CN102091105B - New application of field pennycress and preparation thereof - Google Patents

Abstract

The invention discloses an application of a field pennycress preparation in preparing a medicament for treating senile dementia, and also comprises the application of the field pennycress preparation in preparing medicaments for improving acetylcholine and central neurotransmitter and treating change of synaptic plasticity.

Description

The new purposes of Herba Patriniae and preparation thereof

Technical field

The present invention relates to the new purposes of a kind of Herba Patriniae and preparation thereof, particularly a kind of Herba Patriniae and preparation thereof belong to field of medicaments in the new purposes of improving aspect cognitive dysfunction prevention and the treatment senile dementia.

Background technology

Alzheimer disease (Alzheimer ' s disease is that a kind of the infringement with cognitive disorder of carrying out property and memory ability is master's central nervous system degenerative disease AD).Along with the aggravation of world population aging, its sickness rate is increasingly high, and human life quality's the influence and even the threat of existence are also become clear day by day.

AD had just never stopped its pathogeny and the research of seeking effectively treatment since Alois Alzheimer first report in 1907.A β is considered to after the initiating agent and key link of AD, and is also increasing to the drug research of A β.Although dropped into great amount of manpower and material resources, achievement in research is very limited.

Chinese medicine has abundant theory and practice experience aspect the preventing and treating of slow down aging and diseases associated with senescence, Chinese medicine AD has obtained certain progress in recent years.For this type of AD multi-factor disease; Single target treatment is difficult to obtain promising result, under the situation that the cause of disease and pathogeny to AD are known little about it, can't be made a breakthrough in the recent period, though there is dispute in problems such as the AD cause of disease; But reach common understanding with regard to its treatment: should intervene PD early; If just intervene in the disease later stage, though possibly delay the process of cognitive competence decline, the infringement that has taken place is then irreversible.The development of cognitive dysfunction is a secular process, possibly before clinical, promptly begin by many decades.The method of the old cognitive disorder of current treatment mainly is that the decline rate through the temporary transient improvement and the cognitive function that slows down alleviates patient's symptom, so for old cognitive disorder, early discovery, early diagnosis, early treatment are the most important.Therefore, carry out the best strategy that active intervention is the dull-witted generation of prevention in the cognitive disorder stage and to patient.But regrettably, often there are mistaken ideas in people to the understanding of the cognitive disorder in old age, think that the old people forgets things well to belong to normal, and therefore, the old cognitive disorder patient more than 90% fails to come to light in early days, and have missed optimal treatment and intervention opportunity.

Herba Patriniae claims again that Radix Scrophulariae, pool are lost, Herba Sonchi Oleracei etc., is the dry herb of Valerianaceae plant Herba Patriniae; Cold nature, acrid in the mouth, hardship; Go into stomach, large intestine, Liver Channel; Effect with heat-clearing and toxic substances removing, eliminating carbuncle evacuation of pus, stasis-dispelling and pain-killing.In recent years; Research to Herba Patriniae is very extensive; Herba Patriniae mainly contains multiple compositions such as coumarin, iridoid, Saponin, sterol and glycoside thereof, has very high medical value and is widely used, and mainly shows the antiinflammatory aspect; As treat chronic pelvic inflammatory disease, colitis, prostatitis etc., wherein the application to chronic pelvic inflammatory disease is the most extensive and evident in efficacy.But the molecular level mechanism of action to Herba Patriniae onset composition is done the less of deep and system exploration, and superficial.The present invention confirms that first Herba Patriniae can improve cognitive dysfunction, effectively prevents and treats senile dementia.

Summary of the invention

The objective of the invention is to disclose the new purposes of a kind of Herba Patriniae and preparation thereof.

The present invention provides the application of Herba Patriniae in preparation treatment medicine for senile dementia.

The present invention provides Herba Patriniae to have the application in the medicine that improves acetylcholine and central neurotransmitter effect in preparation.

The present invention provides Herba Patriniae to have the application in the medicine that changes the synaptic plasticity effect in preparation.

The present invention provides the application of patrinia preparation in preparation treatment medicine for senile dementia.

The present invention provides patrinia preparation to have the application that improves in acetylcholine and the central neurotransmitter drugs with function in preparation.

The present invention provides patrinia preparation to have the application in the medicine that changes the synaptic plasticity effect in preparation.

Patrinia preparation according to the invention is to be active component with the Herba Patriniae extract, adds conventional adjuvant, according to common process, processes pharmaceutically acceptable oral liquid, tablet, capsule, injection, the various dosage forms of pill.

Described Herba Patriniae can be directly through commercially available purchase;

Patrinia preparation of the present invention is to be active component with the Herba Patriniae extract, can prepare according to conventional method, includes but not limited to following method for preparing:

Water extraction method: accurately take by weighing Herba Patriniae powder (Herba Patriniae crushing fine powder) 2-3g, add water 15-25 altogether doubly, divide and boil for three times, extraction time is respectively 1-3h, 1-2h, 0.5-1.5h, and it is subsequent use to be settled to 200-300ml behind the merge extractive liquid, sucking filtration;

The organic solvent extraction method: accurately take by weighing Herba Patriniae powder 2-3g, add 70% ethanol 15-25 altogether doubly, divide three times reflux, extract,, extraction time is respectively 1-3h, 1-2h, and 0.5-1.5h, it is subsequent use to be settled to 200-300ml with distilled water behind the merge extractive liquid, sucking filtration;

Cable-styled extraction method: accurately take by weighing Herba Patriniae powder 2-3g, add 70% ethanol 15-25 altogether doubly, in cable type extractor according, extract 3-5h, it is subsequent use that extracting solution sucking filtration adding distil water is settled to 200-300ml;

Ultrasonic extraction: accurately take by weighing Herba Patriniae powder 2-3g, add 70% ethanol 15-25 altogether doubly, carry out ultrasonic extraction three times, extraction time is 35-55min, and it is subsequent use to be settled to 200-300ml behind the merge extractive liquid, sucking filtration;

The method for preparing of patrinia preparation of the present invention is preferably:

Water extraction method: accurately take by weighing Herba Patriniae powder 2.5g, add 20 times in water altogether, divide and boil for three times, extraction time is respectively 2h, 1.5h, 1h, and it is subsequent use to be settled to 250ml behind the merge extractive liquid, sucking filtration;

The organic solvent extraction method: accurately take by weighing Herba Patriniae powder 2.5g, add 20 times of 70% ethanol altogether, divide three times reflux, extract,, extraction time is respectively 2h, 1.5h, and 1h, it is subsequent use to be settled to 250ml with distilled water behind the merge extractive liquid, sucking filtration;

Cable-styled extraction method: accurately take by weighing Herba Patriniae powder 2.5g, add 20 times of 70% ethanol altogether, in cable type extractor according, extract 4h, it is subsequent use that extracting solution sucking filtration adding distil water is settled to 250ml;

Ultrasonic extraction: accurately take by weighing Herba Patriniae powder 2.5g, add 20 times of 70% ethanol altogether, carry out ultrasonic extraction three times, extraction time is 45min, and it is subsequent use to be settled to 250ml behind the merge extractive liquid, sucking filtration.

Herba Patriniae method for preparing of the present invention can also for: accurately take by weighing Herba Patriniae different parts root, stem, leaf, each 2-3g of herb powder respectively, extract by the said extracted method that to be settled to 200-300ml behind the total flavones merging filtrate sucking filtration subsequent use.Be preferably: accurately take by weighing patrima villosa different parts root, stem, leaf, each 2.5g of herb powder respectively, subsequent use by being settled to 250ml behind the said extracted method extraction total flavones merging filtrate sucking filtration.

The present invention through Herba Patriniae and preparation thereof to quick brain aging Mus cognitive dysfunction test, to the injection of A β 1-42 Hippocampus cause the influence experiment of AD rat cognitive function and synapse, to the influence experiment of acetylcholine (Ach) content in the quick aging Mus brain, maincenter monoamine neurotransmitter in the quick aging Mus brain influenced series of experiment research such as experiment; Proved that Herba Patriniae and preparation thereof can improve cognitive dysfunction, improve the synaptic plasticity change that A β is caused; Improve ability of learning and memory; Reduce A β deposition; Regulate NGF signal survival mechanisms, regulate monoamine transmitters level in the brain; Help to improve AD patient and old people's ability of learning and memory, improve cognitive dysfunction and prevention and the dull-witted purpose of treatment thereby reach.

Following experimental example and embodiment further prove but are not limited to the present invention.

Experimental example 1: patrinia preparation improves the experimentation of quick brain aging Mus cognitive dysfunction

1. materials and methods

1.1 object of study

15 of the aging Mus of healthy SAMR1, as matched group, the SAMP88 monthly age 45 of aging Mus, male and female half and half are divided into aricept (The First Affiliated Hospital of Tianjin University of Traditional Chinese Medic's purchases) treatment group at random, patrinia preparation (being prepared by embodiment 1 method) treatment group.

1.2 behavioristics's test

1.2.1Morris the composition of water maze (Morris water maze)

Water maze is made up of round pool, platform and recording system three parts.Pool diameter 90cm, high 50cm arbitrarily is divided into four quadrants (northeast, the southeast, southwest and Northwest Quadrant) with the pond.Experiment beginning every day, water filling 30cm is dark in the pond, and adds 1 jin of milk powder, makes water become opaque milky, and water temperature remains on about 24 ℃ ± 1 ℃.Have abundant space object of reference (door, lamp, tables and chairs, photographic head and experimenter etc.) around the pond, and the position remains unchanged, for the mice locating platform.Cylindrical bar diameter 9cm, high 28cm places arbitrary quadrant central, and the plane is the 2cm in the underwater not.One photographic head places about 2m place, top of pond central authorities; Automatically gather animal swimming image; Collected signal is directly imported computer; Automatically gather with analytical system (institute of Materia Medica,Chinese Academy of Medical Sciences provides) by image and to analyze automatically and to handle, during length, the search strategy that comprises the time of staying, initial angle and the swimming route of escape latency, the swimming path of animal, different quadrants reaches, outer shroud swims apart from parameters such as percentage ratios.

1.2.2 experiment content

(1) hidden platform test (Hidden Platform Trial)

1d before the test lets animal free swimming 90s in the pond that does not contain platform, the morning, afternoon each once, make it be familiar with the labyrinth environment.The position of platform immobilizes during test, places Northeast Quadrant central authorities, and the platform mid point is from pool wall 22.5cm.The platform offside select two equidistant with it as place of entry; During training animal is faced pool wall and put into water gently; The record mice from entry to the swimming route that finds platform length and find the time (escape latency of platform; Escape latency), let mice on platform, stop 10s then.If can not find platform in the 90s, be designated as 90s incubation period, and mice is placed rest 10s on the platform.Respectively train 1 time at 2 place of entry every day, carries out statistical analysis with twice preclinical arithmetic equal value as the achievement of this day.All experiment mices all carry out hidden platform test 5d, and counter-test 3d and visualisation platforms test 1d are to estimate the variation of different treatment group ability of learning and memory.

(2) counter-test (Reversal Trial)

Operation just moves to opposite quadrant (central authorities of Southwest Quadrant, the platform mid point is from pool wall 22.5cm) with the position of platform basically with hidden platform test.

(3) visualisation platforms test (Visible Platform Trial)

For getting rid of the influence to the space learning memory of sensation, vision or dyskinesia, last 1d carries out the visualisation platforms test.Let the position of platform 2cm that surfaces, and card goes up yellow adhesive tape, all the other are operated with hidden platform test.

(5) date processing

All data are represented with mean ± standard deviation

, and are handled with the SPSS10.0 statistical software.The two-way analysis of variance of the The data repeated measurement data that is obtained in hidden platform test and the counter-test (two-way ANOVA with repeated measures), as factor between group, different training natural law is as the group intrinsic factor with group.In exploratory experiment and visualisation platforms test, relatively use one factor analysis of variance (one-way ANOVE) between sample.Inspection level is decided to be P＜0.05 has significance for difference.4 kinds of common search strategys are seen accompanying drawing 1 in the Morris water maze, and experimental result is seen table 1.

Table 1Morris water maze laboratory result

Annotate: compare with SAMp8 ^*P＜0.05, ^*P＜0.01

Japan bamboo professor Tian Junnan prolongs the foster a kind of natural quick aging mice that obtains of being commissioned to train through AKR/J natural variation mice being carried out inbreeding; SAM-P/8 in the many strains of this family shows tangible learning and memory function and goes down, and is in a kind of low anxiety, low terrified dull-witted state.Wherein SAM-P/8 possesses many characteristics of AD; (β-CIGS) spongy variation, astrocyte reaction etc. appear in extensively calm, reticular formation of brain stem maxicell crowd drosal part, are one of best models of present research cognitive dysfunction, ability of learning and memory and senile dementia of generally acknowledging in the world such as cortex atrophy, cortex and the decline of Hippocampus cone neurocyte number, A β appearance granule.

Can be found out by last table 1: hidden platform experiments and reverse experiment, patrinia preparation treatment group, positive control drug aricept treatment group steadily improve in the 3rd day school grade of training, with model group significant difference are arranged relatively.Visualisation platforms test: with the comparison of monthly age SAMP8 model group and SAMR1 matched group and each treatment group escape latency there are no significant difference; Experimental result shows that the difference of animal aspect sensation, vision or motor function does not produce significantly influence to its space learning memory, has guaranteed the reliability of experimental result.Morris water maze presentation of results patrinia preparation treatment group can be improved the space learning dysmnesia that SAMP8 shows, and the acquisition of learning and memory, maintenance reach each item index that learning capacity is again gone down.

Experimental example 2: Herba Patriniae is to A β _1-42The Hippocampus injection causes the influence of AD rat cognitive function and synapse

1 material

1.1 laboratory animal

50 of the male Sprague-Dawley of secondary (SD) rats (Mus 8～October of age; Body weight 380～400g) is provided by Beijing dimension tonneau China Experimental Animal Center, and placing SPF environment (Specific pathogenfree) is that barrier system (barrier system) is raised; Animal freely ingests and drink water (art before fasting except) in the experimentation; 20 ℃～22 ℃ of room temperatures, relative humidity is 60%～70%, periodicity of illumination is 12h (7:00～19:00 illumination; 19:00～7:00 is dark).

1.2 medicine and preparation

Medicine: patrinia preparation is prepared by embodiment 1 method;

Aricept is bought in The First Affiliated Hospital of Tianjin University of Traditional Chinese Medic.

A β _1-42, synthetic by Xuan Wu hospital polypeptide chamber, the high performance liquid chromatogram chemical analysis identifies that purity is 98%.

Antibody: an anti--A β _1-40, postsynaptic density albumen 95 (PSD-95), nerve growth factor (NGF), the former activated protein kinase of mitogen (MAPK), p-CREB is by the synthetic related peptides section of the U.S. 431A of PE company type full-automatic polypeptide synthetic instrument solid phase method; The HPLC purification; Process polyclonal antibody with hemocyanin crosslinked back immune mouse or rabbit, ELISA detects antibody titer and reaches as high as 1: 16000.TrkA is a Senta Cruz product.

Western-blot blotting used two is anti-to be resisted for the alkali phosphatase enzyme mark sheep anti-mouse igg of Beijing Zhong Shan biotech company and goat anti-rabbit igg general two.

Other experiment reagents:

Paraformaldehyde (Paraformaldehyde, PFA), sodium hydrogen phosphate (disodium hydrogenphosphate dodecahydrate, Na ₂HPO ₄12H ₂O), sodium dihydrogen phosphate (sodium dihyfrogenphosphate, NaH ₂PO ₄2H ₂O), sucrose (sucrose), sodium chloride, chromic potassium sulfate (potassiumchromium sulfate), Beijing Chemical Plant's product;

Dimethyl sulfoxide (DMSO), U.S. Sigma Company products;

Isopentane (iso-pentane), 52952 chemical plant, Beijing;

DBA, U.S. Sigma Company products;

30% hydrogen peroxide, the north is with positive Company products;

Triton x-100 (Triton-100), Beijing chemical reagents corporation product.

DAB, sodium lauryl sulphate (SDS), bromjophenol blue (bromphenol blue), trypsin Typsine), and ethylenediaminetetraacetic acid (Ethylene diaminetetraacetic acid, EDTA), benzyl sulphur FA fluorine (PMSF), U.S. Sigma company;

Glycine dye in advance standard molecular weight protein (reference standard albumen size: 176.5KD, 113.7KD, 80.9KD, 63.8KD, 49.5KD, 37AKD, 26.OKD, 19.6KD, 14.9KD, 8.4KD), U.S. GIBCO BRL company;

Folin phenol reagent, sodium potassium tartrate tetrahydrate, copper sulfate (CUS04-5H20), Beijing ancient cooking vessel state biotech company.

Nitrocellulose filter (pure nitrocellulose bloting membrane), Gelman company.

1.3 experiment is with the preparation of reagent

(1) preceding fixative (4%, 1000ml)

Paraformaldehyde (PFA) 40g

0.1M?Na ₂HPO ₄·12H ₂O 28.64g

Said two devices adds ultra-pure water 800ml, places 60 ℃ of stirring and dissolving on the magnetic stirring apparatus.The dissolving back adds fully:

0.1M?NaH ₂PO ₄·2H ₂O 3.12g

Sucrose 30g

After the last liquid dissolving, 4 ℃ of refrigerator overnight, using NaOH or HCL adjustment pH is 7.4, ultra-pure water is settled to 1000ml.

(2) back fixative

Preceding fixative 100ml

Sucrose 20g

(3) 0.1M phosphate buffer (0.1M PBS)

A liquid: 0.1M sodium hydrogen phosphate

0.1M?Na ₂HPO ₄·12H ₂O?35.8g

Deionized water 1000ml

B liquid: 0.1M sodium dihydrogen phosphate

NaH ₂PO ₄·2H ₂O 15.6g

Deionized water 1000ml

0.1M?PBS：

A liquid 800ml

B liquid 200ml

Mixing is transferred pH value to 7.4

(4) 0.01M phosphate buffer (0.01M PBS)

0.1M?PBS 100ml

NaCl 9g

Deionized water is settled to 1000ml.

(5)0.01M?PBST(pH7.4)

0.1M?PBS 1000ml

Triton?X-100?3ml

The two is water-bath to 40 ' C respectively, mixing, and 4 ℃ of refrigerators are subsequent use.

(6) preparation of DAB working solution (existing) with join at present

DAB 6mg

Distilled water 9ml

0.1MPBS?1ml

Last three's mixing filters.Last liquid adds 10ulH ₂O ₂, be working solution.

(7) preparation of brain tissue homogenate's liquid

50mM?Tris-Hcl，pH7.4

0.5M?EDTA，pH8.0

0.1% tryptic peptide

1MM?PMSF

1.4 key instrument

620-E freezing microtome Britain SHANDON company

DIAX900 type electric homogenizer Germany Heidolph company

DYY III type electrophoretic blotting appearance Liuyi Instruments Plant, Beijing

DYY-II12 type voltage stabilization and current stabilization electrophresis apparatus Beijing Medical Equipment Plant

Microfuge R refrigerated centrifuge BECKMAN

VISILOG 5.0 image analysis systems, Noesis Vision company

BS-2 type multifunction electronic thermostat water bath Shanghai Kai Le Electronic Equipment Factory

Ao Lilong 868 type pH appearance ORION

Haimen City, TS-1 type decolorization swinging table Jiangsu kylin medical apparatus factory

SL-2 type numerical control chromatography refrigerator Beijing Fourth Ring scientific instrument factory

Morris water maze appearance and animal institute of the data handling system Chinese Academy of Medical Sciences

Gulf, river I type C Stereotaxic apparatus The 2nd Army Medical College

2 animal models

2.1 choice of animal models

Select A β Hippocampus injection rat model according to document.

2.2 model preparation

(1) animal is selected: with the selected experiment of diving tower experiment screening learning and memory function normal rat.

(2) condensed state A β _1-42Preparation: A β _1-42In 37 ℃ of calorstats, hatch 7 days (dimethyl sulfoxide is no more than 5%) with the inferior maple dissolving of dimethyl back.

(3) model preparation: after the SD rat is fitted and supports a week, carry out intraperitoneal anesthesia with 10% chloral hydrate by the dosage of 0.35ml/kg, brain solid positioner is fixed; Rat brain location collection of illustrative plates is pressed, 3.0mm behind anterior fontanelle in flat cranium head position; 2.0mm place, center line right side opens skull with the dental burr brill, exposes cerebral dura mater; Microsyringe selects rat right side Hippocampus slowly to inject 2 μ lA β from brain Surface Vertical inserting needle 2.8mm _1-42(10 μ g/ μ l are dissolved in the normal saline), every side injection time is 5min, let the acupuncture needle remain at a certain point 5min, slow withdrawal of needle, skin suture after the partly sterilised, intramuscular injection penicillin prevention infection.Sham operated rats is injected isopyknic normal saline (containing and the inferior maple of the dimethyl of model group equal proportion).

2.3 animal divides into groups

Rat is divided physiology saline group, model group, aricept treatment group and patrinia preparation group.Beginning administration in 3 days after modeling, aricept treatment group is pressed clinical administration dosage and is irritated stomach; Patrinia preparation is configured to the concentration of 0.36g/ml, presses the dosed administration of 0.7ml/100g; Model group and blank group are given and isopyknic distilled water, all once a day.The laggard capable behavioristics test of eight weeks is drawn materials.

3 testing indexs

3.1 behavioristics is observed

Study, the memory ability of test rat adopt the test of Morris water maze.

Morris water maze test: with experimental example 1.

3.2Western-blot Western blot

Detect the expression of the NGF of rat cerebral tissue.Rat breaks end under non-narcotization and gets brain, separates cerebral tissue on the ice chest rapidly, weighs, and places the eppendorf pipe.The 0.01M PBS liquid that adds pre-cooling is washed 2 times, according to brain tissue homogenate's liquid of 1: 5 ratio adding pre-cooling, in the ice bath environment, uses electric homogenizer homogenate 2 minutes, ice bath cracking 30 minutes; 15000rpm,, 4 ℃ are centrifugal 30 minutes; Get supernatant, packing ,-70 ℃ of preservations.Folin phenol colorimetry is carried out protein quantification to cerebral tissue.Drain sample to be checked is freezing, be made into and contain the proteinic concentration of 200ug in every 10ul sample, get 10ul sample to be checked and add 10ul 2xSDS gel loading buffer, boil degeneration 5min ,-20 ℃ of preservations, subsequent use.NGF was according to 1: 1000 concentration dilution.All the other steps are identical with step among the materials and methods 2.2.3.5 of first.

Experimental result

1. behavioristics's characteristic

Bilateral Hippocampus CA1 district injection A β _1-40After 4 weeks, swimming continuance time, the swimming path of model group rat in the Morris water maze prolongs, and more all there were significant differences (P＜0.05) with sham operated rats, and mostly search strategy is marginal mode or random mode.After 4 weeks of administration, compare with model group, patrinia preparation group, aricept treatment group rats'swimming persistent period, swimming path all significantly shorten (P＜0.05), and mostly search strategy is the trend formula.The result sees table 2.

The influence

of table 2 pair A β toxic damages rat model space learning memory ability

Annotate: compare #P＜0.05, ##P＜0.01 with sham operated rats; Compare with model group ^*P＜0.05.

2.A β _1-40Expression in Hippocampus CA1 district and cortex changes

Model group Hippocampus CA1 district and cortex A β _1-40The average area of positive cell and AO significantly increase (p＜0.05) than matched group; At hippocampus; Aricept treatment group reduces than model group; But there was no significant difference (p＞0.05), patrinia preparation treatment group significantly descends (p＜0.05) than model group, difference not significantly (p＞0.05) between aricept treatment group and the patrinia preparation; In the cortical area, aricept treatment group, patrinia preparation treatment group are than model group (p＜0.05) (the seeing table 3,4) that significantly descend.

Table 3 A β _1-40Expression in rat hippocampus CA1 district

Annotate: compare with model group: ^*P＜0.05; ^*P＜0.01

Table 4 A β _1-40Expression in rat cortex

Annotate: compare with model group: ^*P＜0.05; ^*P＜0.01.

3.NGF receptor TrkA changes in the expression of Hippocampus CA1 district and cortex

The average area of model group Hippocampus CA1 district and cortex TrkA positive cell and AO significantly reduce (p＜0.01) than matched group, and each treatment group then significantly increases (p＜0.01) (seeing table 5, table 6, accompanying drawing 2) than model group.

Table 5TrkA is in the expression

in rat hippocampus CA1 district

Annotate: compare with model group: ^*P＜0.05; ^*P＜0.01

Table 6TrkA is in the expression in rat hippocampus CA1 district

Annotate: compare with model group: ^*P＜0.01

This shows that A β can cause synaptic plasticity to change, patrinia preparation group and aricept treatment group all can be improved the synaptic plasticity change that A β is caused, and improve ability of learning and memory, reduce A β deposition, regulate NGF signal survival mechanisms.

Experimental example 3: patrinia preparation is to the influence of acetylcholine (Ach) content in the quick aging Mus brain

1, material

1.1 instrument and equipment

582 type binary pump, ACH-3 (5 μ m; 150mm * 3mm ID) immobilized enzyme reactor, 542 type automatic samplers, CH150 type column oven, enclosed pasture array II 5600A type electrochemical detector, the solid-state micropore electricity levels of 5040 types (platinum electrode, solid-state palladium electrode) behind chromatographic column, the preceding immobilized enzyme reactor of post, the post: ESA company, the U.S..

Ultra-pure water cleaning system: Purelab Plus company, the U.S..

Hypervelocity refrigerated centrifuge: 55P-72 type, Hitachi, Ltd, Japan.

-80 ℃ of refrigerators: VXE380 type, Jouan company, France.

Syringe-type nuclepore membrane filter: water system (0.2 μ m), organic system (0.45 μ m), the Tianjin filter factory of rising.

1.2 medicine and reagent

Medicine: patrinia preparation is prepared by embodiment 1 method;

Reagent: chlorination ACh, NE, DA, 2; 4-dihydroxy acetic acid (dihydroxyphenylacetic acid; DOPAC), 5-HT, 5-hydroxyindoleacetic acid (5-hydroxyindole acetic acid, 5-HIAA), 4-hydroxy-3-methoxy-.alpha.-toluic acid. (homovanillic acid, HVA), sodium hydrogen phosphate (Na ₂HPO ₄), tetramethyl ammonium chloride (tetramethylammonium chloride, TMACl), sodium octyl (octanesulfonic acidsodium salt, OSA), sodium dihydrogen phosphate (NaH ₂PO ₄), citric acid, sodium thiosulfate (Na ₂S ₂O ₅), ethylenediaminetetraacetic acid (ethylenediaminetetracetate, EDTA), A β _1-40(HPLC level): Sigma company, the U.S..

Phosphoric acid (85%), perchloric acid, acetonitrile (HPLC level): Fisher Scientific company, the U.S.." MB " reagent: ESA company, the U.S..

1.3 sample preparation

With the quick brain aging Mus sacrificed by decapitation after the experiment completion of experimental example 1 behavioristics, take out full brain on the ice platform rapidly, weigh liquid nitrogen quick freezing ,-80 ℃ of preservations.Full brain is put homogenate 20s in the ice-cold 0.1mol/L perchloric acid (every 0.1g brain heavily adds perchloric acid 1mL) during extraction.Contain 0.04% (w/v) Na in the perchloric acid ₂S ₂O ₅With 0.04% (w/v) EDTA.Brain homogenate is in 4 ℃ of centrifugal (14000 * g) 20min.Supernatant with 0.2 μ m filter membrane filter, packing ,-80 ℃ of cold preservations are used for the detection of ACh.Before detecting, the last appearance of ACh (contains 100mmol/L Na with buffer ₂HPO ₄, 0.5mmol/L TMACl and 2.0mmol/L OSA) by 1: 2 (v/v) dilution.

1.4ACh detection method

Mobile phase:

Na ₂HPO ₄: 100mmol/L; TMACl:0.5mmol/L; OSA:2.0mmol/L; " MB " reagent: 0.005% (v/v);

Heavily boil off the ionized water standardize solution, it is 8.00,0.2 μ m water system membrane filtrations that phosphoric acid (85%) is transferred pH value.Change mobile phase weekly.

Chromatographic condition:

Binary pump system: ESA582 type; Chromatographic column: ESA ACH-3,150 * 3mm I.D.; Enzyme reactor: enzyme reactor, ESA ACH-SPR before the ESA post, 3cm; Sample size: 10 μ L; Flow velocity: 0.35mL/min; Column temperature: 35 ℃;

Testing conditions:

Detector: 5600A type electrochemical detector; Electrode: M5040 type analysis electrode; Working electrode: platinum electrode; Reference electrode: solid-state palladium electrode; Electromotive force :+300mV; Use 0.1mm thickness the Teflon pad so that the electrode reaction volume below 3 μ L.

Outer target preparation:

Take by weighing chlorination ACh 140.06mg, dilute by 1: 10 (v/v), obtain containing the working solution of standard substance, with the mobile phase dilution, obtain 280.12 μ g/L working standard liquid then heavily to boil off ionized water.Because chlorination ACh is easy to the moisture absorption, all working all need carry out fast.

Methodological study:

The standard solution (1400.60,700.30,280.12,140.06 and 28.01 μ g/L) for preparing five kinds of variable concentrations respectively is used to measure the range of linearity; Carry out the response rate, stability and specificity experiment with 280.12 μ g/L ACh standard solution, confirm the lowest detection limit (S/N=3: 1) of system simultaneously.

2 experimental results

Compare with matched group, the full brain ACh of model SHAMP8 mice content significantly reduces (P＜0.01).After 8 weeks of administration, with the model group rat relatively, patrinia preparation group rat whole brain ACh content significantly raise (P＜0.05～0.01); Positive drug aricept treatment group group rat whole brain ACh level has rising trend, but there was no significant difference (P＞0.05) is seen table 7;

ACh is as a kind of important mediator of people and mammal cholinergic nerve of centrum system, physiology, the pathological process of participation in learning and memory, sleep and awakening and multiple nervous system disease such as AD, parkinson etc.One of AD main clinical manifestation is the synthetic minimizing of maincenter ACh, so the ACh assay becomes an important parameter weighing the ability of learning and memory height in the zoopery.This shows that patrinia preparation can improve the aging full brain ACh of Mus content, this medicine improves aging Mus ability of learning and memory, and improving cognitive dysfunction and prevention maybe be relevant therewith with the dull-witted effect of treatment.

Table 7 different pharmaceutical is to the influence

of the full brain ACh of brain aging mouse model content

Annotate: compare with sham operated rats ^##P＜0.01; Compare with model group ^*P＜0.05, ^*P＜0.01.

Experimental example 4: patrinia preparation is to the influence of maincenter monoamine neurotransmitter in the quick aging Mus brain

1, material

1.1 instrument and equipment

582 type binary pump, ACH-3 (5 μ m; 150mm * 3mm ID) immobilized enzyme reactor, 542 type automatic samplers, CH150 type column oven, enclosed pasture array II 5600A type electrochemical detector, the solid-state micropore electricity levels of 5040 types (platinum electrode, solid-state palladium electrode) behind chromatographic column, the preceding immobilized enzyme reactor of post, the post: ESA company, the U.S..

Ultra-pure water cleaning system: Purelab Plus company, the U.S..

Hypervelocity refrigerated centrifuge: 55P-72 type, Hitachi, Ltd, Japan.

-80 ℃ of refrigerators: VXE380 type, Jouan company, France.

Syringe-type nuclepore membrane filter: water system (0.2 μ m), organic system (0.45 μ m), the Tianjin filter factory of rising.

1.2 medicine and reagent

Medicine: patrinia preparation is prepared by embodiment 1 method;

Reagent: chlorination ACh, NE, DA, 2; 4-dihydroxy acetic acid (dihydroxyphenylacetic acid; DOPAC), 5-HT, 5-hydroxyindoleacetic acid (5-hydroxyindole acetic acid, 5-HIAA), 4-hydroxy-3-methoxy-.alpha.-toluic acid. (homovanillic acid, HVA), sodium hydrogen phosphate (Na ₂HPO ₄), tetramethyl ammonium chloride (tetramethylammonium chloride, TMACl), sodium octyl (octanesulfonic acidsodium salt, OSA), sodium dihydrogen phosphate (NaH ₂PO ₄), citric acid, sodium thiosulfate (Na ₂S ₂O ₅), ethylenediaminetetraacetic acid (ethylenediaminetetracetate, EDTA), A β _1-40(HPLC level): Sigma company, the U.S..

Phosphoric acid (85%), perchloric acid, acetonitrile (HPLC level): Fisher Scientific company, the U.S..

" MB " reagent: ESA company, the U.S..

1.3 sample preparation

With the quick brain aging Mus sacrificed by decapitation after the test completion of experimental example 1 behavioristics, take out full brain on the ice platform rapidly, weigh liquid nitrogen quick freezing ,-80 ℃ of preservations.Full brain is put homogenate 20s in the ice-cold 0.1mol/L perchloric acid (every 0.1g brain heavily adds perchloric acid 1mL) during extraction.Contain 0.04% (w/v) Na in the perchloric acid ₂S ₂O ₅With 0.04% (w/v) EDTA.Brain homogenate is in 4 ℃ of centrifugal (14000 * g) 20min.Supernatant with 0.2 μ m filter membrane filter, packing ,-80 ℃ of cold preservations are used for monoamine transmitters and metabolite thereof and detect.

1.4 detection method

Mobile phase:

NaH ₂PO ₄: 90mmol/L; Citric acid: 50mmol/L; OSA:1.7mmol/L; Acetonitrile: 10%; EDTA:100 μ mol/L;

NaH ₂PO ₄, citric acid and OSA behind 0.2 μ m water system membrane filtration, add acetonitrile through 0.45 μ m organic system membrane filtration, add EDTA, heavily boil off the ionized water standardize solution.

Chromatographic condition:

Binary pump system: ESA 582 types; Chromatographic column: C18,150 * 4.6mm, 5 μ m; Sample size: 10 μ L; Flow velocity: 0.6mL/min; Column temperature: room temperature;

Testing conditions:

Detector: 5600A type electrochemical detector; Electrode: M5040 type analysis electrode; Electromotive force :-150 ,+450 ,+500 ,+550mV;

Outer target preparation:

Take by weighing NE, DOPAC, DA, 5-HIAA, HVA, each 10mg of 5-HT and be dissolved in the HClO of 100mL0.1M ₄In as storing solution (100 μ g/mL), packing is stored in-80 ℃ of refrigerators.Get storing solution (100 μ g/mL) 1mL, be settled to 10mL, concentration is 10 μ g/mL.Get diluent 0.5,0.4,0.3,0.2,0.1mL, add 0.1M HClO respectively ₄To 10mL, be made into 500,400,300,200, the hybrid standard liquid of 100ng/mL.

2 experimental results

Compare with sham operated rats, the full brain 5-HT of model group content obviously reduces (P＜0.05), and DA, NE show on a declining curve.With model group relatively, the full brain DA of patrinia preparation group, 5-HT level all significantly raise, but add up meaningless (P＞0.05), the horizontal downward trend of 5-HIAA is obvious, wherein patrinia preparation treatment group HVA significantly descend (P＜0.05).

Main and the ergasia of maincenter monoamine neurotransmitter, emotion, behavior, somatic movement etc. are closely related, with also exist between the learning and memory certain related.Dopamine (DA), norepinephrine (NE), three kinds of monoamine content of serotonin (5-HT) descend and can cause the decline of central excitation property, dull-witted and melancholy symptom occur.NE, DA, 5-HT equal size reduce in the naturally-aged animal brain, and the animal learning memory function descends.The AD pathological changes is often involved nucleus ceruleus and nucleus raphes dorsalis, and also visible DA can system change, and shows as brain NE, the reduction of DA content, 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) lowering of concentration.Regulate monoamine transmitters level in the brain, have the ability of learning and memory that helps improve AD patient and old people.This shows; Patrinia preparation is through delaying Ach, DA, 5-HT degraded; Central DA, 5-HT level are raise relatively, improve monoaminergic system activity in the brain then, regulate monoamine transmitters level in the brain; Have the ability of learning and memory that helps improve AD patient and old people, improve cognitive dysfunction and prevention and the dull-witted purpose of treatment thereby reach.

Table 8 different pharmaceutical is to influence

unit of central neurotransmitter and metabolite content thereof: μ g/L

Annotate: compare with sham operated rats ^#P＜0.05; Compare with model group ^*P＜0.05.

Description of drawings

Fig. 1: 4 kinds of common search strategys in the Morris water maze

The expression of Fig. 2: NGF in each group cerebral tissue changes electrophoretogram

A. matched group; B. model group; C. aricept group; D patrinia preparation group

Following embodiment all can realize the effect of above-mentioned experimental example.

The specific embodiment

Embodiment 1: oral liquid

Accurately take by weighing Herba Patriniae powder 2.5g, add 20 times of 70% ethanol altogether, carry out ultrasonic extraction three times, extraction time is 45min, and it is subsequent use to be settled to 250ml behind the merge extractive liquid, sucking filtration; Get the said extracted thing, add conventional adjuvant,, process oral liquid according to common process.

Embodiment 2: tablet

Accurately take by weighing Herba Patriniae powder 2.5g, add 20 times in water altogether, divide and boil for three times, extraction time is respectively 2h, 1.5h, 1h, and it is subsequent use to be settled to 250ml behind the merge extractive liquid, sucking filtration;

Get the said extracted thing, add conventional adjuvant,, process tablet according to common process.

Embodiment 3: capsule

Accurately take by weighing Herba Patriniae powder 2.5g, add 20 times of 70% ethanol altogether, divide three times reflux, extract,, extraction time is respectively 2h, 1.5h, and 1h, it is subsequent use to be settled to 250ml with distilled water behind the merge extractive liquid, sucking filtration;

Get the said extracted thing, add conventional adjuvant,, process capsule according to common process.

Embodiment 4: pill

Accurately take by weighing Herba Patriniae powder 2.5g, add 20 times of 70% ethanol altogether, in cable type extractor according, extract 4h, it is subsequent use that extracting solution sucking filtration adding distil water is settled to 250ml;

Get the said extracted thing, add conventional adjuvant,, process pill according to common process.

Embodiment 5: injection

Accurately take by weighing patrima villosa stem powder 2.5g respectively, add 25 times of 70% ethanol altogether, carry out ultrasonic extraction three times, extraction time is 50min, and it is subsequent use to be settled to 250ml behind the merging filtrate sucking filtration; Get the said extracted thing, add conventional adjuvant,, process injection according to common process.

Embodiment 6: lyophilized injectable powder

Accurately take by weighing patrima villosa root powder 3g respectively, add 18 times of 70% ethanol altogether, in cable type extractor according, extract 3.5h, it is subsequent use that extracting solution sucking filtration adding distil water is settled to 300ml; Get the said extracted thing, add conventional adjuvant,, process lyophilized injectable powder according to common process.

Embodiment 7: drop pill

Accurately take by weighing each 2g of patrima villosa herb powder respectively, add 25 times of 70% ethanol altogether, divide three times reflux, extract,, extraction time is respectively 3h, 1h, and 0.8h, it is subsequent use to be settled to 300ml with distilled water behind the merge extractive liquid, sucking filtration; Get the said extracted thing, add conventional adjuvant,, process drop pill according to common process.

Claims (4)

1. the application of patrinia preparation in preparation treatment medicine for senile dementia.

2. application as claimed in claim 1 is characterized in that improving acetylcholine and the interior monoamine transmitters level of adjusting brain in the brain.

3. application as claimed in claim 1 is characterized in that improving synaptic plasticity and changes.

4. like the arbitrary described application of claim 1-3; It is characterized in that patrinia preparation is is active component with the Herba Patriniae extract; Add conventional adjuvant,, process pharmaceutically acceptable oral liquid, tablet, capsule, injection, the various dosage forms of pill according to common process.

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