remarkably prolonged, the swimming time and the swimming journey which are needed by finding a safety island by the dementia animals can be remarkably shortened, and the spatial memory of the dementia animals on a platform can be remarkably improved. The clinical first-line medicine for treating dementia, namely donepezil is a positive control medicine in the experiment; and the research result shows that multiple indexes of the therapeutic effect of the JZ-V on learning and memory dysfunction are superior to or equal to those of the donepezil.

Description

The application of JZTX-V in the anti-cognition of preparation, damage in learning and memory medicine

Technical field

The present invention relates to the purposes of peptide material in medicament preparation, the application of particularly a kind of Jingzhao chilobrachys spider toxin-JZTX-V in the anti-cognition of preparation, damage in learning and memory medicine.

Background technology

Chinese patent, ZL200610031864.5 application disclose a kind of novel peptide type neurotoxin that purification obtains from the thick poison of Aranea Jingzhao chilobrachy spider (Chilobrachys jingzhao), i.e. JZTX-V (JZ-V).JZ-V is made up of 29 amino acid residues, and 6 cysteine form 3 pairs of disulfide bond in the molecule, and primary structure is YCQKWMWTCDSKRACCEGLRCKLWCRKII.The CN101428138A patent discloses the application of JZ-V in the preparation analgesic; The CN101543624A patent discloses the application of JZ-V in the preparation anti-additive medicament.So far do not see the relevant report of the application of JZTX-V in the anti-cognition of preparation, damage in learning and memory medicine.

Summary of the invention

The purpose of the embodiment of the invention is the defective to above-mentioned prior art, and the application of a kind of JZTX-V in the anti-cognition of preparation, damage in learning and memory medicine is provided.

The technical scheme taked of the present invention is to achieve these goals:

One aspect of the present invention relates to the purposes that JZ-V improves the mammalian nervous synaptic plasticity.Specifically be meant and significantly improve the inductive Hippocampus long term potentiation of high frequency stimulation HFS (High frequency stimulation) by JZ-V (long-term potentiation LTP), strengthens the inductive phase of LTP.Among the present invention, HFS (5 groups of ripples are wide be the square wave stimulation of 0.2ms for 100HZ, 10 strings) is very near the mode of animal Hippocampus theta rhythm spontaneous activity, the formation that can effectively induce hippocampal dentate LTP.The inductive phase that strengthens LTP was meant before HFS grants 20 minutes and gives JZ-V, can strengthen population spike (population spike, amplitude PS), and keeping at least 1.5 hours greatly.JZ-V can significantly improve mammal cognitive dysfunction and damage in learning and memory through strengthening the inductive phase of LTP, ability of learning and memory such as space learning memory ability of raising Model of Dementia animal and passive avoidance ability.Keeping away dark result of experiment proves: but JZ-V significant prolongation dementia animals error latence reduce errors number; In the Morriss water maze test; The result shows that JZ-V can shorten swimming time and swimming distance that dementia animals searches out safety island; Behind the orientation navigation EOT; Remove platform and carry out in the space exploration experiment, JZ-V can obviously improve the spatial memory of dementia animals to platform, obviously raises in the distance ratio and the time of the quadrant swimming of former safety island place.

One aspect of the present invention relates to the application of JZ-V in the medicine for preparing the disease of preventing, alleviating and/or treating Alzheimer or symptom and can cause cognitive dysfunction and/or damage in learning and memory.

Among the present invention, the prevention in said prevention, alleviation and/or the treatment was meant before Alzheimer occurring, suppressed the generation of cognitive function and learning memory disorder disease; Said alleviation is meant and in the Alzheimer pathogenic process occurring, improves cognitive function and learning memory disorder; Said treatment is meant after Alzheimer occurring or in the pathogenic process, realizes improving cognitive function and damage in learning and memory on the clinical meaning.

More specifically, prevention according to the invention, alleviation and/or treatment Alzheimer or symptom are cognitive function and the learning memory disorders that prevention, alleviation and/or treatment cause to disease.

Another aspect of the present invention; Relate to and a kind ofly be used to prevent, alleviate and/or treat the disease that can cause cognitive dysfunction and/or damage in learning and memory; Like the pharmaceutical composition of Alzheimer or symptom, said medicine contains JZ-V and an amount of optional pharmaceutically acceptable carrier and/or the adjuvant of prevention or treatment effective dose.

In the present invention, according to route of administration, the dosage form of said medicine is selected from solution, suspension agent, Emulsion, pill, capsule, powder agent, sustained release agent or extended release preparation.These preparations will contain the JZ-V of prevention or treatment effective dose, be preferably the JZ-V of purified form, also can be the acceptable salt of its pharmacy, and prepare required an amount of pharmaceutically acceptable carrier of these preparations and/or adjuvant.The selection of dosage form should be suitable for the form to patient's administration.

JZ-V medicine of the present invention can be mixed with various dosage forms with one or more pharmaceutically acceptable carriers and/or excipient through any conventional method; Make it to be applicable to several kinds of approach that the experimenter is used, comprising but be not limited to parenteral, per os, part, Intradermal, intramuscular, subcutaneous, intranasal, maincenter or peripheral nervous and directly give approach.

Therefore, used JZ-V or the acceptable salt of its pharmacy of the present invention can be formulated as the dosage form that for example sucks or be blown into (through port and nose) or transbuccally, parenteral or rectally, local canalis spinalis administration especially.That JZ-V can adopt is charged, the form of the neutral and/or acceptable salt of other pharmacy, comprises hydrochlorate, citrate, the form of the acceptable salt of all pharmacy such as citrate.The pharmaceutically acceptable carrier example includes but not limited to the content described in " Remington ' s Pharmaceutical Sciences (A.R.Gennaro Ed.), 20th edition, Williams & Wilkins PA, USA (2000) ".

Used JZ-V is the white powder product among the present invention, can prepare through known method.

The following document of known method preparation reference:

1. Ceng Xiongzhi, Deng Meichun, Sun Zhenghua, Wang Xianchun, Liang Songping. synthetic, the renaturation and the activity of potassium channels thereof of JZTX-V are identified Chinese biological chemistry and molecular biosciences journal, 2008,24 (5): 463-468;

2. Xiao kindly helps secure the success of, Liang Songping. Hainan catching bird spider toxin-V separation and purification and the research of inhibition tetrodotoxin, TTX responsive type sodium channel activity thereof. and zoological research, 2002,23 (3): 200-205.

The application of the Jingzhao chilobrachy spider toxin extract-JZ-V of chemical method preparation of the present invention in the medicine of behavioristics that improves learning and memory and the effect of raising neural plasticity; Particularly improve the medicine of cognitive function and/or learning and memory function and/or the application in the pharmaceutical composition in preparation; Make this medicine and/or pharmaceutical composition can be used for prevention, alleviate and/or treatment can cause cognitive function and/or damage in learning and memory such as Alzheimer (Alzheimer ' s disease, disease or symptom such as AD).

JZ-V can significantly improve rat hippocampus synapse transmission effects, makes inducing of its synapse transmission path LTP be significance and strengthens.In passive avoidance ability behavioristics test, but JZ-V significant prolongation dementia animals error latence reduce errors number.In the Morriss water maze test; JZ-V can significantly shorten the swimming time and swimming distance that dementia animals searches out safety island; Behind the orientation navigation EOT; Remove platform and carry out the space exploration experiment, JZ-V can obviously improve the spatial memory of dementia animals to platform, obviously raises than A β 1-42 model group in the distance ratio and the time of the quadrant swimming of former safety island place.A clinical line dementia treatment medicine donepezil positive contrast medicine in this experiment, result of study show that JZ-V is superior to for the many index of the therapeutic effect of learning memory disorder or flushes with donepezil.

Description of drawings

Fig. 1 is the index determining figure that brings out current potential;

Fig. 2 mice Morris water maze device sketch map;

Fig. 3 aJZ-V10nmol L ^-1) influence figure to what basic synapse was transmitted;

Fig. 3 bJZ-V100nmol L ^-1) influence figure to what basic synapse was transmitted;

Fig. 4 a is JZ-V 10nmol L ^-1Action diagram to the LTP inductive phase;

Fig. 4 b is JZ-V 100nmol L ^-1Action diagram to the LTP inductive phase;

Fig. 4 c relatively illustrates for the merging of Fig. 4 a and Fig. 4 b.

The specific embodiment

For making the object of the invention, technical scheme and advantage clearer, will combine accompanying drawing that embodiment of the present invention is done to describe in detail further below.

Embodiment 1

JZ-V improves the effect that LTP induces formation

1. experiment material

Laboratory animal: the SD male rat, 250 ± 20g is provided by Nat'l Pharmaceutical & Biological Products Control Institute, certification of fitness SCXK (capital) 2010-008.The operation before with animal feeding in SPF level Animal House, natural lighting, 22 ± 2 ℃ of room temperatures, the drinking-water of freely ingesting underwent surgery after conforming 7 days.Experimental rat is divided into three groups: matched group, JZ-V 10nmol L ^-1+ HFS group and JZ-V 100nmol L ^-1+ HFS group, 6 every group.

Experimental apparatus: VC-11 memory oscilloscope (Nihon Koden, Japan); SEN-7203 three road electronic stimulators (Nihon Koden, Japan); SS-202J stimulus isolator (Nihon Koden, Japan); SR-6N stereotaxic instrument (Narishige, Japan)

2. experimental technique

2.1 the heeling-in of electrode: with weighing body weight 200 ± 20g before the operation of SD rat.With lumbar injection 20% urethane anesthesia (1.0g/kg); Be fixed in and make head maintenance level on the SR-6N stereotaxic instrument,, cut off behind the ear and behind the head hair with 75% alcohol disinfecting skin; Vertically cut skin along the skull median line, push subcutaneous tissue open and use 3%H with absorbent cotton ₂O ₂Remove periosteum, fully expose skull, make sutura clear.With reference to George Paxinos, ChariesWatson rat brain stereotaxic atlas [92], use SR-6N stereotaxic instrument position fixing as bregma after 0.8mm; The other 1.8mm of sagittal suture; 3.5mm under the cerebral dura mater opens skull with bone drill, absorbent cotton is removed bone bits back carefully needle cerebral dura mater with syringe needle.As ventricles of the brain administration inserting needle site.With same method heeling-in stimulating electrode and recording electrode.The stimulating electrode elements of a fix are: AP 7.5-7.8, L 4.4-4.6, H 3.5-4.0; The recording electrode coordinate is: AP 3.7-4.0, L 2.4-2.7, H 3.0-3.5.According to the position of the elements of a fix; Earlier administration basket pipe and recording electrode are put into brain district, place; Stimulate (SEN-7203 three road electronic stimulators, SS-202J stimulus isolator) with strength of fixation then, the population spike that record induces; Slowly adjust the position of stimulating electrode and recording electrode, up to inducing the maximum cluster spike potential.Give the electricity irritation of varying strength then, the action potential that record brings out obtains input-output curve., afterwards stimulating electrode is fixed as detecting stimulus intensity with the stimulating current intensity of the 1/2-1/3 that causes maximum excitatory postsynaptic potential.

2.2 bring out the record of current potential and the computational methods of amplitude:

Bring out the record of current potential: the PS amplitude that calculates in the experiment is as the excitatoty index of DG granular cell crowd.Record PS adopts the extracellular recording method.The testing stimulus frequency is 1/30HZ (SEN-7203 three road electronic stimulators; The SS-202J stimulus isolator); Employing causes the current intensity of 1/2-1/3 of the required stimulus intensity of maximum PS amplitude as testing stimulus intensity, and the testing stimulus intensive parameter is invariable in the whole experiment.Tracer signal is gathered, is amplified through the multi-path physiology signal acquiring processing system, and in multimedia computer, shows and storage.

Referring to Fig. 1, wherein, the starting point of a:PS (end points at first positive peak), the point of b:PS (end points at second positive peak) c:PS, d: the vertical line that process c is ordered and the intersection point of ab point line.The amplitude at dc:PS peak.

PS amplitude computational methods: be observation index with PS amplitude relative value (%) in the experiment, observe the amplification situation after its administration.Its assay method is, with the mensuration of 30min before the HSF mensuration as baseline, each surveys 6 time points (every survey a time point at a distance from 5min), calculates the meansigma methods of 6 time point range value PS amplitudes.As own foundation synapse transmission level (own foundation amplitude), and is 100% with this PS average with the meansigma methods of 6 time point PS range values that 30min write down before the administration.With the PS range value of each time point of surveying after the administration PS meansigma methods, obtain the PS amplitude relative value of each time point divided by 6 time points of baseline.The high frequency stimulation parameter: the high frequency stimulation that brings out LTP is 100HZ, is induced by 10 string high-frequency electrical stimulation, and every train stimulates square wave to form by 5, and ripple is wide to be 0.1ms, and every train is spaced apart 200ms.Stimulus intensity is identical with testing stimulus.If the PS amplitude increases more than 20% than the own control value and the persistent period surpasses 60min, represent that then the LTP phenomenon forms.

2.3 electric Physiological Experiment medication and dosage: JZ-V prepares with normal saline, carries out administration through heeling-in at the conduit of tricorn.The thin plastic pipe that connects microsyringe is imported the basket pipe, after a period of stabilisation, write down PS 30min again, represent the preceding basis of administration to transmit the synapse level.5 μ l medicines or solvent are slowly injected tricorn in 5min, matched group injects 5 μ l normal saline, JZ-V 10nmol L ^-1+ HFS group and JZ-V 100nmol L ^-1+ HFS group is injected 5 μ l 10nmol L respectively ^-1With 5 μ l 100nmol L ^-1JZ-V.In order to prevent interference, carefully mould pipe after administration finishes and still put to stay in the basket pipe to experiment and finish experiment.Medicine is concentration conversion in cerebral tissue: the rat brain volume measurement is 2ml, according to administration volume, original liquid concentration and should reach the drug level in the cerebral tissue in theory, calculates the final concentration [93] of medicine at tricorn.Solvent control group gives the normal saline of respective volume.

2.4 date processing: (mean ± SD) expression adopts the SPSS statistical software to handle to all experimental datas, relatively adopts the t check between group with mean ± standard deviation.P＜0.05 is for difference has significance, and there is the utmost point significance P＜0.01 for difference.

3 results

3.1JZ-V (10,100nmol L ^-1) influence of basic synapse being transmitted level

JZ-V (10,100nmol L ^-1Icv) give through the basket pipe; Horizontal PS range value 30min is transmitted in the basic synapse of record before administration, every 5min record once, with the meansigma methods of the PS range value of these 6 time points as PS amplitude baseline value; Shown in Fig. 3 a and Fig. 3 b, the PS amplitude after the solvent control group administration in the 60min does not change.

3.2JZ-V (10,100nmol L ^-1) influence of HFS being induced DG LTP

Giving J-V 10nmol L ^-1Back 30min gives HFS (100Hz).30min writes down basic synapse and transmits horizontal PS range value before administration, every 5min record once, as PS amplitude baseline value, be 90min observing time after the administration with the meansigma methods of the PS range value of these 6 time points.Shown in Fig. 4 a, Fig. 4 b and Fig. 4 c, the PS amplitude of matched group does not change in 90min, and the PS value of simple high frequency stimulation group is 10,30,60, and 90min is respectively 200% ± 20%, 182% ± 19%, 180% ± 19% and 178% ± 17% (n=6).JZ-V 10nmol L ^-1PS amplitude 10min after giving HFS of+HFS group is 297% ± 21%, is 290% ± 18% during 30min, and the PS amplitude is 296% ± 20% (n=6) during 60min, and JZ-V 100nmol L ^-1The administration group is after giving HFS 10,30, and its PS value of 60min is 348% ± 27%, 333% ± 28%, 335% ± 31% (n=6).Result of study explanation JZ-V is dose dependent and strengthens 100HZ to the inducing of LTP, and significantly improves the Hippocampus synapse of having dashed forward and transmits usefulness.

4, conclusion:

JZ-V can significantly improve the population spike amplitude behind the HFS, strengthens inducing of LTP greatly.Have and strengthen the effect that nerve synapse transmits.

Embodiment 2

JZ-V is to the improvement effect of learning memory disorder due to the A β (amyloid-beta, β-Amyloid Protein)

1 experiment material

1.1 laboratory animal: the C57BL/6 male mice, the SPF level, body weight (23 ± 2) g is provided by Institute of Experimental Animals, Chinese Academy of Medical Sciences, the certification of fitness number: SCXK (capital) 2009-007.With animal feeding in SPF level Animal House, natural lighting, 22 ± 2 ℃ of room temperatures, the drinking-water of freely ingesting, animal conforms in Animal House and underwent surgery in 7 days.

1.2 medicine and reagent: A β 1-42: U.S. Sigma company, 098K4813.Donepezil hydrochloride: defend material medicine company limited, (the accurate word H200550978 of traditional Chinese medicines), lot number: 100223A.JZ-V directly extracts through existing method and obtains.

1.3 experimental apparatus: SR-6N stereotaxic instrument (Narishige, Japan)

2 experimental techniques:

2.1 the animal model preparation: before the operation, the C57 mice is weighed, body weight 25～28g; After mouse peritoneal is injected 4% chloral hydrate anesthesia (1ml/100g), be fixed in and make head maintenance level on the brain solid positioner, with 75% alcohol disinfecting skin; Cut off behind the ear and the head hair after, vertically cut skin along the skull median line, push subcutaneous tissue and periosteum open with absorbent cotton; Fully expose skull, make sutura clear.0.2mm after using the brain solid positioner position fixing as bregma; The other 1mm of sagittal suture, 0.9mm under the cerebral dura mater opens skull with bone drill; Absorbent cotton is removed bone bits back carefully needle cerebral dura mater with syringe needle; With the ophthalmology tweezer vertical the insertion in the aperture of PE pipe of normal saline will be housed, and be fixed on the skull with dentistry cement, injectable drug uses when waiting to test.On the aperture surrounding cranial bone, spilling a small amount of penicillin powder after pipe laying finishes protects from infection.After the operation, animal needs single cage to raise, and recovers to carry out administration after 3 days.After the operation, mice is divided into 4 groups at random, 13 every group, is respectively blank group, A β 1-42 model group, positive drug donepezil hydrochloride administration group and JZ-V administration group.Single cage is raised, and the postoperative animal recovered after 3 days, and modeling is carried out in beginning administration every day in the 4th day.A β 1-42 model group, positive drug donepezil hydrochloride administration group and JZ-V administration group are given the i.e. 5 μ l with A β 1-42400pmol every day, and successive administration carried out modeling in 3 days.

After the modeling, A β 1-42 model group is carried out the Morris water maze test in the last administration after 7 days.After the modeling of positive drug donepezil hydrochloride administration group, irritate stomach in the set time and give donepezil hydrochloride (5mg/kg) every day.After the modeling of JZ-V administration group, intracerebroventricular injection 5 μ l JZ-V every day (0.024 μ M is dissolved in normal saline).Positive drug donepezil hydrochloride administration group and JZ-V administration group are carried out the Morris water maze test behind last administration 1h.

Remove positive drug donepezil hydrochloride administration group set time every day and irritate the positive drug that stomach gives 5mg/kg, all the other each groups are irritated the distilled water (DDW) that stomach waits capacity.JZ-V administration group intracerebroventricular injection JZ-V5 μ l, all the other group tricorns give the normal saline with the capacity of grade.

2.2Morris water maze laboratory: use Morris water maze laboratory device as shown in Figure 2.The Morris water maze laboratory comprises that orientation navigation test and space exploration tests, and preceding four days of Morris water maze laboratory position sea trial.Tested first day, placement platform not lets mice free swimming 2min, to adapt to surrounding.During formal experiment in second day, platform is fixed on NW (pond on average being divided into 4 quadrant: NE, SE, SW, NW by all directions four direction) central authorities, and the liquid level that contains antholeucin in the labyrinth is higher than platform 1.5cm, and water temperature stability is in (22 ± 0.5) ℃.Mice at first is resting on 10s on the step, make its position of understanding platform after, the diagonal quadrant of selecting platform is as entry quadrant (SE); Mice is put into the pond towards pool wall; Make its free swimming, automatic camera system log (SYSLOG) mice seeks the time (escape latency, escape latency) of platform; Setting 60s is the longest escape latency, stops record behind the 60s automatically.If mice is not found platform in 60s, need it is caused platform, at this moment be designated as 60s incubation period.The detection index is incubation period, total distance of swimming, termination type (termination type is for successfully searching out the percentage ratio of platform to the EOT end of test).

The orientation navigation off-test was space exploration test (spatial probe test) in second day; Remove platform, number of times, original platform quadrant distance of swimming ratio and the time ratio (being that animal accounts for the ratio of total distance of swimming and total time in the distance of swimming and the time of original platform quadrant) that in 60s, passes the original platform position through animal estimated the spatial memory ability of animal.The duration of test environment is quiet, and object of reference is constant outside the labyrinth.

During the Morris water maze laboratory, positive drug donepezil hydrochloride administration group and JZ-V administration group still continue administration every day, carry out the Morris water maze laboratory behind positive drug donepezil hydrochloride administration group and the JZ-V administration group last administration 1h.

2.3 keep away dark experiment: after the Morris water maze test finished, animal was kept away dark test after having a rest 1 day.During this period, positive drug donepezil hydrochloride administration group and JZ-V administration group still continue administration every day, keep away dark experimental test behind the last administration 1h.Before the test, let animal in proof box, adapt to 3min first.During training, the mice face is placed bright chamber in the hole dorsad, start self-recording unit simultaneously, timer picks up counting.Animal gets into through the hole and can take out after being shocked by electricity in the darkroom.Official testing behind the 24h, before the 1st entering camera bellows of record mice, in the time (promptly keeping away dark incubation period) that camera-lucida stops, and mice gets into camera bellows among the 5min, the number of times that is shocked by electricity (being the errors number that mice gets into camera bellows).

3 results

3.1Morris the improvement effect of the memory dysfunction that JZ-V (1-42) causes A β 1-42 (β-starch peptide, beta-amyloidpeptide, A β) in the water maze test

Injection A β 1-42 is after one week of modeling in the tricorn; Tangible space learning dysmnesia appear in mice; Show as incubation period than blank group significant prolongation; The success rate (percentage ratio is represented) that mice seeks platform significantly reduces (result is as shown in table 2), and the swimming distance also obviously reduces in the quadrant of platform place.Compare with model group, JZ-V group mice obviously shortens incubation period, and the success rate of seeking platform significantly increases, also showed increased of swimming distance in the quadrant of platform place.

Increase along with frequency of training; Each organizes swimming time, constantly minimizing of swimming distance (incubation period) that mice searches out platform, compares with model group, and all the other respectively organize mice all obviously shortenings (P＜0.01) incubation period; The probability that platform is sought in success constantly increases (P＜0.01), always swims distance and shortens.The result is like table 1,2, shown in 3.

Behind the orientation navigation EOT, remove platform and carry out space exploration experiment and whether formed spatial memory platform with the test mice, the result sees shown in the table 4.The result shows that A β model is compared with blank group mice, and the distance ratio of quadrant swimming obviously descends (P＜0.01) at the original platform place, and the time ratio of quadrant swimming obviously reduces (P＜0.05) at the original platform place.Compare with model group, each organize mice at original platform place the quadrant distance of swimming obviously raise (P＜0.05).

Explanation is in the Morris water maze laboratory, and A β 1-42 has obviously damaged the spatial memory ability of mice, and the damage that prompting JZ-V can obviously resist the mice space learning memory function that is caused by A β 1-42 improves ability of learning and memory.

Table 1.JZ-V seeks preclinical influence to safety island

^#P＜0.05, ##P＜0.01 vs control (contrast); ^*P＜0.05, ^*P＜0.01 vs A β 1-42 group (group)

Table 2.JZ-V seeks the termination type influence of (representing with percentage ratio) to safety island

Table 3.JZ-V seeks total trip distance to safety island influence

P＜0.05，##P＜0.01?vs?control； ^*P＜0.05， ^**P＜0.01?vs?Aβ1-42?group

After table 4. safety island was withdrawn from, JZ-V was to the improvement effect of the inductive learning memory disorder of A β 1-42

^#P＜0.05，##P＜0.01?vs?control； ^*P＜0.05， ^**P＜0.01?vs?Aβ1-42?group

3.2 the improvement effect of the memory dysfunction that JZ-V causes A β 1-42 in keeping away dark test

As shown in table 5, obviously reduce than the blank group the dark incubation period of keeping away of A β 1-42 model group, and errors number obviously increases (P＜0.01).JZ-V administration group and positive drug donepezil hydrochloride administration group (5mg/kg) compare with A β 1-42 model group; Can get into keeping away dark incubation period of darkroom by the significant prolongation mice; Reduce the errors number (P＜0.01) that gets into the darkroom, show that JZ-V and donepezil all can significantly improve the mice passive learning dysmnesia that caused by A β.JZ-V administration group and positive drug donepezil hydrochloride administration group mice are compared, and do not have significant difference.

Table 5.JZ-V in keeping away dark test to the influence of learning memory disorder effect due to β-AP (1-42).

^##P＜0.01?vs?control； ^**P＜0.01?vs?Aβ1-42?group

4. conclusion:

JZ-V water maze with keep away the ability of learning and memory obstacle that β-AP (1-42) of being detected in the dark experiment caused A β and have significance improvement effect, do not have significant difference with positive control medicine donepezil.

Embodiment 3

JZ-V is to the improvement effect of learning memory disorder due to the scopolamine

1 material and method

1.1 laboratory animal: with embodiment 2

1.2 medicine and reagent: scopolamine hydrobromide (Scopolamine hydrobromide, Scop), TOCRIS, BATCHNO:4A/97922, donepezil hydrochloride: defend material medicine company limited, (the accurate word H200550978 of traditional Chinese medicines), lot number: 100223A.JZ-V extracts through existing method and obtains.

1.3 experimental apparatus: with embodiment 22 experimental techniques: except that replacing the A β with scopolamine hydrobromide (consumption 0.5mg/kg), all the other are with embodiment 2

3 results

3.1. the improvement effect of the memory dysfunction that JZ-V causes scopolamine in the Morris water maze test

Morris water maze test result shows; In orientation navigation test, along with the increase of frequency of training, each is organized mice and searches out the required time of platform and reduce gradually; Blank group mice swimming time obviously shortens (P＜0.01) than scopolamine model group mice, shows notable difference.Model group is compared with positive controls, trains beginning in the 2nd day, and positive controls mice swimming time obviously shortens (P＜0.05) than model group mice.Two dose groups of JZ-V are compared with model group, have significantly reduced the needed time of searching platform (P＜0.05).As shown in table 6.

Each organizes the increase of laboratory animal along with frequency of training, and total swimming distance of seeking platform all constantly reduces.Training the 3rd day, the blank group mice distance of always swimming obviously shortened (P＜0.01) than model group mice.The positive drug group is compared with model group, and the mice distance of always swimming obviously shortens (P＜0.05).Two dose groups of JZ-V are compared with model group, have significantly reduced the searching platform distance (P＜0.05) of always swimming.As shown in table 7.

Two dose groups of JZ-V are compared with model group, and high dose and low dosage and donepezil group 5mg/kg all significantly increase the success rate that mice successfully seeks platform.The result is as shown in table 8.

Behind the orientation navigation EOT, remove platform and carry out space exploration experiment and whether formed spatial memory platform with the test mice, the result sees table 9.The space exploration result of the test shows that the scopolamine model is compared with blank group mice, and the distance ratio of quadrant swimming obviously descends (P＜0.05) at the original platform place, and the time ratio of quadrant swimming also obviously reduces (P＜0.05) at the original platform place.Two dose groups of positive drug group mice and JZ-V are compared with model group in original platform place quadrant time ratio and the obviously rising (P＜0.05) of distance of swimming ratio.

Table 6.JZ-V seeks preclinical influence to safety island in the Morris water maze test.

^*P＜0.05， ^**P＜0.01?vs?Scop?group

Table 7.JZ-V seeks total distance of swimming to safety island in the Morris water maze test influence

^*P＜0.05， ^**P＜0.01?vs?Scop?group

Table 8.JZ-V seeks termination type to security platform influence

After table 9. safety island was withdrawn from, JZ-V was to the improvement effect of the inductive learning memory disorder of β-AP (1-42)

^*P＜0.05， ^**P＜0.01?vs?Scop?group

3.2.2 the improvement effect of the memory dysfunction that JZ-V causes scopolamine in keeping away dark test

As shown in table 10, obviously reduce than the blank group the dark incubation period of keeping away of scopolamine model group, and errors number obviously increases (P＜0.01).Two dose groups of JZ-V (high dose and low dosage) and positive drug group (5mg/kg) compare with the scopolamine model group; The incubation period that can the significant prolongation mice gets into the darkroom; Reduce the errors number (P＜0.01) that gets into the darkroom, show that JZ-V and donepezil can significantly improve the mice passive learning dysmnesia that caused by scopolamine.The height of JZ-V and low dose group and positive drug group relatively do not have significant difference.

Table 10. is kept away the influence to the learning memory disorder that scopolamine caused of JZ-V in the dark test

^##P＜0.01?vs?control； ^**P＜0.01，vs?Scop?group

4 conclusions:

JZ-V can significantly improve the mice space learning memory function that caused by scopolamine and the damage of passive avoidance ability, does not have significant difference with positive control medicine donepezil.

Embodiment 4

Under aseptic condition; The JZ-V medicine of chemical method preparation is made with extra care with spray drying method; Make to meet the injection requirement, then with the JZ-V drug powder in direct packaging under the aseptic technique in the bottle or ampoule of cleaning sterilization, jump a queue immediately and seal with aluminium lid.When injection is used, the JZ-V drug powder is dissolved in the glucose, promptly can be made into the solution injection and use.

The toxicity test of medicine: JZ-V toxicity is extremely low, and preliminary study rat intrathecal drug delivery 20mg/kg does not see obvious toxic-side effects, and mouse peritoneal injection 40mg/kg does not see death, does not also show paralysis symptom and the limitation of activity of other sodium ion antagonisies etc.Because synthetic medicine is limited in the early-stage Study, does not measure LD50.

The method for using of medicine: intrathecal drug delivery 20mg/kg, or lumbar injection 40mg/kg.

The above is merely preferred embodiment of the present invention, and is in order to restriction the present invention, not all within spirit of the present invention and principle, any modification of being done, is equal to replacement, improvement etc., all should be included within protection scope of the present invention.

Claims (5)

1. the application of JZTX-V in the anti-cognition of preparation, damage in learning and memory medicine.

2. JZTX-V is used for preventing, alleviate and/or treat the application of the medicine of cognitive dysfunction that Alzheimer causes and/or damage in learning and memory in preparation.

3. application according to claim 1 is characterized in that, said medicine contains prevention or the JZTX-V of treatment effective dose and optional pharmaceutically acceptable carrier and/or adjuvant.

4. application according to claim 1 is characterized in that, the dosage form of said medicine is selected from solution, suspension agent, Emulsion, pill, capsule, powder agent, sustained release agent or extended release preparation.

5. application according to claim 1 is characterized in that, said medicine is through sucking or be blown into mouth and nasal administration, transbuccally administration, parenteral or rectally, local canalis spinalis administration.

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