CN101269179A - Traditional Chinese medicine preparation for treating senile dementia and preparation method thereof - Google Patents

Abstract

The invention discloses a traditional Chinese medicine formulation for curing senile dementia and a preparation method thereof. The traditional Chinese medicine formulation is made of ginseng, milk veteh, epimedium, fumitory, rhizoma gastrodiae, angelica, radid polygoni multflori preparata, sealwort and grassleaf sweelflag rhizome which are performed by an ethanol extracting technology and a poach technology. The traditional Chinese medicine formulation is ground by the theory of traditional Chinese medicine and adopts a scientific theory and an advanced technology to develop the traditional Chinese medicine formulation with good curative effect and fewer side effects and being used for curing the senile dementia. The experimental research shows that the traditional Chinese medicine formulation can improve the learning memory ability of an AD rat model. Meanwhile, the traditional Chinese medicine formulation can improve the antifatigue and anti-stress ability of an organism and has good curative effect on the kidney empty. In addition, the invention also provides an optimized preparation method suitable for the industrial production.

Description

Chinese medicine preparation of treatment senile dementia and preparation method thereof

Technical field

The present invention relates to technical field of Chinese medicines, particularly relate to a kind of Chinese medicine preparation for the treatment of senile dementia and preparation method thereof.

Background technology

Along with global aged tendency of population, and Alzheimer (Aizheimer ' s disease, sickness rate AD) is the trend that rises year by year.Because lack effective treatment means, AD has become the fatal disease of the 4th harm humans health after the heart, cerebrovascular disease and tumor.The aging population sum of China more than 60 years old accounts for about 10% of population according to statistics, has 5% to be AD patient approximately.Therefore, seek the active drug of preventing and treating AD and become problem demanding prompt solution in the life sciences.

Being widely used in both at home and abroad at present has a collection of determined curative effect really, uses the chemical drugs of safety in the clinical anti senile dementia drug, still, as most of chemical medicines, be applied to clinical nervous system chemistry medicine and also show side effect more and more.Fact proved a large amount of AD patients through treatment by Chinese herbs, not only intelligent situation is greatly improved, and can also life lengthening.Adopt Chinese medicine AD worldwide more and more to be accepted by people.Domestic and even world wide is interior from Chinese medicine, natural drug, utilizes the medicine of modern science and technology research and development prevention and treatment alzheimer disease, conforms with the trend of " back to nature " in world today's scope.

Modern medicine is thought: change, gene mutation, free radical, metallic element, nutrition and the dysbolismus of beta amyloid peptide, Tau albumen, apolipoprotein (APOE), neurotransmitter, tumor, hormone, immune system malfunction, chronic viral infection, cerebral trauma etc. all can cause senile dementia.

In clinical practice, people's such as Wang Dehua reinforcing kidney and strengthening resistance method promptly uses stupid clever soup (Radix Aconiti Lateralis Preparata, Herba Epimedii, Cortex Cinnamomi, the Radix Polygoni Multiflori, Rhizoma Acori Graminei etc.) to give birth to marrow with warming the kidney to activate YANG, and clinical symptoms is obviously improved, and mind is gradually clear, reminiscence, and total effective rate reaches 94.1%.Invigorating kidney, promoting blood circulation method such as Shao Nian side is obstructed at blood stasis due to renal deficiency, brain network, with the treatment of having one's ideas straightened out of the kidney invigorating collateral dredging.Treatment back blood fat and part hemorheology index also obviously improve before the treatment.Its side uses Cognex (Radix Polygoni Multiflori, Fructus Lycii, Radix Ginseng, Rhizoma Acori Graminei, Rhizoma Chuanxiong, Radix Paeoniae Rubra).The kidney invigorating eliminating phlegm method of Sun Jiming thinks that this disease is that to decrease with the kidney essense deficiency of vital energy serve as basic, and the expectorant stasis of blood is closed key and is the morbidity key.Proposition control slow-witted must the kidney invigorating, kidney essense foot then brains fills; Control and slow-witted must have one's ideas straightened out, have one's ideas straightened out gods, so with the kidney tonifying recovery gods of eliminating phlegm for resuscitation.Its side uses kidney tonifying eliminating phlegm soup (Radix Rehmanniae Preparata, Herba Cistanches, Fructus Corni, Rhizoma Arisaematis (processed), Bombyx Batryticatus, Rhizoma Pinelliae Preparata, Caulis Bambusae In Taenia).All happy congruent relatively the kidney invigorating Fructus Alpiniae Oxyphyllae sides (being made up of Fructus Lycii, Radix Polygoni Multiflori, Radix Ginseng, Cortex Moutan, Borneolum Syntheticum etc.) and huperzine A are to the influence of Model of Dementia learning and memory in rats ability, results suggest, Chinese medicine the kidney invigorating Fructus Alpiniae Oxyphyllae side treatment rat senile dementia has dosage correlation, and effect is better than huperzine A.

The inventor finds to have the clinical report of a lot of compound of Chinese medicine alzheimer disease when literature survey.In order to inherit and develop Chinese medicine and pharmacy characteristic and advantage, active development has anti-ageing year dementia new drug of independent intellectual property right, intends maintaining secrecy of Guizhou Province's traditional Chinese medical science carried out the research of anti-senile dementia active substance through proved recipe.Effect such as this prescription has invigorating the spleen and replenishing QI, kidney tonifying, essence replenishing, nourishing blood to tranquillize the mind, suppressing the hyperactive liver to relieve the wind syndrome, blood circulation promoting and blood stasis dispelling, eliminate phlegm for resuscitation.Clinically be used for the treatment of diseases such as memory of elderly person power goes down, fatigue, dreaminess.The present invention on the basis of proved recipe, makes full use of scientific theory and advanced technology means at this, with the theory of Chinese medical science is to instruct to optimize prescription the Chinese medicine compound preparation of development prevention of utilization state-of-the-art technology and treatment senile dementia.

Summary of the invention

Technical problem to be solved by this invention is to maintain secrecy on the basis of proved recipe at defying age, is guidance with the theory of Chinese medical science, utilizes scientific theory and advanced technology, develops Chinese medicine preparation of a kind of good effect, treatment senile dementia that toxic and side effects is little and preparation method thereof.

In order to solve the problems of the technologies described above, the present invention adopts following technical scheme:

According to listed as parts by weight, the Chinese medicine preparation of treatment senile dementia is to be prepared from for 100～140 parts by 50～70 parts of raw material of Chinese medicine Radix Ginsengs, 100～140 parts of Radix Astragali Preparatas, 80～100 parts of Herba Epimedii, 80～100 parts of Rhizoma Corydalis, 100～140 parts in Rhizoma Gastrodiae, 80～100 parts of Radix Angelicae Sinensis, 100～140 parts of Radix Polygoni Multiflori Preparatas, 100～140 parts of Rhizoma Polygonatis and Rhizoma Acori Graminei.

Preferably, it is to be prepared from for 120 parts by 60 parts of raw material of Chinese medicine Radix Ginsengs, 120 parts of Radix Astragali Preparatas, 90 parts of Herba Epimedii, 90 parts of Rhizoma Corydalis, 120 parts in Rhizoma Gastrodiae, 90 parts of Radix Angelicae Sinensis, 120 parts of Radix Polygoni Multiflori Preparatas, 120 parts of Rhizoma Polygonatis and Rhizoma Acori Graminei.

The present invention also provides a kind of preferred for preparation method of the Chinese medicine preparation of aforementioned therapies senile dementia, comprises the steps:

(1) alcohol extraction: get Radix Ginseng, Rhizoma Gastrodiae, Radix Astragali Preparata, Herba Epimedii, Rhizoma Corydalis five tastes medical material 85% alcohol reflux, filter, get alcohol extract, decompression recycling ethanol, medicinal liquid is standby;

(2) decocting in water: get Radix Angelicae Sinensis, Radix Polygoni Multiflori Preparata, Rhizoma Polygonati, Rhizoma Acori Graminei four Chinese medicine material and decoct with water, filter, filtrate decompression concentrates standby;

(3) preparations shaping: merge alcohol extraction medicinal liquid and decocting in water medicinal liquid, be concentrated into thick paste, vacuum drying, preparation process is made the various dosage forms on the pharmaceutics meaning routinely.

Be specially:

(1) alcohol extraction: get Radix Ginseng, Rhizoma Gastrodiae, Radix Astragali Preparata, Herba Epimedii, Rhizoma Corydalis five tastes medical material with 8～20 times of amount 85% alcohol reflux 1～3 time, each 1～3h filters, alcohol extract, decompression recycling ethanol, medicinal liquid is standby;

(2) decocting in water: get Radix Angelicae Sinensis, Radix Polygoni Multiflori Preparata, Rhizoma Polygonati, Rhizoma Acori Graminei four Chinese medicine material and decoct 1～3 time with 8～16 times of water gagings, each 1～3h filters, and filtrate decompression concentrates standby;

(3) preparations shaping: merge alcohol extraction medicinal liquid and decocting in water medicinal liquid, be concentrated into thick paste, in 60～80 ℃ of vacuum dryings, preparation process is made the various dosage forms on the pharmaceutics meaning routinely.

Further, the preparation method of the Chinese medicine preparation of treatment senile dementia:

(1) alcohol extraction: get Radix Ginseng, Rhizoma Gastrodiae, Radix Astragali Preparata, Herba Epimedii, Rhizoma Corydalis five tastes medical material with 85% alcohol reflux 2 times, each 2h adds 16 times of amount ethanol for the first time, adds 14 times of amount ethanol for the second time, filter, alcohol extract, decompression recycling ethanol, medicinal liquid is standby;

(2) decocting in water: get Radix Angelicae Sinensis, Radix Polygoni Multiflori Preparata, Rhizoma Polygonati, Rhizoma Acori Graminei four Chinese medicine material water and decoct 3 times, each 2h adds 14 times of water gagings for the first time, adds 12 times of water gagings for the second time and for the third time, filters, and filtrate decompression concentrates standby;

(3) preparations shaping: merge alcohol extraction medicinal liquid and decocting in water medicinal liquid, be concentrated into thick paste, in 70 ℃ of vacuum dryings, preparation process is made the various dosage forms on the pharmaceutics meaning routinely.

The side separates: the Radix Ginseng in this prescription, Radix Polygoni Multiflori Preparata, Rhizoma Gastrodiae, Rhizoma Acori Graminei are monarch drug, have the smart solid and gas of QI invigorating benefit, the merit of endogenous wind stopping resuscitation inducing and mind tranquilizing; Rhizoma Polygonati, Radix Astragali (processed), when being classified as ministerial drug, have the invigorating the spleen and replenishing QI yang invigorating, the effect of nourshing blood and promoting blood circulation silt; Herba Epimedii, Rhizoma Corydalis are adjuvant drug, energy the liver and the kidney tonifying bone strengthening, blood-activating and qi-promoting resolving depression.All medicines share, and making QI and blood get invigorating the liver and kidney must support, and the silt activating QI is capable, ataraxia, and the brain key is satisfied awake.So the energy defying age, cards such as control senile dementia.

The present invention has carried out a large amount of experiments maintaining secrecy on the basis of proved recipe, and is specific as follows:

One, preparation technology and research thereof

1, the design of process route

1.1 process route design

Through to the investigation of the pertinent literature of each activity of drug ingredients position or composition, the comprehensively physicochemical property of the effective ingredient of each flavour of a drug or effective site and extracting method, the extraction and preparation technique of initial setting sky ginseng Cognex can be undertaken by following several design routes:

(1) main active of this each medical material of compound recipe or active site all have certain water solublity, decoct with water together by traditional handicraft, filter, and reclaim to concentrate, and drying gets the extractum sample.

(2) according to the literature in this compound recipe the main active or the extraction ratio of active site in certain pure water of Radix Ginseng, Rhizoma Gastrodiae, Radix Astragali Preparata, Herba Epimedii, Rhizoma Corydalis performance Fructus Alpiniae Oxyphyllae effect best, select Radix Ginseng, Rhizoma Gastrodiae, Radix Astragali Preparata, Herba Epimedii, Rhizoma Corydalis five tastes medical material with certain density pure water extraction, and Radix Polygoni Multiflori Preparata, Radix Angelicae Sinensis, Rhizoma Polygonati, Rhizoma Acori Graminei four Chinese medicine material decoct with water together, alcohol extract and decocting liquid merge after reclaiming respectively and being concentrated into thick paste then, the dry extractum sample that gets.

(3) Radix Ginseng, Rhizoma Gastrodiae, the contained polysaccharide of Radix Astragali Preparata have certain antioxidation, the function of enhancing immunity according to the literature, so select Radix Ginseng, Rhizoma Gastrodiae, Radix Astragali Preparata, Herba Epimedii, Rhizoma Corydalis five tastes medical material earlier with certain density pure water extraction, its medicinal residues decoct with water with Radix Polygoni Multiflori Preparata, Radix Angelicae Sinensis, Rhizoma Polygonati, Rhizoma Acori Graminei four Chinese medicine material again, alcohol extract and decocting liquid merge after reclaiming respectively and being concentrated into thick paste then, the dry extractum sample that gets.

1.2 nootropic effect related activity screening

The inventor prepares sample with reference to above three process routes, carried out the screening active ingredients of the influence of antagonism beta 4 amyloid toxic action, the influence of pair cell cholinesterase activity, pair cell α 7 nicotine receptor protein expressions, result's following (methodology is seen below " the molecular mechanism of action research of capsule treatment AD of the present invention "):

1.2.1 antagonism beta 4 amyloid toxic action

The result shows, each sample all can resist the decline of the MTT percent reduction that the beta 4 amyloid causes in various degree; Its antagonism compares: 2 ≈ 3＞1.Compare * P＜0.05 with the blank group; * P＜0.01 is compared with model group, ^#P＜0.05; ^##P＜0.01.

1.2.2 pair cell cholinesterase activity influence

The result shows that the prepared sample of different process can both suppress the cell cholinesterase activity, and it suppresses active: 2＞3＞1.Compare * P＜0.05 with the blank group; * P＜0.01.

1.2.3 the influence of pair cell α 7 nicotine receptor protein expressions

The result shows, the proteic expression of rise cell α 7 nicotine receptors that the prepared sample of different process all can be in various degree, and the rise ability is followed successively by: 2 ≈ 3＞1.Compare * P＜0.05 with the blank group; * P＜0.01.

1.3 process route is preferred

According to 1.2 nootropic effect related activity The selection result, the sample of technology 2 and 3 preparations is indifference on anti-beta 4 amyloid toxic action, inhibition cell cholinesterase activity ability, rise cell α 7 nicotine receptor protein expression abilities, in conjunction with the practical situation of producing, technology 2 preparation routes are succinct than technology 3.So select to prepare sample by process route 2, i.e. Radix Ginseng, Rhizoma Gastrodiae, Radix Astragali Preparata, Herba Epimedii, the alcohol extraction of Rhizoma Corydalis five tastes medical material in the side, Radix Polygoni Multiflori Preparata, Radix Angelicae Sinensis, Rhizoma Polygonati, Rhizoma Acori Graminei four Chinese medicine material water are together carried.Look out cream situation and experiment practical situation selective finishing technology, and determine its supplementary product kind and addition according to mouldability.

2, process conditions screening

2.1 alcohol extraction process conditional filtering

2.1.1 the investigation of medical material water absorption rate

Take by weighing the medical material of 3 part of 1/10 recipe quantity: Radix Ginseng 6g, Rhizoma Gastrodiae 12g, Radix Astragali Preparata 12g, Herba Epimedii 9g, Rhizoma Corydalis 9g place container, add entry 600mL, are dipped to the heart, with the superfluous water elimination, weigh, and are calculated as follows water absorption rate.The average water absorption rate of the medical material of alcohol extraction part as a result is 218.1%, the results are shown in Table 1, thus when in alcohol extraction process, extracting for the first time than the extraction solvent of adding 2 times of amounts for the 2nd time.

The moisture content test of table 1 alcohol extraction process

2.1.2 the selection of alcohol extraction number of times

Take by weighing 2/50 recipe quantity alcohol extraction medical material, by 16 times of amount 85% alcohol reflux 2.0h, measure 2.0h for 14 times for the second time for the first time, measure 1.5h for 14 times for the third time, each alcohol extract filters separately, and the content and the pure extractum yield of icariin, gastrodine, corydaline measured in gradation, the results are shown in Table 2.

The selection of table 2 alcohol extraction number of times

The backflow number of times	Alcohol extractum yield (%)	Icariin extracted amount (mg)	Gastrodine extracted amount (mg)	Corydaline extracted amount (mg)
					For the second time for the first time,	20.58	23.10	21.43	4.016
For the third time	1.66	0.49	0.57	0.105
					Proportion * (%) for the third time	7.46	2.08	2.59	2.58

* refer to that alcohol extraction for the third time accounts for the ratio of total pure extractum amount, icariin extracted amount, total flavones extracted amount.

Result of the test shows, is index with the yield of extract, and the alcohol extraction proportion is 7.46% for the third time; With the icariin extracted amount serves as to investigate index, and the alcohol extraction proportion is 2.08% for the third time; With the gastrodine extracted amount serves as to investigate index, and the alcohol extraction proportion is 2.59% for the third time; With the corydaline extracted amount serves as to investigate index, and the alcohol extraction proportion is 2.58% for the third time, illustrates that twice alcohol extraction is complete substantially, so the technology of twice of employing alcohol extraction.

2.1.3 the preferred extraction process of orthogonal test

According to documents and materials and result of the test, extraction time, alcohol adding amount, concentration of alcohol are the principal elements that influences extraction ratio, by each factor 3 level, use L ₉(3 ⁴) orthogonal table arrangement test (table 3).With pure extractum yield, icariin extracted amount, gastrodine extracted amount, corydaline extracted amount is evaluation index, and alcohol extraction process is inquired into.

2.1.3.1 material and instrument

Instrument: HP1100 high performance liquid chromatograph (comprising the HP1100 pump, HP1100 UV-detector, HP1100 column oven, HP1100/WND-3D work station), Rotary Evaporators (Tokyo physics and chemistry), uv-spectrophotometric instrument (Tianjin, island UV-2401).

Material: icariin reference substance, gastrodine reference substance (are provided by Nat'l Pharmaceutical ﹠ Biological Products Control Institute.Lot number: 110737-200312,110807-2002205), the corydaline reference substance (is provided by analytical chemistry teaching and research room of Guiyang Medical College pharmaceutical college, be accredited as corydaline through spectral data, measure its purity greater than 98.5% by the HPLC area normalization method, acetonitrile (chromatographically pure), pure water (Robust pure water), methanol (chromatographically pure), other reagent are analytical pure.

2.1.3.2 method and result

Table 3 factor level table

The mensuration of a. pure extractum yield

Press L ₉(3 ⁴) orthogonal table arrangement test, take by weighing 2/50 recipe quantity medical material, extract by the setting scheme, filter, filtrate merges, and measures cumulative volume V _AlwaysThe accurate alcohol extract of drawing 50mL is in the evaporating dish that is dried to constant weight, and water-bath volatilizes, and in 105 ℃ of dryings 3 hours, puts and cools off 30min in the exsiccator, weighs rapidly.Be calculated as follows yield of extract, the results are shown in Table 4.

B. the assay of icariin, gastrodine and corydaline

Press L ₉(3 ⁴) orthogonal table arrangement test, take by weighing 2/50 recipe quantity medical material, extract by the setting scheme, filter, filtrate merges, and is settled to certain volume; (" Chinese pharmacopoeia (33 pages of VID of appendix of version in 2005) is measured the content of icariin, gastrodine, calculates the extracted amount of icariin, gastrodine and corydaline, the results are shown in Table 4 according to high performance liquid chromatography.

Chromatographic condition: 1. icariin: with the octadecylsilane chemically bonded silica is filler, acetonitrile: water (30: 70) is mobile phase, flow velocity: 1mLmin ^-1, wavelength: 270nm; Column temperature: 40 ℃.2. gastrodine: chromatographic column: Diamonsil ^TMC ₁₈(5 μ m, 250 * 4.6mm); Mobile phase: acetonitrile-water (2: 98); Column temperature: 40 ℃; Detect wavelength: 220nm; Flow velocity: 1mLmin ^-1Sample size: 10 μ L.3. corydaline: Diamonsil ^TMC ₁₈Post (5 μ m, 250 * 4.6mm), column temperature: 30 ℃; Detect wavelength: 280nm, mobile phase: 0.1% phosphoric acid (triethylamine is transferred pH=6): second eyeball (48: 52), flow velocity 1mL/min.

The preparation of reference substance solution: icariin reference substance, gastrodine reference substance, corydaline reference substance decided in accurate respectively title, places volumetric flask, dissolving, and standardize solution shakes up, and icariin reference substance solution concentration is 129 μ gmL ^-1, gastrodine reference substance solution concentration is 209 μ gmL ^-1, corydaline reference substance solution concentration is 65.6 μ gmL ^-1

The preparation of sample solution: extract by the orthogonal experiment design, filter, filtrate merges, and is settled to certain volume, promptly gets each sample solution.

Algoscopy: get each sample solution and cross 0.45 μ m microporous filter membrane, filtrate is test sample liquid.Accurate respectively need testing solution and each 10 μ L of reference substance solution of drawing inject chromatograph of liquid, measure, promptly.

Table 4 alcohol extraction process Orthogonal Experiment and Design table and result

Annotate: alcohol extraction process adopts 4 evaluation indexes, adopts comprehensive grading to carry out data analysis, and the icariin weight coefficient is 0.3, and the corydaline weight coefficient is 0.3, and the gastrodine weight coefficient is 0.3, and the yield of extract weight coefficient is 0.1.Comprehensive grading=(icariin/icariin maximum * 0.3+ corydaline/corydaline maximum * 0.3+ gastrodine/gastrodine maximum * 0.3+ yield of extract/yield of extract maximum * 0.1) * 100

Variance analysis

Orthogonal experiments is carried out variance analysis, the results are shown in Table 5.

Table 5 alcohol extraction process evaluation index analysis of variance table

*P＜0.05，F _0.05(2，2)＝19.00

From the analysis of table 4 visual result, the order of each evaluation index in each factor affecting extraction process is A＞B＞C, the having the greatest impact of wherein alcohol extraction concentration; Table 5 The results of analysis of variance shows, is evaluation index with the gastrodine in the extraction process, and concentration of alcohol and ethanol are doubly measured all has the significance meaning (P＜0.05); With the yield of extract is evaluation index, and an ethanol times measurer has significance meaning (P＜0.05); Each evaluation index is carried out overall merit, and concentration of alcohol has significance meaning (P＜0.05); Optimum process condition is A ₃B ₁C ₁

Demonstration test

Take by weighing 2/50 recipe quantity medical material, by preferred plan A ₃B ₁C ₁Carry out demonstration test, result and orthogonal experiments are coincide.This process stabilizing is described, feasible, the results are shown in Table 6.Therefore, determine that alcohol extraction process is 14 times of amount 85% alcohol reflux 2 times 85% ethanol of 16 times of amounts (for the first time with), 2h at every turn.

Table 6 alcohol extraction process checking result

Sequence number	Icariin (mg)	Corydaline (mg)	Gastrodine (mg)	Yield of extract (%)
					1	23.79	4.204	22.43	22.41
2	24.17	4.116	22.38	21.69
					3	24.08	4.243	22.60	22.27
On average	24.01	4.188	22.47	22.12

2.2. decocting boils the process conditions screening

2.2.1 the investigation of medical material water absorption rate

Take by weighing the medical material of 3 part of 1/10 recipe quantity: Radix Angelicae Sinensis 9g, Radix Polygoni Multiflori Preparata 12g, Rhizoma Polygonati 12g, Rhizoma Acori Graminei 12g add 10 times of water gagings in container, be dipped to the heart, with unnecessary water elimination, weigh, and calculate water absorption rate by medical material water absorption rate formula above.The average water absorption rate of decocting in water medical material is 171.9% in the decocting process as a result, the results are shown in Table 7, adds the water of 2 times of amounts when promptly being equivalent to extract for the first time in the decocting process.

The moisture content test of table 7 alcohol extraction process

The sample sequence number	Medical material dry weight (g)	Medicinal residues weight in wet base (g)	Water absorption rate (100%)	Average water absorption rate (100%)
					1	45.2	127.5	182.1	171.9
2	45.3	121.6	168.4
					3	44.8	118.8	165.2

2.2.2 the preferred decocting process of orthogonal test

According to documents and materials and trial test result, decoction number of times, amount of water, decocting time are the principal elements that influences decocting process, by each factor 3 level, use L ₉(3 ⁴) orthogonal table arrangement test (table 8).With total sugar, stilbene glucoside, yield of extract is evaluation index, and decocting process is inquired into.

2.2.2.1 material and instrument

Instrument: the HP1100 high performance liquid chromatograph (comprises the HP1100 pump, the HP1100 UV-detector, the HP1100 column oven, the HP1100/WND-3D work station), Rotary Evaporators (Tokyo physics and chemistry), uv-spectrophotometric instrument (Tianjin, island UV-2401), HHS electric-heated thermostatic water bath (Shanghai medical apparatus and instruments three factories).

Material: D-anhydrous glucose, stilbene glucoside reference substance (are provided by Nat'l Pharmaceutical ﹠ Biological Products Control Institute.Lot number: 110833-200503,0844-200503), other reagent are analytical pure

2.2.2.2 method and result

Table 8 factor level table

A. the mensuration of yield of extract

Press L ₉(3 ⁴) orthogonal table arrangement test, take by weighing 1/25 recipe quantity medical material: Radix Angelicae Sinensis 3.6g, Radix Polygoni Multiflori 4.8g, Rhizoma Polygonati 4.8g, Rhizoma Acori Graminei 4.8g, decoct by the setting scheme, filter, measure filtrate cumulative volume V _AlwaysWith above " pure extractum yield determination method " processing, calculate yield of extract, the results are shown in Table 9.

B. total sugar determination (sulphuric acid phynol method)

Sample treatment takes by weighing 1/25 recipe quantity medical material, presses L ₉(3 ⁴) orthogonal table arrangement test, get decoction liquor, filter, filtrate is measured liquor capacity, is sample liquid, with reference to " Chinese pharmacopoeia (version was an one 58 pages in 2005) fragrant solomonseal rhizome polyoses algoscopy is measured.

The preparation of glucose reference substance solution: precision takes by weighing 0.0305g anhydrous grape saccharide, places the 50mL volumetric flask, and thin up is to scale, shakes up promptly to get (every 1mL contains the 0.61mg glucose).

Standard curve preparation: the accurate respectively standard control storing solution 0mL that draws, 0.5mL, 1.0mL, 1.5mL, 2.0mL, 2.5mL, 3.0mL in the 50mL volumetric flask, adding distil water is diluted to scale, shake up, precision is measured the reference substance solution 2mL of each concentration in tool plug test tube respectively again, the phenol liquid 1mL of accurate adding 4%, add concentrated sulphuric acid 7mL rapidly, shake up, put in 40 ℃ of water-baths and be incubated 30min, take out, put and cool off 5min in the ice bath, taking out, is that the 485nm place measures the trap value at wavelength, gets regression equation to be: Y=0.04074 X+0.0041, r=0.9995, this product is linear in scope.

Sample liquid is measured: the accurate sample liquid 0.1mL that draws, place the 50mL volumetric flask, thin up is to scale, shake up, accurate respectively each sample liquid 2mL of absorption is in tool plug test tube, and method is measured absorbance from " the phenol liquid 1mL of accurate adding 4% " in accordance with the law under the sighting target directrix curve item, retinue is blank, calculates the total sugar amount.The results are shown in Table 9.

C. the assay of stilbene glucoside

Press L ₉(3 ⁴) orthogonal table arrangement test, take by weighing 1/25 recipe quantity medical material, extract by the setting scheme, filter, filtrate merges, and is settled to certain volume; (" Chinese pharmacopoeia (33 pages of VID of appendix of version in 2005) is measured the content of stilbene glucoside, calculates the extracted amount of stilbene glucoside, the results are shown in Table 9 according to high performance liquid chromatography.

Chromatographic condition: Diamonsil ^TMC ₁₈Post (5 μ m, 250 * 4.6mm), column temperature: 30 ℃; Detect wavelength: 320nm, mobile phase: second eyeball: water (25: 75), flow velocity 1mL/min.

The preparation of reference substance solution: the accurate title, decided stilbene glucoside reference substance 0.0106g, places the 50mL volumetric flask, the Diluted Alcohol dissolving, and standardize solution keeps in Dark Place, and reference substance liquid concentration is 212.0 μ gmL ^-1

The preparation of sample solution: extract by the orthogonal experiment design, filter, filtrate merges, and is settled to certain volume, promptly gets each sample solution.

Algoscopy: get each sample solution and cross 0.45 μ m microporous filter membrane, filtrate is test sample liquid.Accurate respectively need testing solution and each 10 μ L of reference substance solution of drawing inject chromatograph of liquid, measure, promptly.

Table 9 decocting process Orthogonal Experiment and Design table and result

Annotate: decocting process adopts 3 evaluation indexes, adopts comprehensive grading to carry out data analysis, and the total sugar weight coefficient is 0.4, and the stilbene glucoside weight coefficient is 0.4, and the yield of extract weight coefficient is 0.2.Comprehensive grading=(total sugar/total sugar maximum * 0.4+ stilbene glucoside/stilbene glucoside maximum * 0.4+ yield of extract/yield of extract maximum * 0.4) * 100

Variance analysis

Orthogonal experiments is carried out variance analysis, the results are shown in Table 10.

Table 10 decocting process evaluation index analysis of variance table

*P＜0.05，F _0.05(2，2)＝19.00

From the analysis of table 9 visual result, be evaluation index with total sugar in the decocting process, the order of each factor affecting is A＞B＞C; With stilbene glucoside in the decocting process is evaluation index, and the order of each factor affecting is A＞B＞C; With yield of extract in the decocting process is evaluation index, and the order of each factor affecting is A＞C＞B; The result of each evaluation index comprehensive grading shows that the order of each factor affecting is A＞B＞C, wherein decocts having the greatest impact of number of times; The The results of analysis of variance of table 10 shows, is evaluation index and each evaluation index comprehensive grading with total sugar, stilbene glucoside, yield of extract in the decocting process respectively, and decocting time number average has significance meaning (P＜0.05), and optimum process condition is A ₃B ₃C ₂

Demonstration test

Take by weighing 2/50 recipe quantity medical material: Radix Angelicae Sinensis 3.6g, Radix Polygoni Multiflori 4.8g, Rhizoma Polygonati 4.8g, Rhizoma Acori Graminei 4.8g press optimised process A ₃B ₃C ₂Carry out demonstration test, result and orthogonal experiments are coincide.This process stabilizing is described, feasible, the results are shown in Table 11.Therefore, determine that extraction process by water is that 14 times of water gagings decoct (being 16 times of water gagings for the first time), each 2 hours 3 times.

Table 11 extraction process by water checking result

Sequence number	Total sugar (g)	Stilbene glucoside (mg)	Yield of extract (%)
				1	8.521	27.248	34.87
2	8.497	27.314	34.29
				3	8.490	27.348	35.11
On average	8.502	27.303	34.76

2.2.3 the selection of decocting liquid impurity removal process

Often contain starch, polysaccharide, protein, pectin etc. in the Chinese traditional medicine water extract, make the solid preparation taking dose that makes excessive, generally tackling extracting solution carries out purification, reduces taking dose, adopts certain density alcohol to carry out the precipitate with ethanol remove impurity more.Therefore, we have carried out purifying process research to decocting liquid, investigated respectively Different concentrations of alcohol carry out precipitate with ethanol, to decocting liquid carry out centrifugal and leave standstill after get impurity-removing methods such as supernatant, by measuring the rate of transform of extractum, stilbene glucoside and total sugar, thereby definite impurity removal process that is adopted the results are shown in Table 12.

Table 12 impurity removal process result

As can be known from Table 12, behind the Different concentrations of alcohol precipitate with ethanol, the rate of transform of total sugar and stilbene glucoside was also reducing when the extractum rate of transform reduced, and the variation of the rate of transform of total sugar is remarkable than stilbene glucoside.When concentration of alcohol was higher than 40%, the rate of transform of its total sugar was lower than 50%, and yield of extract, stilbene glucoside and the total sugar rate of transform have no significant change after centrifuging and settled process processing.And total sugar is one of functional component in this product, and consider in conjunction with the dosage of people's oral administration every day, make finished product as after remove impurity, still adding certain adjuvant, and the starch that will remove in the remove impurity, protein, pectin etc. all can be made natural adjuvant, can reduce the addition of adjuvant.Therefore, on the basis that guarantees drug effect,, do not carry out impurity removal process after decocting liquid is handled after filtration and handle from the angle of economy and the saving of energy input.

2.3 concentrate, drying process preferred

Chinese medicine extraction liquid concentrate, in the dry run because heated time is longer, its contained some effective ingredient may be destroyed to some extent and content is reduced.Concentrating under reduced pressure has the efficient height, the advantage of weak point consuming time, and it is short that same vacuum drying has drying time, and so the loose advantage such as frangible of gained dried product is this process choice concentrating under reduced pressure vacuum drying.The rate of transform with gastrodine, icariin, total sugar is an evaluation index, and the drying process under the condition of different temperatures is investigated.The rate of transform of gastrodine, icariin, total sugar increasing and reduce as a result with temperature, the difference of 60 ℃ and 70 ℃ is less, but 60 ℃ of exsiccant efficient are low than 70 ℃, from production efficiency, determine that 70 ℃ in vacuum carries out drying (vacuum should be controlled at-0.08MPa following)

See Table 13.

Table 13 concentrates, the dry result of investigation

Two, acute toxicity testing

1, handled by the reagent thing

Be configured to the suspension of respective concentration before the test with 5 ‰ sodium carboxymethyl cellulose (CMC)

2, animal subject

Kunming mouse, body weight: 20 ± 2g, male and female half and half are provided by the Guiyang Medical College Experimental Animal Center, the quality certification number: SCXK (Guizhou Province) 2002-0001, the cleaning level, normal diet is raised.

3, experimental technique

Preliminary experiment shows that this preparation toxicity is lower, can not survey LD ₅₀So, carry out maximum tolerance determination.Get 40 of mices (male and female half and half), be divided into 2 groups at random, 20 every group.Fasting (can't help water) 12h.The blank group gives 5 ‰ CMC by 0.4ml/10g and irritates stomach; The preparation group is pressed the 0.4ml/10g gastric infusion, behind the 2h repeat administration once, dosage is 15.2g/kg.1h recovers for food, supplies water after the last administration, observes in the 4h, animal is reported situations and death condition in 14 days, and experiment finishes to put to death animal, dissects animal and observes main organs and change.

4, experimental result

Mice activity in 2h has minimizing slightly after the administration, the movable recovery normally about about 4h.Observing in 14 days, animal appearance, behavior, feed and defecation are normal, weight increase (table 14), and the observation period does not have animal dead.Experiment finishes to put to death animal, dissects the no abnormal change of perusal main organs.The mice maximum tolerated dose is 15.2g/kgd.

The influence of table 14 pair chmice acute toxicity test body weight change (x ± s, n=20)

Group	Before the administration	7 days	14 days	Death toll
					Matched group	20.2±2.6	27.2±2.3	31.5±2.4	0
The preparation group	20.0±1.6	27.4±2.2	31.6±3.4	0

5, conclusion

This experimental studies results shows that maximum tolerated dose is 15.2g/kgd, does not see in the observation period that toxic reaction and death appear in animal, shows this medicine oral medication safety.

Three, pharmacodynamic experiment part:

(1), capsule brain-strengthening experiment of the present invention and and aging relevant enzyme and aging metabolite mensuration

1, experiment material

1.1 laboratory animal

The SD rat: male and female all have, and at the 2-3 monthly age, body weight 200-220g is provided by the Guiyang Medical College Experimental Animal Center, the quality certification number: SCXK (Guizhou Province) 2002-0001, and the cleaning level, normal diet is raised.

1.2 main medicine and reagent

Capsule of the present invention: chocolate brown powder, lot number: 040908.Piracetam: piracetam Piracetam, Tianjin Chasesun Pharmaceutical Co., Ltd, specification: 0.4g/ sheet, lot number: 050102.AlCl ₃.6H ₂O: analytical pure, chemical plant, Gansu Province, west, Shantou, Guangdong, lot number: 040308.ANTI-ChAT: Wuhan BOSTER company, lot number: 050125.ANTI-β-Amyloid (1-40): Wuhan BOSTER company, lot number: 050125.TUNEL method apoptosis test regent box: Wuhan BOSTER company, lot number: 050204.ANTI-Glutamate: Wuhan BOSTER company, lot number: 050125.Instant SABC test kit: Wuhan BOSTER company, lot number: 050308.DAB colour reagent box: Wuhan BOSTER company, lot number: 050401.TBS buffer reagent: Wuhan BOSTER company, lot number: 041116.Antibody diluent: Wuhan BOSTER company, lot number: 040117.The AchE test kit: the biological engineering company limited is built up in Nanjing, lot number 20050513.The SOD test kit: the biological engineering company limited is built up in Nanjing, lot number 20050520.The MDA test kit: the biological engineering company limited is built up in Nanjing, lot number 20050430.Glacial acetic acid: analytical pure, Shanghai chemical reagent company limited, lot number: 041010.

1.3 dosage setting

Capsule of the present invention and positive control drug piracetam dosage all calculate by the Reduced Coefficient Method of laboratory animal and people's clinical medicine dose:

Capsule dosage: chmice acute toxicity test maximum tolerated dose is 15.2g/kg, then the rat equivalent is 10.8g/kg, with 1/10,1/25 of rat equivalent, the 1/50 high, medium and low dosage as rat is respectively: 1.08g/kg, 0.43g/kg, 0.22g/kg.

Piracetam dosage: clinical adult's consumption: each 2-4 sheet (by 3), every day 3 times.Become body weight for humans to calculate with 60kg, the every day consumption of then being grown up is 0.06g/kg, and then rat dosage every day is 0.40g/kg.

2. experimental technique

2.1 modeling

Qualified rat be will screen with the Morris water maze, normal control group (n=10) and modeling group (some) will be divided at random.The normal control group irritates stomach, drinking public water supply for isometric normal saline.The modeling group is given AlCl ₃Solution, concentration are 50g.L ^-1, press 500mg.kg ^-1.d ^-1, every day, non hor am was irritated stomach, continued 2 months.It is 1.6g.L that the modeling group is freely drunk concentration from beginning in three month ^-1AlCl ₃Solution continues 3 months.Regularly carry out water maze laboratory and measure the situation of change of learning and memory in rats.

2.2 grouping and treatment

Through the Morris determined with Morris water, modeling group learning and memory in rats level obviously descends in the time of 5 months in modeling, and there were significant differences with the normal control group.After the modeling success, further modeling group rat is divided at random Model of Dementia group (5 ‰ CMC 0.6mL.kg ^-1.d ^-1), the low (0.22g.kg of capsule of the present invention ^-1.d ^-1), in (0.43g.kg ^-1.d ^-1), high (1.08g.kg ^-1.d ^-1) dosage group and positive controls (piracetam 0.4g.kg ^-1.d ^-1).Normal control group (5 ‰ CMC 0.6mL.kg ^-1.d ^-1), each organizes gastric infusion, once a day, and continuous 3 months.

2.3 detect index and method

2.3.1 ability of learning and memory detects

Index: escape latency, detection range, space exploration time and exploration distance.

Method: before modeling, in the modeling process the 50th day, 100 days and 150 days, and treatment carries out behavioristics when finishing and detect, observe before and after the rat modeling and the learning and memory situation of change of TSYZ after treating.

2.3.2 the cerebral tissue index detects

2.3.2.1 preparation of specimen: after treatment finishes, with 10% chloral hydrate 300mg.kg ^-1Behind the intraperitoneal injection of anesthesia rat, broken end takes out brain rapidly, places on the ice pan, accurately weighs.To lose the shape face is that the boundary is divided into two halves with brain, and wherein half cerebral tissue adds the cold normal saline of 10mL by every gram tissue and makes 10% tissue homogenate with homogenizer, in-80 ℃ of cryogenic refrigerators preservations, is used to detect AChE and SOD activity and MDA content.Second half cerebral tissue, get the crown position brain sheet (comprising Hippocampus) behind the 5mm behind the optic chiasma, spend the night with 4 ℃ of preservations of 4% neutral formalin solution, next day paraffin embedding, do crown section continuously, the thick 3 μ m of sheet, every routine specimen is got continuous 5 sections and is made HE (Hematoxylin-eosin staining) dyeing and immunohistochemical staining respectively, is used for pathological examination and analyzing rat hippocampal neurons A β _1-40, ChAT, Glu expression, the TUNEL method detects rat hippocampus neuronal apoptosis level.

2.3.2.2 the cerebral tissue biochemical indicator detects: detect cerebral tissue AChE and SOD activity and MDA content, all by the test kit description operation, photochemical method is measured.

2.3.2.3 cerebral hippocampus histopathology: hippocampal tissue pathological section HE dyeing, carry out morphological observation.

2.3.2.4 cerebral hippocampus histogenic immunity group index detects

A β _1-40Expression, ChAT activity, Glu content detection: (1) section dewaxing entry; (2) 3%H ₂O ₂Soak 15min, 3 times * 3min of distillation washing; (3) high pressure boils 3min (pH6.0,0.01M citrate buffer), is cooled to room temperature; (4) 3 times * 3min of 0.01MPBS (pH7.2-7.4) flushing adds 5%BSA, room temperature 15min sealing; (5) anti-50 μ l (the A β after the adding dilution _1-401: 50, ChAT 1: 50, GLU 1: 35), in 4 ℃ of refrigerator overnight; (6) 3 times * 3min of 0.01M PBS (pH7.27.4) flushing adds instant two and resists, and hatches 20min in 37 ℃ of wet boxes; (7) 3 times * 3min of 0.01M PBS (pH7.2-7.4) flushing adds 1: 100S _ABCComplex is hatched 20min in 37 ℃ of wet boxes; (8) 3 times * 3min of 0.01M PBS (pH7.2-7.4) flushing, the DAB 10min that develops the color; (9) haematoxylin is redyed; (10) 1% hydrochloride alcohols differentiation 3min, washing; (11) 0.5% ammonia return indigo plant, washing; (12) gradient alcohol dehydration; (13) dimethylbenzene is transparent; (14) neutral gum mounting, mirror is observed down and is analyzed.

Apoptosis detects (TUNEL method): (1) section dewaxing entry; (2) 3%H ₂O ₂Soak 15min, 3 times * 3min of distillation washing; (3) 37 ℃ of trypsinization 10min; (4) 3 times * 3min of 0.01M PBS (pH7.2-7.4) flushing adds labelling buffer 20 μ l, discards more than the labelling buffer, adds mixed liquor 20 μ l (TdT 1 μ l+d-UTP 1 μ l+ labelling buffer 18 μ l), 37 ℃ of reaction 90min in wet box; (5) 3 times * 3min of 0.01M TBS (pH7.2-7.4) flushing adds 5%BSA, room temperature 10min sealing; (6) get rid of section and go up unnecessary liquid, add 1: 100 biotin DigiTAb; (7) 3 times * 3min of 0.01M TBS (pH 7.2-7.4) flushing adds 1: 100S _ABCComplex is hatched 20min in 37 ℃ of wet boxes; (8) 3 times * 3min of 0.01M TBS (pH7.2-7.4) flushing, the DAB 10min that develops the color; (9) haematoxylin is redyed; (10) 1% hydrochloride alcohols differentiation 3min, washing; (11) 0.5% ammonia return indigo plant, washing; (12) gradient alcohol dehydration; (13) dimethylbenzene is transparent; (14) neutral gum mounting, mirror is observed down and is analyzed.

The pathological section graphical analysis: under 10 * 40 optical microscopes, by the SONY photographic head with animal tissue's SABC image acquisition and import the BIOMIAS-99 image analysis system and carry out graphical analysis.

Average gray value (MG) is measured: 5 400 times of visuals field of every section (every routine zoological specimens) picked at random, and 5 positive expression zones are chosen in each visual field, measure MG, meansigma methods with the MG in 5 visuals field is represented its positive expression rate, section statining is shallow more, and MG numerical value is big more, and positive expression rate is low more.

2.3.3 the mensuration of cerebral index (cerebral coefficient)

2.3.4 date processing:

Date processing adopts mean ± standard deviation, and (x ± s) expression, mean relatively adopts the one factor analysis of variance check between group.P＜0.05 is for there being significant difference.

3, experimental result

3.1. normal rat Morris water maze school grade

Carry out the constant-bearing navigation training and write down its school grade screening 60 qualified rats before the modeling, the school grade of rat obviously improves, and through checking its achievement and the 5th day no significant difference, stops training during by the 6th day.

3.2.AlCl ₃Induce the learning and memory level and the hippocampal tissue pathological change of AD rat model

Rat was dyed aluminum after 5 months, escape latency and the detection range of model group in the constant-bearing navigation experiment obviously prolongs, search time and exploration distance at former safety island place quadrant in the space exploration experiment obviously reduce, and with the normal control group significant difference (P＜0.01) are arranged relatively.The modeling success space learning dysmnesia appear, in prompting modeling group rat.(seeing Table 15,16)

Table 15 AlCl ₃Induce achievement (escape latency, detection range, the x ± s) of the constant-bearing navigation of AD rat model

^*P＜0.05， ^**P＜0.01 vs Control

Table 16 AlCl ₃(the space exploration time is explored distance, x ± s) to induce the achievement of AD rat model space exploration

^*P＜0.05， ^**P＜0.01 vs Control

3.3 to AlCl ₃Induce the therapeutical effect of AD rat model

3.3.1 to AlCl ₃Induce the influence of AD rat model learning and memory

Compare with model group, the capsule group was irritated stomach after three months, obviously shortened escape latency and the detection range of rat in the constant-bearing navigation experiment; Rat in exploration time of former safety island quadrant and explore distance and obviously prolong, shows that capsule is to dementia rats ability of learning and memory improve significantly (seeing Table 17,18) in space exploration experiment

Table 17 couple AlCl ₃Induce AD rat model constant-bearing navigation achievement influence (x ± s, n=10)

Group	Dosage (g.kg -1)	Incubation period (second)	Detection range (centimetre)
				Matched group		14.23±2.87	432.86±224.50
Model group		56.38±42.04	1755.9±784.6
				Low dosage	0.22	25.83±15.79	595.73±521.12 #
Middle dosage	0.43	10.39±1.89 #	431.61±362.06 #
				High dose	1.08	9.04±6.14 #	195.45±46.92 #
Piracetam	0.40	13.38±8.01 #	270.13±296.89 #

^*P＜0.05， ^**P＜0.01 vs Control； ^#P＜0.05， ^##P＜0.01 vs Model

Table 18 couple AlCL ₃Induce AD rat model space exploration achievement influence (x ± s, n=10)

Group	Dosage (g.kg -1)	The exploration time (second)	Detection range (centimetre)
				Matched group		43.55±5.43	1327.90±98.66
Model group		36.77±2.51	1172.77±138.64
				Low dosage	0.22	36.96±2.49	1191.79±61.92
Middle dosage	0.43	40.02±3.60	1293.71±65.54
				High dose	1.08	44.31±1.44 #	1348.31±74.56 #
Piracetam	0.40	42.78±2.74 #	1343.40±58.66 #

^*P＜0.05， ^**P＜0.01 vs Control； ^#P＜0.05， ^##P＜0.01 vs Model

3.3.2 to AlCl ₃Induce the influence of AD rat model brain AChE, SOD, MDA

Compare with normal group, the AChE of model group rat cerebral tissue activity obviously increases, and SOD is active to be reduced, and MDA content increases (P＜0.05, P＜0.01).Compare with model group, the AChE activity has decline in various degree in the capsule group rat brain, and SOD is active to raise, and MDA content reduces (P＜0.05, P＜0.01).TSYZ is to AlCl in prompting ₃Cause that the active rising of AChE has the obvious suppression effect and tangible antioxidation (sees Table 19.

Table 19 couple AlCl ₃Induce the influence of AD rat model cerebral tissue SOD, MDA

Group	Dosage (g.kg -1)	SOD(U/mgprot)	MDA(nmol/mgprot)
				Matched group		280.82±41.44	13.92±4.70
Model group		200.30±62.56 *	21.92±4.77 *
				Low dosage	0.22	275.70±64.74 #	15.90±5.77
Middle dosage	0.43	275.15±49.49 #	13.99±3.90 #
				High dose	1.08	289.98±53.35 #	12.78±3.79 ##
Piracetam	0.40	283.61±43.64 #	9.49±4.37 ##

^*P＜0.05， ^**P＜0.01 vs Control； ^#P＜0.05， ^##P＜0.01 vs Model

3.3.3 to AlCl ₃Induce the pathological influence of AD rat model hippocampal tissue

Observe rat hippocampus and organize the HE stained under light microscopic, the result shows:

Normal control group neurocyte size, form and arrangement are normal, and cell space is circle or polygon; Cellularity is complete, and after birth, karyon profile are clear; Neurocyte does not have swelling and necrosis.

Model group neurocyte size, form differ arrangement disorder; Obviously the neurocyte vacuolar degeneration changes: cytoplasm dyeing shoals the circle that differs in size in it or similar round cavity; The performance of tangible neuronal necrosis is arranged: nucleome is long-pending to be increased, and nuclear structure is fuzzy, pyknosis, cracked or disappear.

Basic, normal, high dosage group and piracetam group neurocyte size, form and arrangement recover normal, and cellularity is complete, and after birth, karyon clear-cut can be distinguished; Cell does not have obvious swelling; A small amount of neurocyte vacuolar degeneration; There is not the downright bad feature of obvious neurocyte.

3.3.4 to AlCl ₃Induce the influence of AD rat model Hippocampus ChAT

Measurement result shows: the dyeing of model group hippocampus of rats endochylema is shallow, and a large amount of neuronal structure are destroyed and disappeared, and the MG value obviously increases (P＜0.01) than normal group.The dyeing of capsule group hippocampus of rats endochylema is darker yellowish-brown, and neuronal structure is clear, and the MG value obviously reduces (P＜0.01), and the prompting capsule can effectively activate AD rat hippocampus ChAT activity, to AlCl ₃Cause that active reduction of ChAT has the obvious suppression effect, thereby promote ACh synthetic (seeing Table 20).

Table 20TSYZ is to AlCl ₃Induce the influence of AD rat model Hippocampus ChAT

Group	Dosage (g.kg -1)	MG (hippocampus)
			Matched group		94.48±2.38
Model group		190.78±1.02 **
			Low dosage	0.22	138.901.04 ##
Middle dosage	0.43	113.87±1.49 ##
			High dose	1.08	90.27±1.87 ##
Piracetam	0.40	109.13±2.02 ##

^*P＜0.05， ^**P＜0.01vs Control； ^#P＜0.05， ^##P＜0.01 vs Model

3.5 to AlCl ₃The influence of inducing AD rat model Hippocampus A β 1-40 to express

Show through the graphical analysis measurement result, visible significantly after birth and cytoplasmic staining during model control group is cut into slices, the nucleus structural fuzzy, size, form differ, and neuron disappears, and compares with normal group, and MG obviously reduces (P＜0.01), A β in the hints model group brain _1-40Content increases.Compare with model group, capsule group Hippocampus after birth and endochylema dyeing are more shallow, and the neuron clear-cut is arranged and recovered normal, and MG all obviously increases (P＜0.01), point out capsule group A β _1-40Content reduces, to AlCl ₃A β in the rat brain that causes _1-40Increase and have the obvious suppression effect (seeing Table 21).

Table 21 couple AlCl ₃Induce AD rat model Hippocampus A β _1-10Influence (x ± s, n=10)

Group	Dosage (g.kg -1)	MG(hippocampus)
			Matched group		192.54±1.42
Model group		86.49±1.45 **
			Low dosage	0.22	169.59±1.49 ##
Middle dosage	0.43	179.74±1.45 ##
			High dose	1.08	188.60±1.36 ##
Piracetam	0.40	170.38±1.62 ##

^*P＜0.05， ^**P＜0.01 vs Control； ^#P＜0.05， ^##P＜0.01 vs Model

3.6 to AlCl ₃The influence of inducing AD rat model Hippocampus Glu to express

The result shows that the dyeing of model group hippocampus of rats endochylema is darker, and a large amount of neurons disappear, and the MG value obviously reduces (P＜0.01) than normal group, and hints model group rat hippocampus irritability Glu content increases.Compare with model group, the dyeing of capsule group hippocampal neuron endochylema is more shallow, and neuronal structure is clear, arranges and recovers normal, and the MG value all obviously increases (P＜0.01), points out capsule can reduce irritability Glu content in the AD rat hippocampus, to AlCl ₃Glu increases and has the obvious suppression effect (seeing Table 22) in the rat brain that causes.

Table 22TSYZ is to AlCl ₃Induce the influence that AD rat model Hippocampus Glu expresses (x ± s, n=10)

Group	Dosage (g.kg -1)	MG(hippocampus)
			Matched group		191.44±4.14
Model group		91.04±1.59 **
			Low dosage	0.22	160.98±1.46 ##
Middle dosage	0.43	171.74±1.92 ##
			High dose	1.08	171.86±1.41 ##
Piracetam	0.40	177.753.00 ##

^*P＜0.05， ^**P＜0.01 vs Control； ^#P＜0.05， ^##P＜0.01 vs Model

3.7 to AlCl ₃Induce the influence of AD rat model hippocampal neurons apoptosis

The TUNEL method detects the neurocyte result and shows: compare with normal group, the brown yellow granule that is engrain in the model control group section in the neurocyte nuclear, i.e. apoptotic body, neuron disappears, the MG value obviously reduces (P＜0.01), and hints model group neuronal apoptosis increases.Compare with model group, the dyeing of capsule group neurocyte is more shallow, and size, form and arrangement recover normally, and the MG value all obviously increases (P＜0.01), and prompting preparation group neuronal apoptosis reduces, to AlCl ₃Neuronal apoptosis has the obvious suppression effect in the rat brain that causes, sees Table 23.

Table 23TSYZ is to AlCl ₃Induce AD rat model hippocampal neurons apoptosis influence (x ± s, n=10)

Group	Dosage (g.kg -1)	MG(hippocampus)
			Matched group		195.64±1.81
Model group		97.73±2.11 **
			Low dosage	0.22	168.43±1.75 ##
Middle dosage	0.43	169.37±1.52 ##
			High dose	1.08	199.19±45.02 ##
Piracetam	0.40	160.66±3.36 ##

^*P＜0.05， ^**P＜0.01 vs Control； ^#P＜0.05， ^##P＜0.01 vs Model

3.8 to AlCl ₃Induce the influence of AD rat model cerebral index

The result shows that model group rat brain index significantly reduces than normal group, the obvious atrophy of hints model group rat brain, and brain heavily alleviates; Compare with model group, TSYZ can significantly increase cerebral index (P＜0.05), and brain atrophy is had antagonism, thereby cerebral function improvement delays its decline (seeing Table 24).

Table 24 couple AlCl ₃Induce AD rat model cerebral index influence (x ± s, n=10)

Group	Dosage (g.kg -1)	Cerebral index (g/kg)
			Matched group		0.350.063
Model group		0.280.039 *
			Low dosage	0.22	0.310.060
Middle dosage	0.43	0.340.064 #
			High dose	1.08	0.350.070 #
Piracetam	0.40	0.400.131 #

^*P＜0.05， ^**P＜0.01 vs Control； ^#P＜0.05， ^##P＜0.01 vs Model

5, conclusion

Capsule is to AlCl ₃Inductive AD rat model ability of learning and memory has a better role, and its possible mechanism of action for the treatment of AD is: 1. improve the ChAT activity, reduce the AChE activity, thereby improve Ach content, protection and enhancing central cholinergic system function.2. the expression of irritability Glu in the inhibition brain, the neurotoxic effect of antagonism Glu mediation.3. the deposition of A β in the inhibition brain is intervened A β neurotoxicity.4. suppress brain nervous cell apoptosis, neuroprotective cell function.5. removing free radical, anti-oxidation stress.

(2) capsule anti-aging effects Mechanism Study of the present invention

1. experiment material:

1.1. laboratory animal

Kunming mouse: male and female half and half, body weight 22 ± 2g is provided by the Guiyang Medical College Experimental Animal Center, the quality certification number: SCXK (Guizhou Province) 2004-0001.Normal diet is raised, cleaning level, light application time 12/12,2223 ℃ of room temperatures.

1.2 main medicine and reagent

Capsule of the present invention: chocolate brown powder.D-galactose: U.S. Amresco company.Brain-invigorating capsule (JNJN): Qingdao Guofeng Pharmaceutical Co., Ltd..

2. dosage setting

Capsule chmice acute toxicity test maximum tolerated dose is: 0.076g10g ^-1, i.e. 7.6gkg ^-1, get maximum tolerated dose 1/3,1/6,1/12 respectively as high, medium and low dosage (2.5gkg ^-1, 1.25gkg ^-1, 0.63gkg ^-1).

3. statistical method: experimental data is represented with x ± s, relatively adopts the t check between group.

4. experimental technique:

Kunming mouse (female, hero half and half) carries out the Morris water maze laboratory, till constant-bearing navigation experiment achievement is stable (achievement difference did not have significance in continuous two days).To be divided into 6 groups (n=14) at random, normal control group (NS 14mLkg through 84 qualified mices of Morris water maze screening ^-1), model group (NS 14mLkg ^-1), the high, medium and low (2.50gkg of compound recipe ^-1, 1.25gkg ^-1, 0.63gkg ^-1) (brain-invigorating capsule is write a Chinese character in simplified form JNJN, 0.03gkg for dosage group and positive controls ^-1).Except that the normal control group, all the other each groups all adopt subcutaneous injection 5%D-galactose solution 0.5mLkg ^-1Modeling, simultaneously continuous gastric infusion 45 days.Adopt the Morris water maze laboratory, detect ability of learning and memory, the mouse orbit blood sampling, separation of serum is put and is preserved standbyly in-80 ℃ of ultra cold storage freezers, measures mice serum GSH-PX, SOD, AChE, ChAT activity and MDA content.

5. experimental result:

5.1 influence to D-galactose aging model learning and memory of little mouse

There was no significant difference between each administration group and model group, positive controls (P＞0.05).Achievement and the demonstration of space exploration experiment achievement are tested in the rat constant-bearing navigation behind the gastric infusion, and the study memory level improves behind the rat oral gavage, and the shortening of exploration time, detection range reduce, and with model group significant difference (P＜0.05 or 0.01) are arranged relatively.(seeing Table 25)

Mice space exploration experiment achievement behind the table 25 dementia filling stomach (x ± s)

Group	n	Dosage (g/kg)	The exploration time (s)	Explore space/gross space (%)
					The blank group	11	-	17.73±10.42	647.63±476.85
Model group	13	-	57.02±36.31 *	1403.18±1047.37 *
					High dose	11	2.5	24.35±23.39 △	635.21±364.46 △
Middle dosage	14	1.25	25.47±21.68 △	660.97±484.46 △
					Low dosage	11	0.63	12.77±11.33 △△	262.27±206.63 △△
JNJN	11	0.03	46.80±39.16 △	1278.76±1310.17 △△

^*P＜0.05， ^**P＜0.01 vs NS ^△P＜0.05， ^△△P＜0.01 vs Model

5.2 to the active influence of D-galactose aging model mice serum GSH-PX

GSH-PX is active in the model group rat blood serum reduces, and with the normal control group significant difference (P＜0.05) is arranged, and GSH-PX is active in the rat blood serum behind the gastric infusion raises, and with model group significant difference (P＜0.05) is arranged relatively, sees Table 26.

The table 26 pair active influence of D-galactose aging model mice serum GSH-PX (x ± s)

^*P＜0.05， ^**P＜0.01 vs NS ^△P＜0.05， ^△△P＜0.01 vs Model

5.3 the influence of active MDA content in to D-galactose aging model mice serum SOD

Table 27 shows, SOD is active in the model group mouse brain tissue reduces, MDA content raises, with the normal control group significant difference (P＜0.05 or 0.01) is arranged, SOD is active in the mouse brain tissue behind the gastric infusion raises, MDA content reduces, and with model group significant difference (P＜0.05 or 0.01) is arranged relatively.

The influence of SOD activity and MDA content in the table 27 pair D-galactose aging model mice serum (x ± s)

Group	n	Dose(g/kg)	SOD(nmol/mg prot)	MDA(nmol/mg prot)
					The blank group	11	-	108.04±16.41	3.12±0.44
Model group	13	-	93.49±13.40 *	4.67±0.88 **
					High dose	11	2.5	116.00±14.01 △△	3.77±1.04 △
Middle dosage	14	1.25	136.34±24.47 △△	3.61±1.12 △
					Low dosage	11	0.63	126.33±17.88 △△	3.91±0.55 △
JNJN	11	0.03	119.93±14.13 △△	3.05±2.44 △

^*P＜0.05， ^**P＜0.01 vs NS ^△P＜0.05， ^△△P＜0.01 vs Model

5.4 to D-galactose aging model mice serum AChE, the active influence of ChAT

Table 28 shows, ChAT is active in the model group mouse brain tissue reduces, AChE is active to raise, with the normal control group significant difference (P＜0.05 or 0.01) is arranged, ChAT is active in the mouse brain tissue behind the gastric infusion raises, AChE is active to be reduced, and with model group significant difference (P＜0.05 or 0.01) is arranged relatively.

The active influence of AChE, ChAT in the table 28 pair D-galactose aging model mice serum (x ± s)

^*P＜0.05， ^**P＜0.01 vs NS ^△P＜0.05， ^△△P＜0.01 vs Model

6. conclusion: the high, medium and low dosage group of capsule of the present invention learning and memory of little mouse achievement all has in various degree than model group and improves; Serum GSH-PX, SOD and ChAT vigor all raise, and the AChE vigor reduces, and MDA content reduces; Conclusion: the compound capsule agent has the better prevention effect to AD.

(3) capsule the kidney invigorating experimental study of effect of the present invention

1. experiment material:

1.1. laboratory animal

Laboratory animal and experiment condition are with " defying age " experiment

1.2 main medicine and reagent

Capsule of the present invention, chocolate brown powder.Revise the board SHENBAO HEJI, national accurate word: Z36021192, Jiangxi remittance core Pharmaceutical.Dexamethasone injection, lot number 20021016, the full prestige in Luohe South St village pharmaceutical Co. Ltd.

2. dosage setting

With " defying age " experiment

3. statistical method: experimental data is represented with x ± s, relatively adopts the t check between group.

4. experimental technique:

72 mices are divided into 6 groups (n=12) at random, normal control group (NS 14mLkg ^-1), model group (NS 14mLkg ^-1), the high, medium and low (2.50gkg of compound recipe ^-1, 1.25gkg ^-1, 0.63gkg ^-1) dosage group and positive controls (correction board SHENBAO HEJI 0.03gkg ^-1).Except that the normal control group, all the other each groups all adopt the filling stomach to give dexamethasone 0.2mg/10g, set up the mice model of suffering from a deficiency of the kidney in 30 days, gastric infusion 14 days after last is tried thing 30min, is placed on mice on the perspex bar of pole-climbing frame, make its muscle in tension, the record mice is because muscle fatigue stops experiment when from the time that lucite drops down, falling for the 3rd time, and the time that accumulative total is three times is as the pole-climbing time.Continued gastric infusion 1 day, after the last administration 3 minutes, mice is put into the wide mouthed bottle that fills the 15g sodica calx, each 1 every group, with vaseline bottleneck is sealed, timing immediately is the standard mice time-to-live to cease breathing; Experiment finishes, and weighing mice body weight, thymus, spleen, kidney, adrenal gland, uterus, ovary, testis, seminal vesicle weight are also calculated each internal organs coefficient.

5. experimental result:

5.1 influence to mice internal organs coefficient

Mice internal organs coefficient is not had obvious influence, and each administration group and model group be there was no significant difference (P＞0.05) (seeing Table 29) relatively.

The influence of table 29 pair mice internal organs coefficient (mg/10g, x ± s)

Internal organs	Model	Normal control	Low dosage	Middle dosage	High dose	Positive control
							Spleen	0.09±0.02 *	0.41±0.11	0.09±0.040	0.10±0.06	0.09±0.03	0.11±0.04
Kidney	1.14±0.21	1.09±0.18	1.33±0.24	1.35±0.16	1.32±0.12	1.36±0.18
							The adrenal gland	0.01±0.01 *	0.02±0.01	0.02±0.01	0.03±0.01	0.02±0.01	0.02±0.01
Thymus	0.11±0.08 **	0.25±0.10	0.16±0.13	0.10±0.02	0.10±0.05	0.08±0.03
							Testis	0.77±0.19	0.69±0.10	0.61±0.25	0.49±0.17	0.87±0.16	0.72±0.09
Seminal vesicle	0.67±0.30	0.64±0.19	1.10±0.39	0.67±0.10	0.86±0.23	0.54±0.26
							The uterus	0.51±0.38	0.42±0.17	0.31±0.10	0.28±0.18	0.29±0.12	0.23±0.07
Ovary	0.08±0.06	0.13±0.05	0.12±0.09	0.08±0.03	0.12±0.06	0.05±0.01

^*P＜0.05, ^*P＜0.01 vs normal control; ^△P＜0.05, ^{△ △}P＜0.01 vs model group

5.2 influence to the mice pole-climbing time

High dose administration group pole-climbing time lengthening, than the model group significant difference, P＜0.05 (seeing Table 30).

The influence of table 30 pair mice pole-climbing time (x ± s)

Project	Model	Normal control	Low dosage	Middle dosage	High dose	Positive control
							Number of animals (n)	10	11	10	10	11	11
The pole-climbing time (s)	32.72± 22.84	59.36± 29.78 *	48.35± 32.65	67.41± 34.17 △	35.48± 24.51	60.94± 36.49 △

^*P＜0.05, ^*P＜0.01 vs normal control; ^△P＜0.05, ^{△ △}P＜0.01 vs model group

5.3 to the influence of anoxia enduring time-to-live of mice normal pressure

Each administration group normal pressure hypoxia endurance time prolongs, and is very remarkable than model group difference, P＜0.01 (seeing Table 31)

The influence of table 31 pair mice normal pressure anoxia enduring time-to-live (x ± s)

Project	Model	Normal control	Low dosage	Middle dosage	High dose	Positive control
							Number of animals (n)	10	11	10	10	11	11
Hypoxia endurance time (s)	26.30± 3.59	35.82± 12.52 *	34.70± 5.70 △△	34.40±5.08 △△	33.82± 5.02 △△	33.50±8.20 △

^*P＜0.05, ^*P＜0.01 vs normal control; ^△P＜0.05, ^{△ △}P＜0.01 vs model group

6. conclusion: the compound capsule agent can improve the resisting fatigue and the anti-stress ability of body, to the therapeutical effect preferably of having suffered from a deficiency of the kidney.

Four, the molecular mechanism of action research of capsule treatment AD of the present invention

(1), nicotine receptor is expressed and is reduced and the antagonism of cytotoxicity infringement in the neurocyte that the beta amyloid is caused

1 material and main chemical reagent:

Capsule of the present invention.Come from the neuroblastoma cell line (SH-SY5Y cell) (available from GermanCollection of Microorganisms and Cell Cultures company) of human brain.A β _25-35(available from Sigma company).DMEM culture fluid (available from Gibco Introvagen company).Calf serum (available from magnificent company).MTT colorimetry main agents (available from Sigma company).The anti-sheep of goat-anti a3, a7 and mouse-anti b-actin (b-Actin) polyclonal antibody and HRP labelling and anti-Mus two are anti-available from Santa Cruz Biotechnology company.Polyethylene difluoro (PVDF) film (available from Amersham company).ECL-Plus luminescence reagent (available from Amersham company).Efficient developed film (available from Amersham company).Trizol (available from Sigma company).The DNA enzyme of MMLV reverse transcriptase, RNA enzyme inhibitor, no RNA enzyme (available from Premega company).OligodT, primer, TaqDNA polymerase, dNTP etc. (all giving birth to worker company) available from Shanghai.TBA colorimetry test kit (building up company) available from Nanjing.Other chemical reagent (all available from Sigma company).

2 methods

2.1 cell culture:

With containing 10% calf serum, two DMEM that resists (penicillin 25U/ml, streptomycin 25U/ml) as culture medium, 5%CO ₂, saturation vapour, 37 ℃ of constant temperature culture.

2.2MTT colorimetry examination Chinese medicine safe concentration:

In 96 porocyte culture plates, add some (about 1 * 10 ⁴/ hole) cell to be detected treats that the stable back of cell growth adds capsule of the present invention, and final concentration is respectively: 0mg/ml, 0.005mg/ml, 0.025mg/ml, 0.05mg/ml, 0.1mg/ml, 0.25mg/ml, 0.5mg/ml, 1mg/ml, 5mg/ml, 10mg/m, l cultivate and measure the MTT level after 24 hours.

2.3 the cell grouping is induced and corresponding pharmaceutical intervention:

(1) normal control group: without drug treating, culture period is 48 hours.(2) A β processed group: in cultured cells, add 10 μ M A β _25-35, incubation time is 48 hours (A β _25-35After the PBS dissolving, hatched 3～4 hours for 37 ℃).(3) simple VE processed group: add 20 μ M VE, culture period is 48 hours.(4) VE and A β processed group: add 20 μ MVE earlier and cultivate after 24 hours, handle according to the method for Ab processed group.(5) simple Chinese medicine processed group: add Chinese medicine and cultivated 48 hours.(6) Chinese medicine and A β processed group: add the pre-cultivation of Chinese medicine earlier after 24 hours, handle according to Ab processed group method again.

2.4 the MTT percent reduction is measured after the drug treating:

In 96 porocyte culture plates, add some (about 1 * 10 ⁴/ hole) cell to be detected is measured the MTT level behind the grouping drug treating cell.

2.5 expressing, the nicotine receptor subunit protein measures:

Extract epicyte protein and detect nicotine receptor subunit α 3, α 7 subunit protein levels with reference to the method for Guan etc. with Western blot (Western blotting) method.With the Labworks software analysis as a result the time with β-Actin protein band as internal reference, calculate the relative level ratio that α 3, α 7 protein bands and the percent value of β A-ctin protein band pixel grey scale are expressed as α 3, α 7 gene proteins.

2.6 the expression of nicotine receptor subunit mRNA is measured:

The Trizol method is extracted cell total rna, and with the dnase digestion DNA impurity (carrying out according to the reagent description) of no RNA enzyme.Adopt reverse-transcription polymerase chain reaction (RT-PCR) method to measure the mRNA level.With the internal reference of ciclosporin Avidin (Cyc) gene, calculate the relative level of the percent value of α 3, α 7 genes and Cyc gene amplification band expression pixel grey scale during with the Labworks software detection as α 3, α 7 gene mRNA expressions as the RT-PCR product.NAChR receptor subunits a3, a7 and ciclosporin Avidin primer sequence see Table 32.

Table 32.RT-PCR primer sequence

Subunit	Primer sequence	Fragment length
			α3	The upstream: 5 '-TTCAGCCGTGCAGACTCCA-3 ' downstream: 5 '-GGCAACGTACTTCCAATCATC-3 '	271bp
α7	The upstream: 5 '-CGCCACATTCCACACTAAC-3 ' downstream: 5 '-ACCTTTCACTCCTCTTGCC-3 '	257bp
			β2	The upstream: 5 '-GCCTGCGCCTGCGGCGACGCC-3 ' downstream: 5 '-CCTCACTCACGCTCTGGTCAT-3 '	270bp
Cyc	The upstream: 5 '-Agaaaattttcgtgctctgagca-3 ' downstream: 5 '-Aacacaagtcaaactttattcgag-3 '	482bp

2.7 level of lipid is measured:

Collect the culture fluid of cultivating neurocyte, measure malonaldehyde (intermediate product of lipid peroxidation) content, operation is carried out according to the test kit description.

2.8 statistical analysis:

Every group of gained result represented that with mean ± standard deviation the result carries out one factor analysis of variance with the SPSS12.0 statistical software.

3. result

3.1. Chinese medicine safe concentration examination:

Downward trend appears in SH-SY5Y neurocyte MTT percent reduction during capsule concentration of the present invention＞0.5mg/ml, and cell MTT percent reduction obviously reduces during concentration＞1mg/ml, shows the toxic effect of this concentration pair cell.Therefore select the safe concentration of 0.25mg/ml as this research capsule of the present invention.

3.2 the MTT percent reduction is measured after the drug treating:

The MTT percent reduction is measured and is found 10mM A β after the drug treating _25-35Cause that the MTT percent reduction obviously reduces after handling cell; 0.25mg/ml handling, capsule and 20 μ M VE do not influence cell function; And with the toxic action that can obviously weaken the Ab pair cell behind capsule and the VE pretreatment cell, the difference between A β group is organized with VE+A β group and X+A β has statistical significance (P＜0.01); Capsule and VE resist the Cytotoxic action intensity of Ab roughly the same, difference not statistically significant therebetween.

3.3 nicotine receptor protein expression level

Nicotine receptor α 7 and α 3 sub-unit protein expression semi-quantitative results show 10mM A β _25-35Cause that nicotine receptor α 7 and α 3 sub-unit protein expressions obviously reduce after handling cell; Simple 0.25mg/ml capsule is handled cell can raise nicotine receptor α 7 sub-unit protein expressions; And 20 μ M VE processing does not influence this receptor protein level; With the downward modulation effect that can weaken Ab pair cell α 7 and α 3 sub-unit protein expressions behind capsule and the VE pretreatment cell, the difference between A β group and VE+A β group and the X+A β group has statistical significance (P＜0.01 or P＜0.05); The effect that capsule antagonism A β reduces α 7 sub-unit protein expressions is better than VE, and the difference between VE+A β group and the X+A β group has statistical significance (P＜0.05).The downward modulation α 3 sub-unit protein expression effects that capsule and VE antagonism A β causes roughly the same, the difference not statistically significant between VE+A β group is organized with X+A β.

3.4 nicotine receptor mRNA expression

Nicotine receptor α 7 and α 3 subunit mRNA expression semi-quantitative results show 10mM A β _25-35Cause that nicotine receptor α 7 and α 3 subunit mRNA expressions obviously reduce after handling cell; 0.25mg/ml handling, capsule and 20 μ M VE do not influence this receptor mRNA expression; With the downward modulation effect that can weaken Ab pair cell α 7 and α 3 subunit mRNA expressions behind capsule and the VE pretreatment cell, the difference between A β group and VE+A β group and the X+A β group has statistical significance (P＜0.01); The α 7 subunit mRNA expression downward modulation effects that capsule and VE antagonism A β cause are suitable, the difference not statistically significant between VE+A β group and the X+A β group.And do not have this effect with α 3 subunits that capsule antagonism A β causes, and the difference between A β group and the X+A β group does not have statistical significance, and the difference between VE+A β group and the X+A β group has statistical significance (P＜0.05).

3.5 lipid peroxidation metabolite MDA level:

The result shows, 10mM A β _25-35Cause that the cytolipin levels of peroxide obviously raises after handling cell; Do not influence MDA content with 0.25mg/ml capsule and 20 μ M VE processing; Raise and can weaken the caused level of lipid of Ab behind capsule and the VE pretreatment cell, but this kind effect of capsule than VE a little less than (table 33).

Table 33.TBA method mensuration MDA (x ± s)

Group	Matched group	Aβ	VE	VE+Aβ	Capsule	Capsule+A β
							MDA(nmol /ml)	4.19± 0.44	5.825± 0.70**	4.16± 0.46	4.38± 0.43	4.26± 0.29	4.93± 0.46*

4, conclusion

The capsule of safe concentration has the rise effect to α 7 at protein expression level; Can resist the α 3 that A β causes and α 7 nicotine receptor sub-unit protein levels reduce and α 7 subunit mRNA are expressed low but α 3 subunits are not had obvious influence; Can weaken cell injury and level of lipid rising that A β causes.The prompting capsule can raise nicotine receptor α 7 subunits at protein level, and the nicotine receptor that antagonism A β causes is expressed and reduced and neurotoxic effect.

(2), to the influence of SH-SY5Y neurocyte acetylcholine esterase

1. material and reagent

The compound capsule agent; The SH-SY5Y cell strain is purchased in German Collection of Micro organisms and CellCultures company (Germany); Calf serum is available from magnificent company; DMEM culture medium, no phenol red DMEM culture medium are available from HyClone company; A β _1-42Available from Sigma company; Coomassie brilliant blue method protein quantification reagent builds up company available from Nanjing; Acetylcholine esterase is measured reagent acetylthiocholine (ATC), sulfo-BuCh (BTC), 5,5 '-Lian sulfo--two-2-nitrobenzoic acid (DTNB), BW284C51 (inhibitor of AChE), iso-OMPA (inhibitor of BuChE) are all available from Sigma company; Cell protein extracts reagent phenylmethane sulfonyl fluoride (PMSF), compound protein enzyme inhibitor (Complete completion) available from Sigma company.

2. cell culture and packet transaction

Cell culture is the same, changes no phenol red DMEM processing 12 hours 70% the time approximately at the bottom of the culture bottle when cell is long, is further divided into 5 groups of processing: A and organizes: the blank group; B group: A β individual processing group: in cultured cells, add A β _1-42, its final concentration is 10mmol/L, incubation time is 48 hours; The C group: compound preparation individual processing group, its final concentration are 0.5g/L, cultivate 24 hours; D group: add the compound preparation pretreatment earlier and add A β again _1-42Processed group: add compound preparation earlier and handle after 24 hours, again according to the operation of B group; E group: add A β earlier _1-42Pretreatment adds the compound preparation processed group again: add A β earlier _1-42Handle after 48 hours, again according to the operation of C group.

3. cell protein is quantitative

When disposing, collects in cell culture medium and cell respectively in 1.5mL EPP pipe.Add about 300mL of cell pyrolysis liquid and PMSF and compound protein enzyme inhibitor in the pipe of collecting cell in the centrifugal 40 minutes (12000g after 1 hour of cracking on ice, 4 ℃), collect supernatant, adopt the Coomassie brilliant blue method that each group cell is carried out protein quantification, operate according to the test kit description.

4. the mensuration of cell culture based cholinesterase

The mensuration of ChE makes the culture medium of adding contain identical protein gram number with reference to Fllman colorimetry and list of references during mensuration.

5. statistical procedures

Every group of gained result represents that with x ± s the result carries out statistical analysis with the one factor analysis of variance method, thinks that with P＜0.05 difference has statistical significance.

6. result

6.1 each processed group AChE activity value

A β _1-42Individual processing group AChE activity value (24.69 ± 1.41nmolmin ^-1ML ^-1) than blank group (16.86 ± 0.41nmolmin ^-1ML ^-1) raising 46.44%, there is statistical significance (P＜0.05) in difference; Add the compound capsule agent earlier and add A β again _1-42Processed group (17.73 ± 0.59nmolmin ^-1ML ^-1), add A β earlier _1-42Add compound capsule agent processed group (17.02 ± 1.13nmolmin again ^-1ML ^-1) respectively with A β _1-42The individual processing group is compared, and its AChE activity value reduces by 39.26%, 45.06% respectively, and there is statistical significance (P＜0.05) in difference; Compound capsule agent individual processing group (11.08 ± 1.05nmolmin ^-1ML ^-1) also than the reduction of blank group, there is statistical significance (P＜0.05) in difference to the AChE activity value.Illustrated that compound preparation has the function of cholinesterase inhibitor.

6.2. each processed group BuChE activity value

Each processed group BuChE activity value is respectively: blank group 11.88 ± 0.33nmolmin ^-1ML ^-1, A β _1-42Individual processing group 13.21 ± 2.49nmolmin ^-1ML ^-1, compound capsule agent individual processing group 10.36 ± 1.83nmolmin ^-1ML ^-1, add the compound capsule agent earlier and add A β again _1-42Processed group 12.45 ± 2.54nmolmin ^-1ML ^-1, add A β earlier _1-42Add compound capsule agent processed group 10.98 ± 0.93nmolmin again ^-1ML ^-1, and BuChE activity value difference not statistically significant (P＞0.05) between each group.

7. conclusion

The compound capsule agent can reduce the beta induced class AD cell AChE activity of A, has the function of similar cholinesterase inhibitor.

Compared with prior art, the present invention maintains secrecy on the basis of proved recipe at defying age, is guidance with the theory of Chinese medical science, utilize scientific theory and advanced technology, developed the Chinese medicine preparation of a kind of good effect, treatment senile dementia that toxic and side effects is little, proved that through experimentation this Chinese medicine preparation is to AlCl ₃Inductive AD rat model ability of learning and memory has a better role, and AD is had the better prevention effect, can improve the resisting fatigue and the anti-stress ability of body, to the therapeutical effect preferably of having suffered from a deficiency of the kidney.Molecular mechanism of action to its treatment AD is discovered, this Chinese medicine can raise nicotine receptor α 7 subunits at protein level, the nicotine receptor that antagonism A β causes is expressed and is reduced and neurotoxic effect, weaken cell injury and level of lipid rising that A β causes, reduce the beta induced class AD cell AChE activity of A.

The present invention simultaneously also provides a kind of industrial, preferred manufacturing procedure that is fit to.

The specific embodiment

Embodiment 1: Radix Ginseng 60g, Radix Astragali Preparata 120g, Herba Epimedii 90g, Rhizoma Corydalis 90g, Rhizoma Gastrodiae 120g, Radix Angelicae Sinensis 90g, Radix Polygoni Multiflori Preparata 120g, Rhizoma Polygonati 120g and Rhizoma Acori Graminei 120g

Preparation method: (1) alcohol extraction: get Radix Ginseng, Rhizoma Gastrodiae, Radix Astragali Preparata, Herba Epimedii, Rhizoma Corydalis five tastes medical material with 85% alcohol reflux 2 times, each 2h adds 16 times of amount ethanol for the first time, add for the second time 14 times of amount ethanol, filter, get alcohol extract, decompression recycling ethanol, medicinal liquid is standby;

(2) decocting in water: get Radix Angelicae Sinensis, Radix Polygoni Multiflori Preparata, Rhizoma Polygonati, Rhizoma Acori Graminei four Chinese medicine material water and decoct 3 times, each 2h adds 14 times of water gagings for the first time, respectively adds 12 times of water gagings for the second time and for the third time, filters, and filtrate decompression concentrates standby;

(3) preparations shaping: merge alcohol extraction medicinal liquid and decocting in water medicinal liquid, be concentrated into thick paste, in 70 ℃ of vacuum dryings, promptly get the active component of the Chinese medicine preparation for the treatment of senile dementia, adding starch is an amount of in active component, granulates, encapsulated, make 1000 of capsules, every 0.35g.

Function cures mainly: QI invigorating brain-strengthening, strengthening the body resistance, the Fructus Alpiniae Oxyphyllae of having one's ideas straightened out, blood circulation promoting and blood stasis dispelling; Be used for the prevention and the treatment senile dementia due to hypomnesis.

Usage and dosage: oral, 3 times on the one, one time 4.

Embodiment 2: Radix Ginseng 50g, Radix Astragali Preparata 100g, Herba Epimedii 100g, Rhizoma Corydalis 100g, Rhizoma Gastrodiae 140g, Radix Angelicae Sinensis 80g, Radix Polygoni Multiflori Preparata 100g, Rhizoma Polygonati 100g and Rhizoma Acori Graminei 140g

(1) alcohol extraction: get Radix Ginseng, Rhizoma Gastrodiae, Radix Astragali Preparata, Herba Epimedii, Rhizoma Corydalis five tastes medical material with 20 times of amount 85% alcohol reflux 1 time, extract 3h, filter, alcohol extract, decompression recycling ethanol, medicinal liquid is standby;

(2) decocting in water: get Radix Angelicae Sinensis, Radix Polygoni Multiflori Preparata, Rhizoma Polygonati, Rhizoma Acori Graminei four Chinese medicine material and decoct 3 times with 10 times of water gagings, each 1h filters, and filtrate decompression concentrates standby;

(3) preparations shaping: merge alcohol extraction medicinal liquid and decocting in water medicinal liquid, be concentrated into thick paste,, promptly get the active component of the Chinese medicine preparation for the treatment of senile dementia, in active component, add adjuvant in 60 ℃ of vacuum dryings, mixing, tabletting is made 1000 in tablet.

Usage and dosage: oral, 3 times on the one, each 4.

Embodiment 3: Radix Ginseng 70g, Radix Astragali Preparata 140g, Herba Epimedii 80g, Rhizoma Corydalis 80g, Rhizoma Gastrodiae 100g, Radix Angelicae Sinensis 100g, Radix Polygoni Multiflori Preparata 140g, Rhizoma Polygonati 140g and Rhizoma Acori Graminei 1000g

(1) alcohol extraction: get Radix Ginseng, Rhizoma Gastrodiae, Radix Astragali Preparata, Herba Epimedii, Rhizoma Corydalis five tastes medical material with 10 times of amount 85% alcohol reflux 3 times, each 1h filters, alcohol extract, decompression recycling ethanol, medicinal liquid is standby;

(2) decocting in water: get Radix Angelicae Sinensis, Radix Polygoni Multiflori Preparata, Rhizoma Polygonati, Rhizoma Acori Graminei four Chinese medicine material with 16 times of water gagings decoctions 1 time, decoct 3h, filter, filtrate decompression concentrates standby;

(3) preparations shaping: merge alcohol extraction medicinal liquid and decocting in water medicinal liquid, be concentrated into thick paste, in 80 ℃ of vacuum dryings, promptly get the active component of the Chinese medicine preparation for the treatment of senile dementia, add adjuvant in active component, technology is made 1000 of soft capsules routinely.

Usage and dosage: oral, 3 times on the one, each 4.

Claims (5)

1. Chinese medicine preparation for the treatment of senile dementia, it is characterized in that: according to listed as parts by weight, it is to be prepared from for 100～140 parts by 50～70 parts of raw material of Chinese medicine Radix Ginsengs, 100～140 parts of Radix Astragali Preparatas, 80～100 parts of Herba Epimedii, 80～100 parts of Rhizoma Corydalis, 100～140 parts in Rhizoma Gastrodiae, 80～100 parts of Radix Angelicae Sinensis, 100～140 parts of Radix Polygoni Multiflori Preparatas, 100～140 parts of Rhizoma Polygonatis and Rhizoma Acori Graminei.

2. according to the Chinese medicine preparation of the described treatment senile dementia of claim 1, it is characterized in that: according to listed as parts by weight, it is to be prepared from for 120 parts by 60 parts of raw material of Chinese medicine Radix Ginsengs, 120 parts of Radix Astragali Preparatas, 90 parts of Herba Epimedii, 90 parts of Rhizoma Corydalis, 120 parts in Rhizoma Gastrodiae, 90 parts of Radix Angelicae Sinensis, 120 parts of Radix Polygoni Multiflori Preparatas, 120 parts of Rhizoma Polygonatis and Rhizoma Acori Graminei.

3. the preparation method of the Chinese medicine preparation of claim 1 or 2 described treatment senile dementias comprises the steps:

(1) alcohol extraction: get Radix Ginseng, Rhizoma Gastrodiae, Radix Astragali Preparata, Herba Epimedii, Rhizoma Corydalis five tastes medical material 85% alcohol reflux, filter, get alcohol extract, decompression recycling ethanol, medicinal liquid is standby;

(2) decocting in water: get Radix Angelicae Sinensis, Radix Polygoni Multiflori Preparata, Rhizoma Polygonati, Rhizoma Acori Graminei four Chinese medicine material and decoct with water, filter, filtrate decompression concentrates standby;

(3) preparations shaping: merge alcohol extraction medicinal liquid and decocting in water medicinal liquid, be concentrated into thick paste, vacuum drying, preparation process is made the various dosage forms on the pharmaceutics meaning routinely.

4. according to the preparation method of the Chinese medicine preparation of the described treatment senile dementia of claim 3, comprise the steps:

(1) alcohol extraction: get Radix Ginseng, Rhizoma Gastrodiae, Radix Astragali Preparata, Herba Epimedii, Rhizoma Corydalis five tastes medical material with 8～20 times of amount 85% alcohol reflux 1～3 time, each 1～3h filters, alcohol extract, decompression recycling ethanol, medicinal liquid is standby;

(2) decocting in water: get Radix Angelicae Sinensis, Radix Polygoni Multiflori Preparata, Rhizoma Polygonati, Rhizoma Acori Graminei four Chinese medicine material and decoct 1～3 time with 8～16 times of water gagings, each 1～3h filters, and filtrate decompression concentrates standby;

(3) preparations shaping: merge alcohol extraction medicinal liquid and decocting in water medicinal liquid, be concentrated into thick paste, in 60～80 ℃ of vacuum dryings, preparation process is made the various dosage forms on the pharmaceutics meaning routinely.

5. according to the preparation method of the Chinese medicine preparation of the described treatment senile dementia of claim 4, comprise the steps:

(1) alcohol extraction: get Radix Ginseng, Rhizoma Gastrodiae, Radix Astragali Preparata, Herba Epimedii, Rhizoma Corydalis five tastes medical material with 85% alcohol reflux 2 times, each 2h adds 16 times of amount ethanol for the first time, adds 14 times of amount ethanol for the second time, filter, alcohol extract, decompression recycling ethanol, medicinal liquid is standby;

(2) decocting in water: get Radix Angelicae Sinensis, Radix Polygoni Multiflori Preparata, Rhizoma Polygonati, Rhizoma Acori Graminei four Chinese medicine material water and decoct 3 times, each 2h adds 14 times of water gagings for the first time, adds 12 times of water gagings for the second time and for the third time, filters, and filtrate decompression concentrates standby;

(3) preparations shaping: merge alcohol extraction medicinal liquid and decocting in water medicinal liquid, be concentrated into thick paste, in 70 ℃ of vacuum dryings, preparation process is made the various dosage forms on the pharmaceutics meaning routinely.