CN102397526B - 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 the medicine preparation, the particularly application of 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 comprised 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 disclosure JZ-V in the application of preparation in the analgesic; The CN101543624A patent disclosure JZ-V in the application of preparation in the anti-additive medicament.The relevant report of the application of JZTX-V that so far there are no 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 for 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.Refer to that specifically JZ-V can significantly improve the Hippocampus long term potentiation (long-term potentiation, LTP) that high frequency stimulation HFS (High frequency stimulation) induces, strengthen the phase of inducing of LTP.Among the present invention, HFS (the wide square waves for 0.2ms of 5 groups of ripples stimulate 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.Strengthen inducing of LTP and referred to mutually before HFS grants to give JZ-V in 20 minutes, can greatly strengthen the amplitude of population spike (population spike, PS), and keep at least 1.5 hours.JZ-V can significantly improve mammal cognitive dysfunction and damage in learning and memory by strengthening the phase of inducing of LTP, the ability of learning and memory such as the space learning memory ability of raising Model of Dementia animal and passive avoidance ability.The result who keeps away dark 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, after the orientation navigation test finishes, removing platform carries out in the space exploration experiment, JZ-V can obviously improve dementia animals to the spatial memory of platform, obviously raises in distance ratio and the time of the swimming of former safety island place quadrant.

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 described prevention, alleviation and/or the treatment referred to before Alzheimer occurring, suppressed the generation of cognitive function and learning memory disorder disease; Described alleviation refers to improve cognitive function and learning memory disorder in the Alzheimer pathogenic process occurring; Described treatment refers to 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 of the present invention, alleviation and/or treatment Alzheimer or symptom are cognitive function and the learning memory disorders that prevention, alleviation and/or medical needle cause disease.

Another aspect of the present invention, relate to a kind of disease that can cause cognitive dysfunction and/or damage in learning and memory for prevention, alleviation and/or treatment, such as the pharmaceutical composition of Alzheimer or symptom, described 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 described medicine is selected from solution, suspension agent, Emulsion, pill, capsule, powder agent, Controlled release formulation 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 the 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 by any conventional method, make it to be applicable to several 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 (by mouth 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 include but not limited to " Remington ' s Pharmaceutical Sciences (A.R.Gennaro Ed.), 20th edition, Williams ﹠amp; Wilkins PA, USA (2000) " described in content.

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

Known method preparation is with reference to Publication about Document:

1. Ceng Xiongzhi, Deng Meichun, Sun Zhenghua, Wang Xianchun, Liang Songping. synthetic, 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 Toxic extraction thing-JZ-V of chemical method preparation of the present invention in the medicine of the 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 disease or the symptoms such as cognitive function and/or damage in learning and memory such as Alzheimer (Alzheimer ' s disease, 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 Behavior Test, but JZ-V significant prolongation dementia animals error latence reduce errors number.In the Morriss water maze test, JZ-V can significantly shorten swimming time and the swimming distance that dementia animals searches out safety island, after the orientation navigation test finishes, remove platform and carry out the space exploration experiment, JZ-V can obviously improve dementia animals to the spatial memory of platform, obviously raises than A β 1-42 model group in distance ratio and the time of the swimming of former safety island place quadrant.A clinical line dementia treatment medicine donepezil is positive contrast medicine in this experiment, and result of study shows that JZ-V is better than 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) affect figure to what basic synapse was transmitted;

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

Fig. 4 a is JZ-V 10nmol L ^-1LTP is induced the action diagram of phase;

Fig. 4 b is JZ-V 100nmol L ^-1LTP is induced the action diagram of phase;

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

The specific embodiment

For making the purpose, technical solutions and advantages of the present invention clearer, embodiment of the present invention is described further in detail below in conjunction with accompanying drawing.

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 was performed the operation 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: will weigh body weight 200 ± 20g before the SD rat operation.With lumbar injection 20% urethane anesthesia (1.0g/kg), be fixed in and make head maintenance level on the SR-6N stereotaxic instrument, with 75% alcohol disinfecting skin, cut off behind the ear and behind the head hair, 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 carefully needled cerebral dura mater with syringe needle after bone is considered to be worth doing.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, first administration basket pipe and recording electrode are put into brain district, place, then stimulate (SEN-7203 three road electronic stimulators with fixing intensity, the SS-202J stimulus isolator), the population spike that record induces, slowly adjust the position of stimulating electrode and recording electrode, until induce the maximum cluster spike potential.Then give the electricity irritation of varying strength, 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 group.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 experimentation.Tracer signal gathers, amplifies through the multi-path physiology signal acquiring processing system, and shows in multimedia computer 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 calculation method: take PS amplitude relative value (%) as observation index, observe the amplification situation after its administration in the experiment.Its assay method is, with the mensuration of 30min before the HSF mensuration as baseline, each surveys 6 time points (surveying a time point every 5min), calculates the meansigma methods of 6 time point range value PS amplitudes.The meansigma methods of 6 time point PS range values that 30min before the administration is recorded is as own foundation synapse transmission level (own foundation amplitude), and take this PS average as 100%.With the PS range value of each time point of surveying after the administration PS meansigma methods divided by 6 time points of baseline, obtain the PS amplitude relative value of each time point.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 by heeling-in at the conduit of tricorn.The thin plastic pipe that connects microsyringe is imported the basket pipe, after a period of stabilisation, record again PS 30min, represent the front basis of administration and 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 respectively 5 μ l 10nmol L ^-1With 5 μ l 100nmol L ^-1JZ-V.In order to prevent the interference to experiment, carefully mould pipe after administration finishes and still put to stay in the basket pipe to experiment and finish.Medicine is concentration conversion in cerebral tissue: the rat brain volume measurement is 2ml, according to administration volume, original liquid concentration and should reach in theory drug level in the cerebral tissue, calculates medicine at the final concentration [93] of tricorn.The solvent control group gives the normal saline of respective volume.

2.4 date processing: (mean ± SD) expression adopts the SPSS statistical software to process 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) impact of basic synapse being transmitted level

JZ-V (10,100nmol L ^-1Icv) give by 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 administration of solvent control group in the 60min does not change.

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

Giving J-V 10nmol L ^-1Rear 30min gives HFS (100Hz).30min records 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 respectively 200% ± 20% at 10,30,60,90min, 182% ± 19%, 180% ± 19% and 178% ± 17% (n=6).JZ-V 10nmol L ^-1The PS amplitude was 296% ± 20% (n=6) when the PS amplitude of+HFS group was 290% ± 18%, 60min when 10min is 297% ± 21%, 30min after giving HFS, 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, greatly strengthens inducing of LTP.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 performed the operation 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 by existing method and obtains.

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

2 experimental techniques:

2.1 animal model preparation: before the operation, the C57 mice is weighed, after body weight 25～28g, mouse peritoneal inject 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, open skull with bone drill, absorbent cotton is removed the bone bits carefully needle cerebral dura mater with syringe needle afterwards, with ophthalmic tweezers 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 to be tested.Spilling a small amount of penicillin powder at the aperture surrounding cranial bone 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, give donepezil hydrochloride (5mg/kg) in the set time gavage 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 the positive drug that positive drug donepezil hydrochloride administration group set time every day gavage gives 5mg/kg, all the other respectively organize the distilled water (DDW) that gavage 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 front four days of Morris water maze laboratory position sea trial.The experiment first day, placement platform not allows mice free swimming 2min, to adapt to surrounding.When second day is formally tested, platform is fixed on NW (pond on average being divided into 4 quadrant: NE, SE, SW, NW by all directions four direction) central authorities, 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, after making its position of understanding platform, select the diagonal quadrant of platform as entry quadrant (SE), mice is put into the pond towards pool wall, make its free swimming, automatic camera system record mice seeks the time (escape latency, escape latency) of platform, setting 60s is the longest escape latency, automatically stops record behind the 60s.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 end of test).

Orientation navigation off-test second day is space exploration test (spatial probe test), 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 passes the original platform position by animal in 60s estimated the spatial memory capacity 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, allow animal in proof box, adapt to 3min first.During training, the mice face is placed bright chamber in the hole dorsad, start simultaneously self-recording unit, timer begins timing.Animal can be taken out after entering the darkroom and shocked by electricity by the hole.Official testing behind the 24h, before the record mice entered camera bellows the 1st time, in the time (namely keeping away dark incubation period) that camera-lucida stops, and mice entered camera bellows, the number of times that is shocked by electricity (being the errors number that mice enters camera bellows) among the 5min.

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, obvious space learning dysmnesia appear in mice, show as incubation period than blank group significant prolongation, the success rate (percentage ratio represents) 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, compare with model group, 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 shown in table 1,2,3.

After orientation navigation test finishes, remove platform and carry out space exploration and test to test mice and whether formed spatial memory to platform, the results are shown in Table shown in 4.The result shows that A β model is compared with blank group mice, and the distance ratio of swimming at original platform place quadrant obviously descends (P＜0.01), and the time ratio of swimming at original platform place quadrant obviously reduces (P＜0.05).Compare with model group, each organizes mice in the original platform place 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 capacity 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 impact 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 impact of (representing with percentage ratio) to safety island

Table 3.JZ-V seeks total trip distance on safety island impact

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

After table 4. safety island is withdrawn from, the improvement effect of the learning memory disorder that JZ-V induces 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 the significant prolongation mice enter keeping away of darkroom dark incubation period, reduce the errors number (P＜0.01) that enters 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 on the impact 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:

The ability of learning and memory obstacle that β-AP (1-42) that JZ-V detects in the dark experiment at water maze and keeping away causes A β has significance improvement effect, does 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 materials and methods

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 by existing method and obtains.

1.3 experimental apparatus: with embodiment 22 experimental techniques: except 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 dosage 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 laboratory animal along with the increase of 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 dosage 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 dosage 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.

After orientation navigation test finishes, remove platform and carry out space exploration and test to test mice and whether formed spatial memory to platform, the results are shown in Table 9.The space exploration result of the test shows, the scopolamine model is compared with blank group mice, distance ratio in the swimming of original platform place quadrant obviously descends (P＜0.05), in the also obviously minimizing (P＜0.05) of time ratio of original platform place quadrant swimming.Two dosage 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 impact 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 on safety island in the Morris water maze test impact

^*P＜0.05， ^**P＜0.01?vs?Scop?group

Table 8.JZ-V seeks termination type on security platform impact

After table 9. safety island is withdrawn from, the improvement effect of the learning memory disorder that JZ-V induces 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 dosage 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 enters the darkroom, reduce the errors number (P＜0.01) that enters 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. keep away JZ-V in the dark test on scopolamine cause the impact of learning memory disorder

^##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, namely 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 has no obvious toxic-side effects, and it is dead that mouse peritoneal injection 40mg/kg has no, and does not also show the paralysis symptom of other sodium ion antagonisies and limitation of activity etc.Because medicine synthetic in the early-stage Study is limited, does not measure LD50.

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

The above only is preferred embodiment of the present invention, and is in order to limit the present invention, within the spirit and principles in the present invention not all, any modification of doing, 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. application according to claim 1 is characterized in that, the application of described JZTX-V in the medicine of the cognitive dysfunction that causes for the preparation of prevention, alleviation and/or treatment Alzheimer and/or damage in learning and memory.

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

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

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

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