CN102671007B - Medicament for treating senile dementia and preparation method thereof - Google Patents

Medicament for treating senile dementia and preparation method thereof Download PDF

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CN102671007B
CN102671007B CN 201110061899 CN201110061899A CN102671007B CN 102671007 B CN102671007 B CN 102671007B CN 201110061899 CN201110061899 CN 201110061899 CN 201110061899 A CN201110061899 A CN 201110061899A CN 102671007 B CN102671007 B CN 102671007B
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CN102671007A (en
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王平
孔明望
刘玲
石和元
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Hubei College of Chinese Medicine
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Abstract

The invention discloses a medicament for treating senile dementia and a preparation method thereof. The medicament consists of active ingredients or pharmaceutically acceptable carriers. The medicament is prepared from the following active ingredients in part by weight: 1 to 15 parts of pinellia tuber, 1 to 15 parts of bamboo shavings, 1 to 15 parts of immature bitter orange, 1 to 15 parts of grassleaf sweelflag rhizome, 1 to 15 parts of thinleaf milkwort root-bark, 1 to 10 parts of ginseng, 1 to 15 parts of ligusticum wallichii and 1 to 20 parts of prepared rehmannia rhizome. The medicament is also prepared from one or more raw materials in part by weight: 1 to 15 parts of Japanese ginseng, 1 to 15 parts of tuckahoe, 1 to 15 parts of dried orange peel, 1 to 10 parts of white mustard seed, 1 to 10 parts of leech and 1 to 20 parts of prepared multiflower knotweed tuber. The medicament can be prepared into formulations such as powder, tablets, capsules, dispersible tablets, pellets, injection, oral liquid and granules. The preparation method comprises the following steps of: chopping raw materials into small pieces or crushing the raw materials into coarse powder, mixing extracts of the raw materials, which are obtained by using water or ethanol, or continuing concentrating to form dry extractum, or mixing the mixture of the extracts and the carriers. Clinical application proves that the medicament is an effective medicament for treating alzheimer's diseases and improving the memory, cognitive disorder and living abilities of patients.

Description

A kind of medicine for the treatment of senile dementia and preparation method thereof
Technical field
The present invention relates to a kind of medicine for the treatment of senile dementia and preparation method thereof, more particularly, relate to and a kind ofly take treatment prepared as raw material by Chinese herbal medicine and take medicine that Alzheimer is main senile dementia and preparation method thereof.
Background technology
Senile dementia comprises the types such as Alzheimer (Alzheimer ' s disease, AD) and vascular dementia (vascuIar dementia, VaD).Along with China's aged tendency of population is accelerated, the sickness rate of senile dementia improves constantly, and has a strong impact on aged's health.Because pathogenesis understanding is also insufficient, up to now, there is no desirable Therapeutic Method.The medicine of U.S. FDA approval has three kinds of cholinesterase inhibitor, example hydrochloric acid donepezil (DoneDezil), galantamine (Galantamine) and Exelon (Rivastigmine) and a kind of excitatory amino acid receptor antagonists, example hydrochloric acid memantine (Memantine), they can short-term improve the symptom of dementia patients but can not delay disease progression.
AD belongs to the categories such as the traditional Chinese medical science " dementia ", " dementia ".Chinese medicine prevention AD is better with its curative effect, toxic and side effects is little, by increasing doctor, is approved, has become in recent years the focus of domestic and international research, has carried out a large amount of Clinical and experimental studies, comprises Chinese medicine compound and single medicinal material, and has obtained larger achievement.
The senile dementia disease position is at brain, and its pathogenesis always belongs to deficiency in origin and excess in superficiality, and the clinical deficiency and excess that mostly is is interlocked, the disease complexity.Its deficiency in origin is the vital essence deficiency of kidney, the brain virtual loss, and its mark is hoodwinked the brain key in the turbid blood stasis of expectorant, impatency brain network really.Deficiency of kidney essence, the vital essence deficiency, expectorant stasis of blood impatency is two kinds of different pathological changes in the senile dementia genesis, and the two both connected each other, influenced each other again, and common causing a disease, form vicious cycle, so that the course of disease is touching, sees the disease multiterminal.
Wherein turbid phlegm blocking the clear orifices plays an important role in the AD morbidity.The patient belongs to the deficient people with the passing of time of internal organs more, its gas degradation, and the body fluid metabolism is not normal, causes water to wet and stops gathering, the turbid interior life of expectorant.The brain key is closed in the turbid resistance of expectorant, causes the brain muddiness, and sudden inspiration is not transported, refreshing machine imbalance, and dementia is arisen at the historic moment." dialectical record " explicitly points out that " accumulation of phlegm, in brain, is illegally occupied outside the heart, makes the unclear dementia that forms of gods." Chen Shiduo illustrates further dementia degree and the turbid relation of expectorant, " the expectorant gesture is contained most, and stupidity is the darkest " in " the secret record in stone chamber ".The control of tcm clinical practice to primary disease, also be placed on important position eliminating the phlegm.Domestic doctor analyzes alzheimer disease effective 40 first prescriptions having reported in 1992-1997, during the compatibility of finding all effective prescriptions forms, the flavour of a drug of eliminating phlegm for resuscitation is all arranged.The verified classical side's WEIDANTANG treatment alzheimer disease of reducing phlegm of Japan North Japan Institute for Medical Research obtains sure curative effect.
Summary of the invention
The purpose of this invention is to provide a kind of medicine for the treatment of senile dementia and preparation method thereof.Described senile dementia comprises Alzheimer, especially belongs to the Alzheimer of deficiency of kidney-essence, expectorant stasis blocking key card, the memory and cognition obstacle, and viability is low.
To achieve these goals, technical scheme of the present invention be take Chinese medicine as basis, carries out Chinese medicine active component compatibility.Theory according to tcm treatment according to syndrome differentiation, whole medication, adopt the Therapeutic Principle of " the kidney invigorating of reducing phlegm, benefiting QI for activating blood circulation, the Fructus Alpiniae Oxyphyllae of having one's ideas straightened out ", at waking up the patient from unconsciousness by dissipating phlegm, when the Fructus Alpiniae Oxyphyllae of invigorating blood circulation takes stopgap measures, tonify Qi of the kidney and effect a permanent cure, thus make the kidney essense abundance brain must support, QI and blood is unobstructed, the expectorant stasis of blood must be changed key, and to open network smooth, reaches the effect of improving ability of learning and memory.
The present invention be take experiment in clinical research, experiment in vitro and body and, as basis, is added and subtracted on the basis of classical Huatan Formula WEIDANTANG, forms a new compound.Herbal mixture material of the present invention source mainly contains: the Rhizoma Pinelliae, Caulis Bambusae In Taenia, Fructus Aurantii Immaturus, Rhizoma Acori Graminei, Radix Polygalae, Radix Ginseng, Rhizoma Chuanxiong, Radix Rehmanniae Preparata.
Wherein the Rhizoma Pinelliae is hot warm, and drying dampness to eliminate phlegm, be monarch drug.
Caulis Bambusae In Taenia is sweet and be slightly cold, removing heat-phlegm.The Rhizoma Pinelliae and Caulis Bambusae In Taenia, a temperature one is cool, and the stomach function regulating relieving restlessness of reducing phlegm, bring out the best in each other; The Rhizoma Acori Graminei refreshment of having one's ideas straightened out, removing dampness to restore normal function of the stomach, the beneficial will of allaying excitement, Radix Polygalae mind tranquilizing and the heart calming; Eliminate the phlegm and have one's ideas straightened out, the merit that both equal tools eliminate the phlegm and have one's ideas straightened out, so Rhizoma Acori Graminei is relatively hot loose to declare its phlegm-damp, and Radix Polygalae is partial to hardship and is fallen to determine the phlegm-damp of superinverse.The two same use, Ji is proved effective mutually, make gas from along and stop up from opening, clear key is not covered in the turbid dissipation of expectorant, has a clear mind.Radix Rehmanniae Preparata is enriched blood moist, and beneficial essence is filled out marrow; The Radix Ginseng strongly invigorating primordial QI, invigorating the spleen to benefit the lung, the beneficial will of calming the nerves." book on Chinese herbal medicine is newly organized ": Rhizoma Acori Graminei, happy key must be helped with Radix Ginseng; Fructus Aurantii Immaturus sending down the abnormal ascending QI intestinal stasis relieving, expectorant is except painful abdominal mass, gas along and expectorant from disappearing, increase the power of monarch-minister drug materialization expectorant.The Rhizoma Chuanxiong promoting flow of QI and blood, be gas medicine in blood, on the smooth sharp vim and vigour of kind energy, reach the head, sensible brain-strengthening.
A kind of Chinese medicine composition for the treatment of senile dementia of the present invention, it is by effective ingredient or also have pharmaceutically acceptable carrier to form, it is characterized in that, its contained effective ingredient is mainly made by the raw material of Chinese medicine of following weight proportioning: Rhizoma Pinelliae 1-15 part, Caulis Bambusae In Taenia 1-15 part, Fructus Aurantii Immaturus 1-15 part, Rhizoma Acori Graminei 1-15 part, Radix Polygalae 1-15 part, Radix Ginseng 1-10 part, Rhizoma Chuanxiong 1-15 part, Radix Rehmanniae Preparata 1-20 part.
Be preferably: 10 parts of the Rhizoma Pinelliaes, 12 parts of Caulis Bambusae In Taenia, 10 parts of Fructus Aurantii Immaturuss, 10 parts of Rhizoma Acori Graminei, 10 parts of Radix Polygalaes, 6 parts of Radix Ginsengs, 10 parts of Rhizoma Chuanxiongs, 12 parts, Radix Rehmanniae Preparata.
Chinese medicine composition of the present invention is except the raw material of Chinese medicine of the above, also comprise one or more combinations of raw material of Chinese medicine that are selected from following weight proportion: Rhizoma Panacis Japonici 1-15 part, Poria 1-20 part, Pericarpium Citri Reticulatae 1-15 part, Semen Sinapis Albae 1-10 part, Hirudo 1-10 part, Radix Polygoni Multiflori Preparata 1-25 part.Preferably: 10 parts of Rhizoma Panacis Japonicis, 15 parts, Poria, 10 parts of Pericarpium Citri Reticulataes, 6 parts of Semen Sinapis Albaes, 6 parts of Hirudos, 20 parts of Radix Polygoni Multiflori Preparata.
Medicine of the present invention all available its extract replaces.
Chinese medicine composition of the present invention is by the raw material of Chinese medicine of above-mentioned weight proportion or also has said dosage form on any pharmaceutics that pharmaceutically the acceptable carrier is made, comprise decoction, powder, tablet, capsule, dispersible tablet, micropill, injection, oral liquid and granule.
On pharmaceutics of the present invention, the acceptable carrier includes but not limited to: excipient, as starch and derivant thereof, dextrin, calcium hydrogen phosphate, magnesium stearate, differential silica gel etc.; Disintegrating agent, as search sodium carboxymethylcellulose pyce, through propyl cellulose etc.; Lubricant, as magnesium stearate etc.; The sugar coating material, as sucrose, Pulvis Talci, gelatin, pigment, river wax etc.; Thin film coating material, as stomach dissolution type water, pure coating material etc.
The dosage form of medicine of the present invention is preferably oral formulations, for example tablet, capsule, granule, pill or decoction, and wherein preparing the method for described preparation and the pharmaceutically suitable carrier used and/or excipient is the routine techniques of the art.
The preparation method of Chinese medicine composition of the present invention, be the mixture by described each drug extract that for raw material of Chinese medicine, different process, Different Extraction Method obtain, or medicine is divided into the mixture of the extract of several different components after extracting respectively, or total extract, the preparation of making.
The preparation method of described Chinese medicine composition of the present invention, it comprises described raw material of Chinese medicine directly is cut into small pieces or is ground into coarse powder, the mixing of each drug extract that water or ethanol extraction obtain again, or continue to be condensed into dry extract, or with acceptable carrier on pharmaceutics, mix again.
The preparation method of Chinese medicine composition of the present invention, comprise the Rhizoma Pinelliae, Caulis Bambusae In Taenia, Fructus Aurantii Immaturus, Rhizoma Acori Graminei, Radix Polygalae, Radix Ginseng, Rhizoma Chuanxiong, Radix Rehmanniae Preparata, and water decocts 3 times, each 20 minutes, filter merging filtrate, be evaporated to every 1 milliliter and be equivalent to raw medicinal herbs amount 1 gram, obtain decoction; Or, again through sterilizing, filling bottle, obtain oral liquid; Or be concentrated into thick paste, and continue drying under reduced pressure to dry cream, add starch, pulverize, mix, cross 80 mesh sieves, with 95% ethanol wet granulation, dry below 80 ℃, granulate, granulation agent; Or add magnesium stearate, and mix, tabletting, coating, obtain tablet; Or add excipient, and incapsulate, obtain capsule.
The preparation method of Chinese medicine composition of the present invention; Comprise Fructus Aurantii Immaturus, Rhizoma Acori Graminei, Radix Polygalae, are refluxed with the 75-85% ethanol of 20-25 times of quality after 12 hours at twice by soak with ethanol, each micro-rear backflow 1～1.5 hour of boiling, filter, and reclaims ethanol to without the alcohol flavor, obtains extract; Get the Rhizoma Pinelliae, Caulis Bambusae In Taenia, Radix Ginseng, Rhizoma Chuanxiong, Radix Rehmanniae Preparata decoction 40-50 minute, filter, filtrate is concentrated into the 1/6-1/2 volume, with extract, merges, and amalgamation liquid is evaporated to every 1 milliliter and is equivalent to raw medicinal herbs amount 1 gram, obtains decoction; Or make as stated above oral liquid, granule, tablet, capsule.
The administering mode of medicine of the present invention is conventional, and dosage is judged by the attending doctor according to factors such as age of the character of disease, patient, body weight, the state of an illness, administering modes.Each representative Chinese drug-treated group in the kidney invigorating, QI invigorating, the method for the treatment of of reducing phlegm, invigorate blood circulation is become to medicine of the present invention, effect with treating both the principal and secondary aspects of a disease, strengthening vital QI to eliminate pathogenic factors, be different from single method for the treatment of, clinical and experimental results show that its treatment is dull-witted, effect of cognition and memory impairment significantly is better than chemical drugs, has advantages of that effective site is clear and definite, curative effect is sure, flavour of a drug are few, cost is little.
Technical scheme of the present invention can realize one or more purpose of the present invention.
The present invention has carried out pharmacological effect research from cell experiment, zoopery and three aspects of clinical experiment to medicine of the present invention respectively, has obtained better effects.Observed clinically the improvement effect of medicine of the present invention to the old dementia patients learning and memory.The experimentation aspect; on model respectively with amino-(3-hydroxy-5-isoxazolyl)acetic acid. (Ibotenicacid; IBO) the AD cell model that damage Meynert core plan AD rat model, IBO+A β 25-35 damage Meynert core plan AD rat model, A β 25-35 induce PC12 cell and NG108 cell and glutamic acid to bring out the neural cell injury model of the former culture of rat hippocampal is object of study; observed the neurovirulent protective effect of medicine of the present invention to animal pattern and cell, systematically be studied from the many target spots of a plurality of links simultaneously.
Annotate: the ethanol % used in the present invention all refers to concentration expressed in percentage by volume.
The accompanying drawing explanation
Fig. 1 be the invention medicine to AD rat model orientation navigation experiment trajectory diagram, wherein: a1 is normal group, and b1 is blank group, and c1 is model group, and d1 is the Western medicine group, and e1 is low dose group, and f1 is middle dosage group, and g1 is high dose group.
Fig. 2 be the invention medicine to AD rat model space search experimental traces figure, wherein: a2 is normal group, b2 is blank group, c2 is model group, d2 is the Western medicine group, e2 is low dose group, f2 is middle dosage group, g2 is high dose group.
Fig. 3 is the impact of invention medicine on AD rat model cerebral tissue senile plaque, and wherein: a3 is normal group, and b3 is blank group, and c3 is model group, and d3 is the Western medicine group, and e3 is low dose group, and f3 is middle dosage group, and g3 is high dose group.
Fig. 4 is the impact of invention medicine on AD rat model cerebral tissue neurofibrillary tangles, and wherein: a4 is normal group, and b4 is blank group, and c4 is model group, and d4 is the Western medicine group, and e4 is low dose group, and f4 is middle dosage group, and g4 is high dose group.
Fig. 5 is the impact of invention medicine on AD rat model cerebral tissue Ultrastructural pathology, and wherein: a5 is normal group, and b5 is blank group, and c5 is model group, and d5 is the Western medicine group, and e5 is low dose group, and f5 is middle dosage group, and g5 is high dose group.
Fig. 6 is that the invention medicine is to rat hippocampus Ub protein level RT-PCR result.
Fig. 7 is that the invention medicine is to rat hippocampus E1 protein level western blot result.
Fig. 8 is that the invention medicine is to rat hippocampus E2-EPF protein level western blot result.
Fig. 9 is that the invention medicine is to rat hippocampus PGP9.5 protein level western blot result.
Figure 10 is that the invention medicine is to rat Phosphorylated tau western blot result.
Figure 11 is that the invention medicine is to rat cdk5RT-PCR testing result.
Figure 12 be the invention medicine to PC12 cell situation under Contained Serum and blank serum effect, wherein: a12 is 5% blank serum, and b12 is 10% blank serum, c12 is 20% blank serum, d12 is 5% Contained Serum, and e12 is 10% Contained Serum, and f12 is 20% Contained Serum.
Figure 13 condition diagram to its protection that is the invention medicine to A β 25-35 damage PC12 cell and Contained Serum; wherein: a13 is PC12+20 μ MA β+5% blank serum; b13 is PC12+20 μ MA β+5% Contained Serum; c13 is PC12+20 μ MA β+10% blank serum; d14 is PC12+20 μ MA β+10% Contained Serum; e13 is PC12+20 μ MA β+20% blank serum, and f13 is PC12+20 μ MA β+20% Contained Serum.
Figure 14 is the streaming result of invention medicine to AD model cell apoptosis rate, wherein: a14 is normal control, b14 is model group, c14 is 5% blank serum, d14 is 10% blank serum, e14 is 20% blank serum, and f14 is 5% Contained Serum, and g14 is that 10% Contained Serum h14 is 20% Contained Serum.
Figure 15 invention medicine is to AD model cell caspase-3 ImmunohistochemistryResults Results (10X10), wherein: a15 is the complete culture solution group, and b15 is A β 25-35group, c15 is A β 25-35+ 5% blank serum, d15 is A β 25-35+ 10% blank serum, e15 is A β 25-35+ 20% blank serum, f15 is A β 25-35+ 5% Contained Serum, g15 is A β 25-35+ 10% Contained Serum, h15 is A β 25-35+ 20% Contained Serum.
Figure 16 invention medicine is to AD model cell form result (10x20), wherein: a16 is blank group, and b16 is model group, and c16 is blank serum group, and d16 is the Contained Serum group, and e16 is blank cerebrospinal fluid group, and f16 is pastille cerebrospinal fluid group.
The impact of Figure 17 invention medicine pastille cerebrospinal fluid on AD model cell apoptosis rate, wherein: a17 is blank group, and b17 is model group, and c17 is blank serum group, and d17 is the Contained Serum group, and e17 is blank cerebrospinal fluid group, and f17 is pastille cerebrospinal fluid group.
The impact of Figure 18 invention medicine on Bcl-2, Bax protein expression, wherein: a18 is blank group bax (40 * 10), b18 is model group bcl-2 (40 * 10), c18 is blank serum group bax (40 * 10), d18 is blank cerebrospinal fluid group bcl-2 (40 * 10), e18 is Contained Serum group bax (40 * 10), and f18 is pastille cerebrospinal fluid group bcl-2 (40 * 10).
The specific embodiment
Below EXPERIMENTAL EXAMPLE be used for illustrating preparation and the performance thereof of medicine of the present invention, in any case but they can not be construed as limiting the invention.
Preparation Example
Raw material of Chinese medicine is purchased from medical expert's consultation section of Hubei university of TCM.
Embodiment 1
1, get by weight 10 parts of the Rhizoma Pinelliaes, 12 parts of Caulis Bambusae In Taenia, 10 parts of Fructus Aurantii Immaturuss, 20 parts of Radix Polygoni Multiflori, 10 parts of 6 parts of Radix Ginseng Rubra, 10 parts of Rhizoma Chuanxiongs, 6 parts of Hirudos, 10 parts of Rhizoma Acori Graminei and Rhizoma Panacis Japonicis;
2, the Rhizoma Pinelliae, Caulis Bambusae In Taenia, Fructus Aurantii Immaturus, Radix Polygoni Multiflori, Radix Ginseng Rubra, Rhizoma Chuanxiong, Hirudo, Rhizoma Acori Graminei water are decocted 3 times, each 20 minutes, make medicinal liquid leach as far as possible, merge water extraction liquid, being evaporated to density is the thick paste of 1.0-3.0 (50 ℃), continues drying under reduced pressure to dry cream, add starch, pulverize, mix, cross 80 mesh sieves, with 95% ethanol wet granulation, dry below 80 ℃, granulate, granulation agent.
Embodiment 2
1, get by weight 10 parts of the Rhizoma Pinelliaes, 12 parts of Caulis Bambusae In Taenia, 10 parts of Fructus Aurantii Immaturuss, 20 parts of Radix Polygoni Multiflori, 10 parts of 6 parts of Radix Ginseng Rubra, 10 parts of Rhizoma Chuanxiongs, 6 parts of Hirudos and Rhizoma Acori Graminei;
2, the Rhizoma Pinelliae, Caulis Bambusae In Taenia, Fructus Aurantii Immaturus, Radix Polygoni Multiflori, Radix Ginseng Rubra, Rhizoma Chuanxiong, Hirudo, Rhizoma Acori Graminei water are decocted 3 times, each 20 minutes, make medicinal liquid leach as far as possible, merge water extraction liquid, being evaporated to density is the thick paste of 1.0-3.0 (50 ℃).
Embodiment 3
1, get by weight 20 parts of Radix Polygoni Multiflori Preparata, 10 parts of Rhizoma Acori Graminei, 10 parts of Rhizoma Panacis Japonicis, 15 parts, Poria, 10 parts of Pericarpium Citri Reticulataes, 10 parts of the Rhizoma Pinelliaes, 6 parts of Semen Sinapis Albaes;
2, Radix Polygoni Multiflori Preparata, Rhizoma Acori Graminei, Rhizoma Panacis Japonici, Poria, Pericarpium Citri Reticulatae, the Rhizoma Pinelliae, Semen Sinapis Albae water are decocted 3 times, each 20 minutes, make medicinal liquid leach as far as possible, merge water extraction liquid, being evaporated to density is the thick paste of 1.0-3.0 (50 ℃).
Embodiment 4
1, get by weight 10 parts of Rhizoma Pinelliae (processed with Rhizoma Zingiberis Recens)s, 12 parts of Caulis Bambusae In Taenia, 10 parts of Fructus Aurantii Immaturuss; Rhizoma Pinelliae (processed with Rhizoma Zingiberis Recens), Caulis Bambusae In Taenia, Fructus Aurantii Immaturus water are decocted 3 times, each 20 minutes, make medicinal liquid leach as far as possible, merge water extraction liquid, being evaporated to density is the thick paste of 1.0-3.0 (50 ℃).
2, get by weight 10 parts of Rhizoma Pinelliae (processed with Rhizoma Zingiberis Recens)s, 12 parts of Caulis Bambusae In Taenia, 10 parts of Fructus Aurantii Immaturuss, 10 parts of Pericarpium Citri Reticulataes, 15 parts, Poria, 6 parts, Radix Glycyrrhizae; Rhizoma Pinelliae (processed with Rhizoma Zingiberis Recens), Caulis Bambusae In Taenia, Fructus Aurantii Immaturus, Pericarpium Citri Reticulatae, Poria, Radix Glycyrrhizae water are decocted 3 times, each 20 minutes, make medicinal liquid leach as far as possible, merge water extraction liquid, being evaporated to density is the thick paste of 1.0-3.0 (50 ℃).
3. get by weight 10 parts of Rhizoma Pinelliae (processed with Rhizoma Zingiberis Recens)s, 12 parts of Caulis Bambusae In Taenia, 10 parts of Fructus Aurantii Immaturuss, 12 parts, Radix Rehmanniae Preparata, 20 parts of Radix Polygoni Multiflori Preparata; Rhizoma Pinelliae (processed with Rhizoma Zingiberis Recens), Caulis Bambusae In Taenia, Fructus Aurantii Immaturus, Radix Rehmanniae Preparata, Radix Polygoni Multiflori Preparata water are decocted 3 times, each 20 minutes, make medicinal liquid leach as far as possible, merge water extraction liquid, being evaporated to density is the thick paste of 1.0-3.0 (50 ℃).
4, get by weight 10 parts of Rhizoma Pinelliae (processed with Rhizoma Zingiberis Recens)s, 12 parts of Caulis Bambusae In Taenia, 10 parts of Fructus Aurantii Immaturuss, 10 parts of Radix Polygalaes, 10 parts of Rhizoma Acori Graminei, 6 parts of Semen Sinapis Albaes; Rhizoma Pinelliae (processed with Rhizoma Zingiberis Recens), Caulis Bambusae In Taenia, Fructus Aurantii Immaturus, Radix Polygalae, Rhizoma Acori Graminei, Semen Sinapis Albae water are decocted 3 times, each 20 minutes, make medicinal liquid leach as far as possible, merge water extraction liquid, being evaporated to density is the thick paste of 1.0-3.0 (50 ℃).
5, get by weight 10 parts of Rhizoma Pinelliae (processed with Rhizoma Zingiberis Recens)s, 12 parts of Caulis Bambusae In Taenia, 10 parts of Fructus Aurantii Immaturuss, 10 parts of Rhizoma Chuanxiongs; Rhizoma Pinelliae (processed with Rhizoma Zingiberis Recens), Caulis Bambusae In Taenia, Fructus Aurantii Immaturus, Rhizoma Chuanxiong water are decocted 3 times, each 20 minutes, make medicinal liquid leach as far as possible, merge water extraction liquid, being evaporated to density is the thick paste of 1.0-3.0 (50 ℃).
6, get by weight 10 parts of Rhizoma Pinelliae (processed with Rhizoma Zingiberis Recens)s, 12 parts of Caulis Bambusae In Taenia, 10 parts of Fructus Aurantii Immaturuss, 6 parts of Radix Ginsengs; Rhizoma Pinelliae (processed with Rhizoma Zingiberis Recens), Caulis Bambusae In Taenia, Fructus Aurantii Immaturus, Radix Ginseng water are decocted 3 times, each 20 minutes, make medicinal liquid leach as far as possible, merge water extraction liquid, being evaporated to density is the thick paste of 1.0-3.0 (50 ℃).
7, get by weight 10 parts of Rhizoma Pinelliae (processed with Rhizoma Zingiberis Recens)s, 12 parts of Caulis Bambusae In Taenia, 10 parts of Fructus Aurantii Immaturuss, 12 parts, Radix Rehmanniae Preparata, 20 parts of Radix Polygoni Multiflori Preparata, 10 parts of Radix Polygalaes, 10 parts of Rhizoma Acori Graminei, 6 parts of Semen Sinapis Albaes; Rhizoma Pinelliae (processed with Rhizoma Zingiberis Recens), Caulis Bambusae In Taenia, Fructus Aurantii Immaturus, Radix Rehmanniae Preparata, Radix Polygoni Multiflori Preparata, Radix Polygalae, Rhizoma Acori Graminei, Semen Sinapis Albae water are decocted 3 times, each 20 minutes, make medicinal liquid leach as far as possible, merge water extraction liquid, being evaporated to density is the thick paste of 1.0-3.0 (50 ℃).
8, get by weight 10 parts of Rhizoma Pinelliae (processed with Rhizoma Zingiberis Recens)s, 12 parts of Caulis Bambusae In Taenia, 10 parts of Fructus Aurantii Immaturuss, 12 parts, Radix Rehmanniae Preparata, 20 parts of Radix Polygoni Multiflori Preparata, 10 parts of Rhizoma Chuanxiongs; Rhizoma Pinelliae (processed with Rhizoma Zingiberis Recens), Caulis Bambusae In Taenia, Fructus Aurantii Immaturus, Radix Rehmanniae Preparata, Radix Polygoni Multiflori Preparata, Rhizoma Chuanxiong water are decocted 3 times, each 20 minutes, make medicinal liquid leach as far as possible, merge water extraction liquid, being evaporated to density is the thick paste of 1.0-3.0 (50 ℃).
9, get by weight 10 parts of Rhizoma Pinelliae (processed with Rhizoma Zingiberis Recens)s, 12 parts of Caulis Bambusae In Taenia, 10 parts of Fructus Aurantii Immaturuss, 12 parts, Radix Rehmanniae Preparata, 20 parts of Radix Polygoni Multiflori Preparata, 6 parts of Radix Ginsengs; Rhizoma Pinelliae (processed with Rhizoma Zingiberis Recens), Caulis Bambusae In Taenia, Fructus Aurantii Immaturus, Radix Rehmanniae Preparata, Radix Polygoni Multiflori Preparata, Radix Ginseng water are decocted 3 times, each 20 minutes, make medicinal liquid leach as far as possible, merge water extraction liquid, being evaporated to density is the thick paste of 1.0-3.0 (50 ℃).
10, get by weight 10 parts of Rhizoma Pinelliae (processed with Rhizoma Zingiberis Recens)s, 12 parts of Caulis Bambusae In Taenia, 10 parts of Fructus Aurantii Immaturuss, 10 parts of Radix Polygalaes, 10 parts of Rhizoma Acori Graminei, 6 parts of Semen Sinapis Albaes, 10 parts of Rhizoma Chuanxiongs; Rhizoma Pinelliae (processed with Rhizoma Zingiberis Recens), Caulis Bambusae In Taenia, Fructus Aurantii Immaturus, Radix Polygalae, Rhizoma Acori Graminei, Semen Sinapis Albae, Rhizoma Chuanxiong water are decocted 3 times, each 20 minutes, make medicinal liquid leach as far as possible, merge water extraction liquid, being evaporated to density is the thick paste of 1.0-3.0 (50 ℃).
11, get by weight 10 parts of Rhizoma Pinelliae (processed with Rhizoma Zingiberis Recens)s, 12 parts of Caulis Bambusae In Taenia, 10 parts of Fructus Aurantii Immaturuss, 10 parts of Radix Polygalaes, 10 parts of Rhizoma Acori Graminei, 6 parts of Semen Sinapis Albaes, 6 parts of Radix Ginsengs; Rhizoma Pinelliae (processed with Rhizoma Zingiberis Recens), Caulis Bambusae In Taenia, Fructus Aurantii Immaturus, Radix Polygalae, Rhizoma Acori Graminei, Semen Sinapis Albae, Radix Ginseng water are decocted 3 times, each 20 minutes, make medicinal liquid leach as far as possible, merge water extraction liquid, being evaporated to density is the thick paste of 1.0-3.0 (50 ℃).
12, get by weight 10 parts of Rhizoma Pinelliae (processed with Rhizoma Zingiberis Recens)s, 12 parts of Caulis Bambusae In Taenia, 10 parts of Fructus Aurantii Immaturuss, 10 parts of Rhizoma Chuanxiongs, 6 parts of Radix Ginsengs; Rhizoma Pinelliae (processed with Rhizoma Zingiberis Recens), Caulis Bambusae In Taenia, Fructus Aurantii Immaturus, Rhizoma Chuanxiong, Radix Ginseng water are decocted 3 times, each 20 minutes, make medicinal liquid leach as far as possible, merge water extraction liquid, being evaporated to density is the thick paste of 1.0-3.0 (50 ℃).
13, get by weight 10 parts of Rhizoma Pinelliae (processed with Rhizoma Zingiberis Recens)s, 12 parts of Caulis Bambusae In Taenia, 10 parts of Fructus Aurantii Immaturuss, 12 parts, Radix Rehmanniae Preparata, 20 parts of Radix Polygoni Multiflori Preparata, 10 parts of Radix Polygalaes, 10 parts of Rhizoma Acori Graminei, 6 parts of Semen Sinapis Albaes, 10 parts of Rhizoma Chuanxiongs; Rhizoma Pinelliae (processed with Rhizoma Zingiberis Recens), Caulis Bambusae In Taenia, Fructus Aurantii Immaturus, Radix Rehmanniae Preparata, Radix Polygoni Multiflori Preparata, Radix Polygalae, Rhizoma Acori Graminei, Semen Sinapis Albae, Rhizoma Chuanxiong water are decocted 3 times, each 20 minutes, make medicinal liquid leach as far as possible, merge water extraction liquid, being evaporated to density is the thick paste of 1.0-3.0 (50 ℃).
14, get by weight 10 parts of Rhizoma Pinelliae (processed with Rhizoma Zingiberis Recens)s, 12 parts of Caulis Bambusae In Taenia, 10 parts of Fructus Aurantii Immaturuss, 12 parts, Radix Rehmanniae Preparata, 20 parts of Radix Polygoni Multiflori Preparata, 10 parts of Radix Polygalaes, 10 parts of Rhizoma Acori Graminei, 6 parts of Semen Sinapis Albaes, 6 parts of Radix Ginsengs; Rhizoma Pinelliae (processed with Rhizoma Zingiberis Recens), Caulis Bambusae In Taenia, Fructus Aurantii Immaturus, Radix Rehmanniae Preparata, Radix Polygoni Multiflori Preparata, Radix Polygalae, Rhizoma Acori Graminei, Semen Sinapis Albae, Radix Ginseng water are decocted 3 times, each 20 minutes, make medicinal liquid leach as far as possible, merge water extraction liquid, being evaporated to density is the thick paste of 1.0-3.0 (50 ℃).
15, get by weight 10 parts of Rhizoma Pinelliae (processed with Rhizoma Zingiberis Recens)s, 12 parts of Caulis Bambusae In Taenia, 10 parts of Fructus Aurantii Immaturuss, 12 parts, Radix Rehmanniae Preparata, 20 parts of Radix Polygoni Multiflori Preparata, 10 parts of Rhizoma Chuanxiongs, 6 parts of Radix Ginsengs; Rhizoma Pinelliae (processed with Rhizoma Zingiberis Recens), Caulis Bambusae In Taenia, Fructus Aurantii Immaturus, Radix Rehmanniae Preparata, Radix Polygoni Multiflori Preparata, Rhizoma Chuanxiong, Radix Ginseng water are decocted 3 times, each 20 minutes, make medicinal liquid leach as far as possible, merge water extraction liquid, being evaporated to density is the thick paste of 1.0-3.0 (50 ℃).
16, get by weight 10 parts of Rhizoma Pinelliae (processed with Rhizoma Zingiberis Recens)s, 12 parts of Caulis Bambusae In Taenia, 10 parts of Fructus Aurantii Immaturuss, 10 parts of Radix Polygalaes, 10 parts of Rhizoma Acori Graminei, 6 parts of Semen Sinapis Albaes, 10 parts of Rhizoma Chuanxiongs, 6 parts of Radix Ginsengs; Rhizoma Pinelliae (processed with Rhizoma Zingiberis Recens), Caulis Bambusae In Taenia, Fructus Aurantii Immaturus, Radix Polygalae, Rhizoma Acori Graminei, Semen Sinapis Albae, Rhizoma Chuanxiong, Radix Ginseng water are decocted 3 times, each 20 minutes, make medicinal liquid leach as far as possible, merge water extraction liquid, being evaporated to density is the thick paste of 1.0-3.0 (50 ℃).
17, get by weight 10 parts of Rhizoma Pinelliae (processed with Rhizoma Zingiberis Recens)s, 12 parts of Caulis Bambusae In Taenia, 10 parts of Fructus Aurantii Immaturuss, 12 parts, Radix Rehmanniae Preparata, 20 parts of Radix Polygoni Multiflori Preparata, 10 parts of Radix Polygalaes, 10 parts of Rhizoma Acori Graminei, 6 parts of Semen Sinapis Albaes, 10 parts of Rhizoma Chuanxiongs, 6 parts of Radix Ginsengs; Rhizoma Pinelliae (processed with Rhizoma Zingiberis Recens), Caulis Bambusae In Taenia, Fructus Aurantii Immaturus, Radix Rehmanniae Preparata, Radix Polygoni Multiflori Preparata, Radix Polygalae, Rhizoma Acori Graminei, Semen Sinapis Albae, Rhizoma Chuanxiong, Radix Ginseng water are decocted 3 times, each 20 minutes, make medicinal liquid leach as far as possible, merge water extraction liquid, being evaporated to density is the thick paste of 1.0-3.0 (50 ℃).
EXPERIMENTAL EXAMPLE
The impact experiment of the embodiment 5 medicine of the present invention plan AD rat model learning and memory level that injection is induced on IBO
1 experiment material
1.1 animal
60 of Wistar rats, male and female half and half, body weight 300 ± 50g, provided by Tongji Medical College, Huazhong Science and Technology Univ.'s Experimental Animal Center.Under the laboratory steady-state conditions, raise after one week, with the scalping of Y type water maze method, in all 15 tests, correct number of times reaches more than 12 times the person for qualified.
1.2 medicine and preparation
Preparation Example 2 medicines.Piracetam (piracetam) tablet, (by Northeast Pharmaceutical Factory production, lot number: 990618), water pulverizes and adds distilled water and be made into the 10mg/ml suspension 400mg/ sheet.IBO (Sigma company).
1.3 main agents and instrument
The automatic experimental record instrument of treated rats in Morris water maze performance, the program control shuttle box of rat, being the Chinese Academy of Medical Sciences provides.
2 experimental techniques
2.1AD model manufacturing
After animal via pentobarbital sodium (40mg/kg) intraperitoneal injection of anesthesia, be fixed in brain solid positioner, the scalp preserved skin, routine disinfection field of operation skin, aseptic lower operation, do the 2cm otch along the calvarium center line, with wet cotton balls, separates periosteum, uses the dental burr sphenotresia.With reference to the three-dimensional elements of a fix of brain, ear bar+0.2, center line is outer 0.3, under skull 7.0, advances miniature injector entry needle, every deep propylhomoserin (IBO) 0.5 μ l (1ug/1ul) of injection goose cream, continue 2-3min inject time, with non-proliferation.Slowly after the withdraw of the needle, with hydrogen peroxide, clean wound surface, sandwich rubber seals the skull hole, sews up scalp, and intramuscular injection penicillin 100,000 u are anti-to be infected.Animals survived was injected with the infringement of same coordinate row offside after one week.Blank group is got same coordinate, injection equivalent normal saline after inserting needle.
2.2 grouping and administration
Rat is divided into 6 groups at random: normal group, sham operated rats (blank group), model group, piracetam group (abbreviation matched group), invention medicine low dose group (the abbreviation amount is hanged down group), invention medicine high dose group (abbreviation is measured high group).Every group 10.After modeling 3 days again screening once, in all 15 tests correct number of times than modeling before decline be the modeling success.Start administration after success, each administration group all adopts the administration of gavage method, and high dose group gives invention medicine water decoction by 20g/kg.d.Low dose group gives invention drug dilution liquid by 10g/kgd, and matched group gives the piracetam suspension by 100mg/kgd, and normal group, blank group, model group all give the normal saline gavage of isometric(al) (1ml/100gd); Continuous 28 days.All animal vias are crossed processing to before getting brain, and normal group, blank group, model group, matched group, the low group of amount, the high group of amount rat are survived respectively 10,9,7,8,9 and 9.
2.3Morris water maze test
The temperature control of a orientation navigation test water, 25 ℃ of left and right, morning and afternoon every day each 4 times, divides four quadrants to put into water towards pool wall rat, records its time that searches out platform in 2min (escape latency).If rat is not found platform in 2min, with hands, draw it to platform, allow rat stop 10s, then put back in cage, calculate 120s its incubation period.Last 5d.
B space exploration is tested 6d and is removed platform, and optional 1 place of entry of rat is put into, and records the number of times of crossing over the original platform position in its 2min.Two tests are also recorded rat and are always accounted for apart from percentage ratio in the swimming of platform quadrant distance, and pool wall 10% and 30% zone swimming are apart from percentage ratio.
2.4 rat shuttle box experiment
After administration finishes, carry out the shuttle box test.Experiment is divided into training and official testing two parts. training period 4d, 5d carries out official testing, setting electric stimulating time 15s, buzzing time 5s, interval time 2min, electric current 20mA trains 20 circulations, once a day at every turn.
2.5 all results of statistical procedures are with mean ± standard deviation
Figure BSA00000451126400081
mean.Adopt SPSS 10.0 statistical package statistical analysiss.Comprise the t check.
3 results
3.1 the general performance of animal
Relatively, animal is owed gloss by hair for model group animal and matched group, and diet reduces, and movable the minimizing takes a turn for the better to some extent after the treatment of Chinese medicine and western medicine gavage, and dead rat wound all occurs suppurating waiting and infects sign.
3.2Morris water maze test
3.2.1 the rat orientation navigation is tested to preclinical impact
From preclinical impact, two groups of rats train descend rapidly a few days ago incubation period, since the 3rd day, tend to be steady, and substantially maintain a constant level.For the analytical information acquisition capability, measured total incubation period and the training end rear incubation period of 3 days of training in 5 days, the results are shown in Table 2-1.
3.2.2 the impact on the test of rat orientation navigation and space search test learning and memory
The swimming distance of rat orientation navigation test and space search test platform quadrant always accounts for apart from percentage ratio, and swimming apart from percentage ratio in pool wall 10% and 30% zone, the results are shown in Table 2-2,3.
3.2.3 rat is striden across to the impact of original platform position number of times
Respectively organize rat in the space search test and stride across original platform position number of times relatively in Table 2-4.Above result all unanimously shows, normal group, sham operated rats, the low group of amount, the high group of amount rat can rely on spatial cues to find position of platform, and its movement locus is positioned at most the original platform quadrant, matched group more at the original platform quadrant adjacent left and right sides quadrant find.But the model group rat is substantially around pool wall swimming, less trip is near original platform, and its movement locus is and is randomly distributed among all quadrants.No matter from the reflection space learning, remember the orientation navigation test of acquisition capability, or test from the space search of reflection information storage ability, matched group, amount are hanged down group, the high group of amount relatively has significant difference (P<0.01) with other three groups.Measure high group than the obvious better ability (P<0.05) that shows of matched group.Yet indices does not all reach the level of normal group, sham operated rats.
The impact of table 2-1 medicine of the present invention on rat orientation navigation test average latency
Figure BSA00000451126400091
Figure BSA00000451126400092
Annotate: with the normal group ratio △ △p<0.01 and model group ratio ▲ ▲p<0.01
With the matched group ratio ★ ★p<0.01
The impact of table 2-2 medicine of the present invention on rat orientation navigation test learning and memory
Figure BSA00000451126400093
Figure BSA00000451126400094
Annotate: with the normal group ratio △p<0.05 △ △p<0.01 and model group ▲ ▲p<0.01
With the matched group ratio ★p<0.05 ★ ★p<0.01
The impact of table 2-3 medicine of the present invention on rat space search test learning and memory
Figure BSA00000451126400095
Figure BSA00000451126400096
Annotate: with the normal group ratio △ △p<0.01 and model group ratio ▲ ▲p<0.01
With the matched group ratio ★p<0.05 ★ ★p<0.01
Table 2-4 medicine of the present invention is the impact through original platform position number of times on the test of rat space search
Figure BSA00000451126400097
Figure BSA00000451126400098
Annotate: with the normal group ratio △ △p<0.01 and model group ratio ▲ ▲p<0.01
With the matched group ratio ★p<0.05
3.3 rat shuttle box experiment
3.3.1 active avoidance response rate
In the training of first 3 days, treating each group, to be electrically shocked number of times less, but unknown significance difference between 3 groups of active avoidance response rates.Since the 4th day, model group still maintained former level substantially, shows obvious behavior disorder.Other active avoidance response rates of respectively organizing rat obviously increase, and with model group, significant difference (P<0.01) are relatively arranged, but do not reach yet the normal group level.The low group of amount compares there was no significant difference (P>0.05) with matched group.To the 5th day official testing result, with above-mentioned consistent, just difference was more obvious.The results are shown in Table 2-5.
Table 2-5 medicine of the present invention is initiatively avoided the impact of accuracy on rat
Figure BSA00000451126400101
Figure BSA00000451126400102
Annotate: with the normal group ratio △p<0.05 △ △p<0.01 and model group ratio ▲ ▲p<0.01
With the matched group ratio ★ ★p<0.01
3.3.2 the passive escape time, the passive escape time of matched group, the low group of amount, the high group of amount, than the obvious shortening of model group (P<0.01), continued to shorten (P<0.01) on the 5th day since the 4th day.Model group continues the passive escape time that keeps long.It is consistent with the active avoidance response rate that each organizes result.In Table 2-6
The impact of table 2-6 medicine of the present invention on the passive escape of the rat time
Figure BSA00000451126400103
Figure BSA00000451126400104
Annotate: with the normal group ratio △ △p<0.01 and model group ratio ▲ ▲p<0.01
With the matched group ratio ★ ★p<0.01
4 discuss
Experimental result shows, the above-mentioned detection index of medicine high and low dose group treated rats in Morris water maze performance of the present invention all obviously is better than model group, and shows as the trend that the high dose group outline is better than low dose group; Show that the learning and memory function that medicine of the present invention causes the AD rat model of Meynert core damage to IBO improves significantly.Experimental result also shows: medicine high and low dose treatment group of the present invention also has a certain distance to the improvement effect of AD learning and memory in rats and normal group and sham operated rats, illustrate that the invention medicine can only partly improve the ability of learning and memory of AD, and can not make it recovery from illness, this is also similar with embodiment 4 clinical manifestations.
The impact experiment of the plan AD rat cholinergic system that embodiment 6 medicine of the present invention is induced IBO
1 material
1.1 animal, medicine and preparation are with embodiment 5.
1.2 detectable and instrument
ChAT, Ach radioimmunoassay kits are purchased from Beijing Zhong Shan biotechnology research institute.
2 methods
2.1 modeling, grouping and administration are with embodiment 5.
2.2 rat hippocampus district ChAT and Ach determination of activity
Separate hippocampal tissue, weigh, homogenate, make 10% homogenate.The employing radioimmunology carries out, active ρ mol number (the ρ mol Ach/minmg brain) expression that catalyzes and synthesizes Ach with ChAT in every milligram of weight in wet base cerebral tissue per minute of ChAT, the Ach that the Ach activity produces with every milligram of weight in wet base cerebral tissue means (ρ mol/mg brain).Product description is shown in concrete operations.
2.3 statistical procedures
All results are with mean ± standard deviation
Figure BSA00000451126400111
mean.Adopt SPSS 10.0 statistical package statistical analysiss.
3 results
3.1 the impact of medicine of the present invention on hippocampus ChAT activity.
This measuring respectively organize the ChAT activity of rat hippocampus, as can be seen from Table 7, model group hippocampus ChAT compares with each group all and significantly to reduce (P<0.01), measure low, measure high group and control rats hippocampus ChAT is active with model group, increase (all P<0.01) in various degree relatively.Measure high group and its activity of the low group of amount and be respectively model group 1.5 and 1.3 times.The ChAT in the high group of amount rat hippocampus district is close to blank group of level, but does not reach yet the normal group level.The results are shown in Table 3-1.
The impact of table 3-1 medicine of the present invention on hippocampus ChAT activity
Figure BSA00000451126400112
Figure BSA00000451126400113
Annotate: with the normal group ratio △p<0.05 △ △p<0.01 and model group ratio ▲ ▲p<0.01
With the matched group ratio ★ ★p<0.01
3.2 the impact of medicine of the present invention on rat hippocampus district Ach activity
This measuring respectively organize the Ach activity of rat hippocampus, result shows, model group hippocampus Ach compares remarkable minimizing (P<0.01) with each group.Measure low, high group and control rats, hippocampus Ach activity has increase (P<0.01) in various degree.Measure high group and its activity of the high group of amount and be respectively model group 1.6 and 1.9 times.The Ach that measures low, high group rat sea area raises, and than matched group, there were significant differences (P<0.05～0.01), and the high group of amount has approached normal group level (P<0.01).The results are shown in Table 3-2
The impact of table 3-2 medicine of the present invention on rat hippocampus district Ach activity
Figure BSA00000451126400121
Figure BSA00000451126400122
Annotate: with the normal group ratio △ △p<0.01 and model group ratio ▲ ▲p<0.01
With the matched group ratio ★p<0.05 ★ ★p<0.01
4 discuss
Medicine of the present invention can improve AD rat model Hippocampus CHAT activity, thereby improves Ach activity in cerebral tissue, reaches the purpose of improving learning and memory, and the invention medicine is described, and to central cholinergic system, infringement has repair.Analyze its mechanism, the invention medicine may increase the memory synapse of rat downright bad marginal zone because modeling damages, and the number of cholinergic synapse, increase its density, thereby makes the remaining larger effect of neuron performance; Make the memory synapse gap turn narrow, accelerated the conduction function between synapse; Simultaneously, have the bypass of some nerve conductions in brain, medicine of the present invention may cause neural impulse, through peripheral nervous, conducts to nervus centralis (brain), bypass through working less at ordinary times stimulates cerebral cortex, thereby causes the change of downright bad marginal zone synapse number and structure; Postsynaptic density matter thickness is thickened, may cause that the change of its molecular configuration is relevant with the phosphorylation process of some enzyme and substrate protein thereof, this may be the morphological base that function changes.
Experimental result shows, medicine of the present invention can improve AD rat model Hippocampus ChAT activity, suppresses too high AchE activity, thereby improves Ach activity in cerebral tissue, reaches the purpose of improving learning and memory.The invention medicine is described, and to central cholinergic system, infringement has protective effect.
The protective effect of plan AD neurons of rats and mechanism thereof that embodiment 7 medicine of the present invention is induced IBO
1 material
1.1 animal, medicine and preparation are with embodiment 5.
1.2 detectable and instrument
INOS, NGF and nNOS monoclonal antibody, P38 in situ hybridization test kit SABC test kit SABC, DAB colour reagent box, all purchased from Wuhan Boster Biological Technology Co., Ltd... flow cytometer (U.S. company BD, FACS) test.
2 methods
2.1 modeling, grouping and administration are with embodiment 5.
2.2 neuronal apoptosis detects
Fluidic cell detects---and the propidium iodide staining, the collection and treatment hippocampal cell, in centrifuge tube, is counted as 1 * 10 6cell, 800～1000r/min, centrifugal 5min, use blank re-suspended cell, centrifugal, abandon supernatant, after fixedly spending the night with 4 ℃ of 1ml 70% ethanol, centrifugal, 800r/min, 8min, abandon supernatant, and PBS washes, centrifugal again, retain 0.2ml liquid, add RNA enzyme 10 μ l (final concentration is 50g/l) and shake up, 37 ℃ of 45min.After adding propidium iodide (Promideiodine PI) 50 μ l (final concentration is 50g/l), add PBS to 0.5ml, shake up.4 ℃ of 1h that keep in Dark Place.The 300 orders nylon wire that sieves filters, flow cytometer (U.S. company BD, FACS) test.
2.3 rat hippocampus district P38 detects
Adopt hybridization in situ technique, concrete experimental implementation is carried out with reference to description.
2.4 the detection of hippocampus NOS, NGF
After administration finishes, each organize rat all with 4% paraformaldehyde, pour in fixing cerebral tissue, conventional paraffin embedding, 20 of cortex and the thick serial section of Hippocampus position 4 μ m, get 1 every 4.Adopt the SABC Immunohistochemical Staining to detect, product description is shown in concrete operations.
2.5 image analysis processing
Every rat got 5 sections at random, selects at random respectively each 4 visual field statistics n NOS positive cell numbers of right side cortex and Hippocampus under light microscopic, calculates the mean in each visual field.
2.6 all testing results of statistical procedures are with mean ± standard deviation
Figure BSA00000451126400131
mean.Adopt SPSS 10.0 statistical package statistical analysiss.
3 results
3.1 the impact of medicine of the present invention on rat cortex and hippocampus nNOS expression
Under light microscopic, visible positive material accumulates in endochylema and projection, and painted cell size does not wait, painted deep mixed, is mostly oval, and minority is circular.Model group nNOS positive cell number is starkly lower than normal group and blank group (P<0.01), and cell is painted very shallow.The expression of amount high group rat cortex and Hippocampus Nei Ge district nNOS cell is similar to normally, and cell is painted darker.The low group of amount and matched group nNOS positive cell number also have raising in various degree, the results are shown in Table 4-1.
The impact of table 4-1 medicine of the present invention on cortex and hippocampus nNOS positive cell number
Figure BSA00000451126400132
Figure BSA00000451126400133
Annotate: with the normal group ratio △ △p<0.01 and model group ratio ▲p<0.05 ▲ ▲p<0.01
With the matched group ratio ★p<0.05
3.2 the impact of medicine of the present invention on rat cortex and hippocampus iNOS expression
Microscopic observation iNOS positive material accumulates in endochylema and projection, is pale brown dyeing, and painted cell size does not wait, out-of-shape.Result is as table 4-2, and normal group and blank group iNOS seldom express; Model group iNOS positive cell number is apparently higher than normal group and blank group (P<0.01).The expression of the high group of amount rat iNOS cell is significantly fallen (P<0.01) than model group.The low group of amount and matched group iNOS positive cell number, also have and improve in various degree (P<0.01), but there is no difference (P>0.05) between two groups.The high group of amount reduces significantly (P<0.01).
The impact that table 4-2 medicine of the present invention is expressed iNOS
Figure BSA00000451126400142
Annotate: with the normal group ratio △p<0.05 △ △p<0.01 and model group ratio ▲ ▲p<0.01
With the matched group ratio ★ ★p<0.01
3.3 the impact of medicine of the present invention on rat cortex and hippocampus P38 expression
The demonstration of this experimentation, model group hippocampus and the P38 of cortical areas obviously reduce.Normal group hippocampus and cortical areas all have more widely expresses, and can see diffuse synaptic protein and distribute, and dyes darker.Difference tool statistical significance (P<0.01).Each treatment group Meynert core and the synaptic protein compared with normal group minimizing of limit week thereof, but be significantly increased than model group, significance (P<0.01), to measure high group the most obviously (P<0.01).The results are shown in Table 4-3.
The impact of table 4-3 medicine of the present invention on AD rat model cortex and hippocampus P38 expression
Figure BSA00000451126400143
Figure BSA00000451126400144
Annotate: with the normal group ratio △ △p<0.01 and model group ratio ▲ ▲p<0.01
With the matched group ratio ★ ★p<0.01
3.4 the impact of medicine of the present invention on rat cortex and hippocampus NGF expression
As it is more to show 4-4 normal group, blank group rat cortex and the distribution of hippocampus NGF positive neuron, model group NGF positive neuron quantity is compared remarkable minimizing (P<0.01) with each group.Measure low, measure high and control rats NGF and express increase (P<0.01) is in various degree arranged.Measure high, its positive neuron quantity of the low group of amount and be respectively model group 6.5 and 4.5 times.The high group of amount rat NGF positive neuron approaches normal group level (P<0.01), and its expression increasing action to rat cortex and hippocampus NGF is the most obvious.
The impact of table 4-4 medicine of the present invention on rat NGF
Figure BSA00000451126400145
Annotate: with the normal group ratio △ △p<0.01 and model group ratio ▲ ▲p<0.01
With the matched group ratio ★ ★p<0.01
3.5 the impact of medicine of the present invention on rat cortex and hippocampus neuronal apoptosis rate
Flow cytometry is found, occurs the peak type of hypodiploid cell mass on the left of model group neurocyte G1 peak.The feature of this feature during with apoptosis conforms to, and illustrates that IBO can make neuronal apoptosis.Normal group and rats in sham-operated group neurocyte loseed the hypodiploid peak are arranged in the front G1 phase, showed that nothing or minute quantity phenomena of apoptosis occur.The high group of amount neurocyte has a little hypodiploid peak as seen in the front G1 phase, shows and only has a small amount of apoptosis, and damage has protective effect to IBO to show high dose invention medicine.The neurocyte of the low group of amount and matched group also has a little hypodiploid peak as seen in the front G1 phase, but trial of strength Gao Zugao, demonstration also has a small amount of apoptosis, and damage also has protective effect to IBO to show low dosage invention medicine.With normal group, compare, the model group apoptosis rate obviously increases (P<0.01); With model group relatively, matched group, measure high group, the low group of amount apoptosis rate obvious minimizing (P<0.01) also arranged, the apoptosis that medicine energy part antagonism IBO of the present invention induces is described, best with the high dose effect.The results are shown in Table 4-5.
The impact of table 4-5 medicine of the present invention on neuronal apoptosis
Figure BSA00000451126400151
Figure BSA00000451126400152
Annotate: with the normal group ratio △p<0.05 △ △p<0.01 and model group ratio ▲ ▲p<0.01
With the matched group ratio ★ ★p<0.01
4 discuss
This experiment confirms, the intervention effect of medicine of the present invention to the infringement of experimental rat cholinergic is by regulating the cholinergic neurotransmitter level, and the factors such as synaptic protein content, inhibition neuronal apoptosis that improve realize.Below its concrete mechanism of action:
4.1 improve rat brain nNOS neuron and reduce iNOS content and activity
This experimental result shows, medicine of the present invention can make the quantity of rat cerebral cortex and hippocampus nNOS cell increase, and reduce the expression of iNOS, and local neuron is impaired lighter.Medicine energy inhibition rat cerebral cortex of the present invention and hippocampus NOS neuronal damage, the low group of the high group trial of strength of amount better effects if.The effect of pointing out thus medicine of the present invention to improve learning and memory in rats, be wherein by improving rat brain nNOS neuronal activity, suppressing the expression of iNOS, and protection local neuron realizes.
4.2 promote new synapse to form, increase rat synapse quantity
Experiment shows: medicine of the present invention can effectively promote new synapse to form, and increases rat model synapse quantity, improves the learning and memory level, and this shows that its effect to Hippocampus synaptic plasticity mechanism may be that it improves one of neurobiology basis of learning and memory.Previously having the increase that studies confirm that cholinergic fiber to can be synapse provides material base.In addition, may be also to improve neuroprotective, promote that the synthetic increase of synapse auxin is relevant, this remains further to be studied.
4.3 suppress the cranial nerve cell apoptosis
This experimental result shows, rat model cranial nerve cell apoptosis obviously increases, and medicine of the present invention can obviously reduce the cranial nerve cell apoptosis, illustrates that piracetam and medicine of the present invention have the effect that suppresses the cranial nerve cell apoptosis, best with the high dose effect.Its inhibited apoptosis effect of medicine of the present invention may be by removing free radical in neurocyte, and has played the effect of neuroprotective cell, has reached the effect of inhibited apoptosis.
4.4 in the raising cerebral tissue, NGF expresses
From aforementioned each experimental result, medicine of the present invention all improves significantly to information obtaining ability and the information storage ability of experimental rat, and it is consistent with its action learning level respectively to organize rat endogenous NGF expression, illustrates that the raising of rat model endogenous NGF content and experimental rat learning and memory level have substantial connection.Infer that this effect may be because medicine of the present invention has improved endogenous NGF content, NGF promotes the cholinergic fiber quantity in innervation district to increase, and promotes single Nerve Terminals In The Human Skin to form more synapse, and this contributes to the rapid recovery of function after neuronal damage.Its concrete mechanism of action remains further to be inquired into.The impact of embodiment 8 medicine of the present invention on cultured hippocampus neuronal apoptosis and calcium ion
1 material
1.1 reagent
Poly-D-lysine (poly-l-lysine), the Sigma product; Calf serum, Sijiqing Bioengineering Material Inst., Hangzhou City's product; Complete DMEM culture medium, the Gibco product, contain NaHCO in 1L DMEM culture fluid 33.7g, penicillin G sodium 100U/ml, streptomycin 100 μ g/ml and 10% calf serum, pH7.4; Trypsin, the Sigma product; Pidolidone, Shanghai uncle's biotechnology difficult to understand company product; MTT (dimethylthiazole diphenyl Thiazolyl blue tetrazolium bromide salt), the Fluka product; Nimodipine, Tianjin pharmaceutical factory of central authorities product; Other reagent is commercially available analytical pure.Annexin-V-FITC apoptosis detection kit is purchased from Beijing Bao Sai Bioisystech Co., Ltd
1.2 medicament composing prescription of the present invention and preparation
With embodiment 5.
1.3 the preparation containing medicine rat blood serum of the present invention and blank serum
Get 10 of clean level Wistar rats, 190-220g, provided by Tongji Medical College, Huazhong Science and Technology Univ.'s animal center.Be divided at random two groups of A, B, 5 every group.
The A group is fed medicine decocting liquid of the present invention, compare scaling method by the people with the Mus body surface area, calculate to obtain the dosage of rat every day, press again the Sero-pharmacological experiment method, the serum donor be take effective dose (as the clinical 8-10 at people's dosage doubly measures) administration of clinical application amount or block mold animal, therefore the every daily dose of rat is 2g/100g, be 1ml/100g, medicine decocting liquid of the present invention 2 administrations every day (morning 8:00-9:00, afternoon 3:00-4:00) continuous 7 days, 2h after last administration (before administration, 12h prohibits drinking-water), with after pentobarbital anesthesia, blood is got from rat heart by sterile working, centrifuging and taking obtains serum, 5 rat blood serums are mixed, 56 ℃, 30 minutes inactivation treatment.With 0.22 μ m filtering with microporous membrane degerming ,-20 ℃ save backup.Prepared by this kind of method is containing medicine rat blood serum of the present invention.By serum with 20%FBS DMEM/F12 be diluted to respectively 10%, the Contained Serum of 20% 2 kind of variable concentrations.
The B group is fed 0.9% continuous normal saline 7 days, administration time, method and obtain serological method with the A group.Prepared by this kind of method is blank serum.By same method, blank serum is diluted to the Contained Serum of 10% concentration.
1.4 key instrument
2003 type CO 2incubator (U.S. Sheldon company), aseptic operating platform (Suzhou Decontamination Equipment Plant), LG-10 centrifuge (Beijing Medical Centrifugal Machine Factory), thermostatic water tank, enzyme-linked immunosorbent assay instrument (East China Electronics Co., Ltd pipe factory), FACSCaliber flow cytometer (U.S. company BD), CK-2 type inverted phase contrast microscope (Japan), BX-60 fluorescence microscope (Japan), RF-5000 spectrofluorophotometer (Japan), electronic balance, aseptic centrifuge tube, 96 well culture plates, 24 well culture plates.
1.5 the preparation of main solution
PBS liquid: NaCL 8g/L, KCL 0.20g/L, NaHPO 4h 2o 1.56g/L, KH 2pO 40.20g/L packed powder is melted in appropriate distilled water, stir, make it fully to melt, in the liquid subpackage saline bottle, through the disinfection with high pressure steam sterilizing, with aseptic NaHCO 3liquid is regulated PBS liquid, and pH value to 7.2～7.4,4 ℃ of Refrigerator stores are standby.
The DMEM/F12 culture fluid: the triply distilled water by preparation adds warm water to 16-30 ℃; Packed DMEM/F12 dry powder is melted in water, and the dissolving of stirring order adds; Appropriate NaHCO 3; With aseptic NaHCO 3liquid is regulated medium pH value to 7.2～7.4; Filter, pH may influenced use 0.22 μ m or is less than this microporous filter membrane and filters when filtering, packing, be stored in the refrigerator of 4 ℃ standby.
MTT solution: take 250mgMTT, put into small beaker, add 50mlPBS liquid (0.01mol/L, pH7.4) and stir on magnetic stirring apparatus 30 minutes, filter with 0.22 μ m microporous filter membrane, 4 ℃ of packing save backup, effective in two weeks.
The preparation of Annexin V-FITC/PI: prepared by operation instructions.
2 methods
2.1 the former culture of rat hippocampal neurocyte
Get after newborn 1 day rat puts into 75% ethanol sterilization, put in superclean bench, immigration is placed with in the beaker of PBS, soak a moment, taking-up is put and is filled in D-Hanks liquid plate, cut off skin of head, peel off cerebral cortex and be placed in rapidly ice-cold D-Hanks liquid, reject meninges and blood vessel, getting Rat hippocampus moves in the bottle that fills a small amount of D-Hanks liquid, with eye scissors, tissue is cut into to rotten shape, add 0.25% trypsin to cover and organize fragment, 37 ℃ of digestion 30min, piping and druming disperses cerebral tissue repeatedly, the DMEM culture medium added containing 10% calf serum stops digestion, standing, sucking-off upper strata cell suspension is in the graduated centrifuge tube of sterilization, the centrifugal 10min of 800rpm, abandon supernatant, the DMEM that precipitation adds containing 10% calf serum makes cell suspension, be inoculated in 24 orifice plates that are coated with poly-l-lysine in advance, put 37 ℃ of 5%CO 2incubator in cultivate, change liquid after 24h once, within later every 3 days, change liquid once, within the 7th day, change into containing the culture fluid of cytosine arabinoside (final concentration is 5 μ g/ml) to suppress the propagation of the non-neurocytes such as glial cell, change normal culture medium after 48h into, go to drug study.
2.2 the growing multiplication effect of medicine Contained Serum of the present invention to hippocampal neurons.
Select cell density approaches, growth conditions is consistent culture hole for experiment.Cell is divided into immediately: 1, culture fluid group, 2,5% medicine Contained Serum group of the present invention, 3, medicine Contained Serum low dose group of the present invention, 4, invention medicine Contained Serum high dose group, 5, blank serum group.Suck original fluid, use sugar-free Earle ' s liquid to swing and wash twice gently, every hole adds sugar-free Earle ' s liquid 1ml, effect 30min, and medicine Contained Serum group of the present invention adds respectively the medicine Contained Serum of the present invention that final concentration is 5%; Blank serum group add final concentration be 10% without medicine serum.37 ℃, 5%CO 2cultivate 24h.Add MTT (final concentration is 0.5mg/ml), continue to cultivate 4h, suck culture fluid, every hole adds the dimethyl sulfoxide of 200 μ l 100%, after blue particle in hole dissolves fully, measures optical density value (OD) at the 490nm place with enzyme-linked immunosorbent assay instrument.
Figure BSA00000451126400171
2.3 medicine Contained Serum of the present invention is on the apoptotic impact of glutamic acid inducing neural cell injury
Choose cell density approaches, growth conditions is consistent culture hole for experiment.Cell is divided into immediately: 1, model group 2, nimodipine group, 3,10% blank serum group, 4,10% medicine Contained Serum group of the present invention, 5,20% medicine Contained Serum group of the present invention.Suck original fluid, use sugar-free Earle ' s liquid to swing and wash twice gently, every hole adds sugar-free Earle ' s liquid 1ml, effect 30min, the nimodipine group adds the nimodipine that final concentration is 5 * 10-6mol/L, and medicine Contained Serum of the present invention hangs down agent, a large amount group adds respectively the invention medicine Contained Serum that final concentration is 5%, effect 20min, the glutamic acid (Glu) that adds again final concentration 500 μ mol/L, effect 30min; Normal group only adds the Glu that final concentration is 500 μ mol/L; Blank serum group add final concentration be 5% without medicine serum, and Normal group does not add Glu, with D-Hanks, processes 30min.Last each group sucks liquid, with D-Hanks liquid, swings and washes twice gently, and every hole adds DMEM/F12 culture fluid 1ml, and 37 ℃, 5%CO2 cultivates 24h.By Annexin V-FITC/PI method, detect apoptosis rate on flow cytometer.
2.4 the impact of medicine Contained Serum of the present invention on glutamic acid inducing neural cell injury intracellular calcium
Cell divides into groups, processes, cultivates the same.Get the neurocyte of cultivation after cultivation 24h, with D-Hanks liquid suspension cell, adjust cell concentration 105/ml, add Fura-2 in cell suspension, put 37 ℃, 5%CO 2hatch 50min in incubator, take out cell suspension, with D-Hanks liquid washed cell twice, the Fura-2 in the extracellular fluid of place to go.Use D-Hanks liquid suspension cell, cell concentration is adjusted to 105/ml again, and the survival condition of observation of cell, with spectrofluorophotometer with 340 and the 380nm dual wavelength excite, the 505nm wavelength emission, measure the fluorescence intensity of intracellular free calcium under different experimental conditions, and proofread and correct the cell autofluorescence.According to formula: calcium ion (n mol/L)=Kd (F-F min)/(F max-F) to calcium ion concentration, (F is the fluorescence intensity while not adding Triton-100, F in calculating maxfor maximum fluorescence intensity, F minminimum fluorescence intensity, Kd=224nmol/L).
3 results
3.1MTT testing result
The invention medicine on former culture sea God through cell affect result as shown in Table 5-1, invention medicine Contained Serum high dose group is obvious through Growth of Cells, proliferation function to the sea God, with matched group, significant difference (p<0.05) is relatively arranged.Blank serum group, invention medicine Contained Serum low dose group matched group be there was no significant difference (p>0.05) relatively.
Table 5-1 respectively organizes OD value and Growth of Cells, rate of increase
Figure BSA00000451126400181
Figure BSA00000451126400182
Annotate: compare △ p<0.05 with the culture fluid group
3.2 contain medicine rat blood serum of the present invention to apoptotic variation
As show 5-2 and show, neuronal apoptosis is the highest in model group, blank serum group is taken second place, drug serum group of the present invention is minimum, brain-care is low, high dose group and model group relatively, significant significant difference (P<0.05) is arranged, and blank serum group and model group compare, there was no significant difference (P>0.05).
The impact of table 5-2 medicine rat blood serum of the present invention on neuronal apoptosis
Figure BSA00000451126400183
Figure BSA00000451126400184
Figure BSA00000451126400191
Annotate: △ compares with the culture fluid group, p<0.05
3.3 the impact containing medicine rat blood serum of the present invention on Neuronal Calcium
As show 5-3 and show, neuronal apoptosis is the highest in model group, blank serum group is taken second place, drug serum group of the present invention is minimum, brain-care is low, high dose group and model group relatively, significant significant difference (P<0.05) is arranged, and blank serum group and model group compare, there was no significant difference (P>0.05).
The impact of table 5-3 medicine rat blood serum of the present invention on Neuronal Calcium
Figure BSA00000451126400192
Figure BSA00000451126400193
Annotate: compare △ p<0.05 with the culture fluid group, △ △ p<0.01
4 discuss
4.1 medicine of the present invention can suppress neuronal apoptosis
This experimental result shows that medicine Contained Serum of the present invention can obviously promote neurocyte to increase, also can suppress neural apoptosis.A large amount of achievements in research class Chinese medicine that shows to invigorate blood circulation has the effect of removing interior free yl; so its inhibited apoptosis effect of medicine of the present invention is by removing free radical in neurocyte; and played the effect of neuroprotective cell; reached the effect of inhibited apoptosis; in addition; medicine of the present invention can reduce the Neuronal Calcium overload, also can play the effect of inhibited apoptosis.4.2 medicine of the present invention can alleviate the neurocyte Calcium overload
This experiment adds glutamic acid and causes the intracellular calcium overload on the neurocyte that utilizes cultivation, and utilize the agent of calcium fluorescence detection to detect the effect of invention medicine to the neurocyte Calcium overload, result shows, the invention medicine has obvious inhibitory action to the neurocyte Calcium overload.The effect that shows thus medicine inhibition neuronal apoptosis of the present invention may be relevant with inhibition neurocyte Calcium overload, medicine of the present invention has the antagonism calcium ion to enter neurocyte, alleviate the damage of Calcium overload to cell, reached the effect of inhibited apoptosis.
Embodiment 9 medicine of the present invention is to IBO+A β 25-35induce the impact experiment of intending AD rat model learning and memory level
1 experiment material
1.1 laboratory animal
15 70 of months aged Wistar rats, male and female half and half.Body weight 450 ± 50g, provided by Animal Experimental Study center, Hubei Province.
1.2 medicine, reagent and instrument
Preparation Example 3 medicines, high, medium and low dosage group is equivalent to respectively containing crude drug 2g/ml, 1g/ml and 0.5g/ml.Huperzine A-Zhulin Antun, pharmaceutical factory in Henan all living creatures Pharmacy stock Co., Ltd Henan.The deep propylhomoserin (Ibotenicacid, IBO) of goose cream and A β 25-35all from Sigma company, order.
The automatic experimental record instrument of treated rats in Morris water maze performance, the Chinese Academy of Medical Sciences.Brain solid positioner Beijing large hunting park Science and Technology Ltd.
2 experimental techniques
2.1AD model construction
By A β 25-35peptide section 1mg is dissolved in 100 μ l sterile salines, and concentration is 10 μ g/ μ l.After sealed membrane is sealed, be placed in 37 ℃ and hatch 96h, make its gathering, aging.Get IBO 1mg and be dissolved in wherein, be made into IBO and A β 25-35mixed liquor, matching while using.
Model group: with EXPERIMENTAL EXAMPLE 5 composite model groups.
Sham operated rats: under location, give the disposable injection 1 μ l sterile saline of rat negative contrast.
Normal group: be left intact.
2.2 animal grouping
15 70 of months aged Wistar rats, be divided at random 7 groups, be normal group, sham operated rats (being called for short blank group), model group, Huperzine A-Zhulin Antun treatment group (being called for short the Western medicine group), the high, medium and low dosage treatment group of invention medicine (being called for short high, medium and low amount group), 10 every group.
2.3 administration
Intracerebral injection administration after 2 weeks, dosage is all determined by people and rat body surface area coefficient ratio, with clinical people's medication dose,equivalent, as middle dosage, presses and determines high, medium and low dosage at 4: 2: 1.3 groups, Chinese medicine is respectively to this liquid medicine decocting liquid of rat oral gavage; And Western medicine group gavage Huperzine A-Zhulin Antun suspension; Normal group, blank group, model group all give isometric normal saline gavage.Each organizes rat all by the 1ml/100gd gavage.Continuous 28 days.
2.4Morris water maze laboratory is with embodiment 5.
2.5 statistical disposition
Ditto.
3 results
3.1Morris water maze behavioristics test result
3.1.1 rat average escape latency
In experiment a few days ago, each treated animal is all substantially around pool wall swimming, and less trip is near platform, and its movement locus is and is randomly distributed among all quadrants.But the propelling along with experiment, normal group, rats in sham-operated group rely on spatial cues to find the time of position of platform to shorten gradually, its movement locus also slowly is positioned at the platform quadrant, or adjacent left and right sides quadrant is found at the platform quadrant, and descend its incubation period rapidly.But the model group rat is except only a few can find platform, and the situation while substantially keeping a few days ago, slightly be improved.For the analytical information acquisition capability, measured total incubation period and the training end rear incubation period of 2 days of training in 4 days.From table 6-1, the model group average latency obviously extends than normal group and blank group, and significant difference (P<0.01) is arranged.The average latency of Western medicine group and high, medium and low dosage group obviously shortens than model group, with the model group ratio, significant difference (P<0.01) is arranged.Further between group, relatively find that high dose group and Western medicine group ratio have significant difference (P<0.01).Middle high dose group is than significant difference (P<0.01) is also arranged.Referring to Fig. 1.
First 4 days of table 6-1 rat orientation navigation experiment and latter 2 days average incubation periods result
Figure BSA00000451126400211
Figure BSA00000451126400212
Annotate: with the normal group ratio △ △p<0.01 and model group ratio ▲ ▲p<0.01
With Western medicine group ratio ★p<0.05 ★ ★p<0.01 and middle amount group ratio ☆ ☆p<0.01
The distance 3.1.2 rat is on average swum
Model group rat quadrant percentage ratio compared with normal group and blank group rat obviously shorten.The quadrant percentage ratio of high, middle dosage group is than model group be significantly improved (P<0.01).The quadrant percentage ratio of low dose group is than model group zero difference (P>0.05).Further between group, relatively find that low dose group has significance (P<0.01) in the difference of Western medicine group.There is obvious minimizing (P<0.01) in 20%, 40% zone of high, medium and low dosage group than model group, and, with the increase of dosage, its value is corresponding to be reduced.But between middle high dose group relatively except the also zero difference (P>0.05) in 20% zone.With Western medicine group more also zero difference (P>0.05).There is significant difference (P<0.01) in low dose group 20%, 40% zone than Western medicine group.The results are shown in Table 6-2.
Table 6-2 rat orientation navigation experiment learning and memory result
Figure BSA00000451126400213
Figure BSA00000451126400214
Annotate: with the normal group ratio △p<0.05 △ △p<0.01 and model group ratio ▲ ▲p<0.01
With Western medicine group ratio ★ ★p<0.01 and middle amount group ratio ☆p<0.05
3.1.3 rat search platform variation tendency
From table 6-3, can find out, model group rat space search ability significantly descends, and its quadrant percentage ratio compared with normal group and blank group obviously shorten (P<0.01), and 20%, 40% regional compared with normal group and blank group obviously increase (P<0.01).Zero difference (P>0.05) between normal group and blank group.The quadrant percentage of high, medium and low dosage group obviously raises, and has compared significant difference (P<0.01) with model group.The quadrant percentage ratio of high dose group, 20% zone have significance (P<0.01 or P<0.05) with the difference of Western medicine group.In 20% zone, between high dose group, there were significant differences (P<0.01).Dosage group variant (P<0.05) during 40% zone is low.Referring to Fig. 2.
Table 6-3 rat space search experimental learning memory result
Figure BSA00000451126400215
Figure BSA00000451126400216
Figure BSA00000451126400221
Annotate: with the normal group ratio △ △p<0.01 and model group ratio ▲ ▲p<0.01
With Western medicine group ratio ★p<0.05 ★ ★p<0.01 and middle amount group ratio ☆p<0.05 ☆ ☆p<0.01
3.1.4 the number of times of rat spanning platform
Each group rat is compared through the number of times of original platform position, can find out from table 6-4, the model group rat is respectively organized rat than other and strides across original platform position number of times and obviously descend, and compares with other two groups that there were significant differences (P<0.01).Each group of Chinese medicine is compared with model group, and difference all has statistical significance (P<0.01).Western medicine group and middle high dose group more variant (P<0.01 or P<0.05), with low dosage, than zero difference (P>0.05), high dose and middle dose ratio be variant (P<0.05) also.
Table 6-4 rat through original platform position number of times result (
Figure BSA00000451126400222
inferior)
Figure BSA00000451126400223
Annotate: with the normal group ratio △ △p<0.01 and model group ratio ▲ ▲p<0.01
With Western medicine group ratio ★p<0.05 ★ ★p<0.01 and middle amount group ratio ☆p<0.05
4 discuss
This experimental result shows, the model group rat is going down of basic space orientation memory owing to take vision, to position of platform, can not produce memory, still blindly finds means of escape, therefore its swimming track mainly is distributed in the outer shroud (pool wall 20% zone) without platform.Its space orientation memory ability is subject to grievous injury, and prompting can not be set up memory by the training of obtaining, and shows that the animal model intelligence that the method causes obviously descends, and animal has necessarily " dementia " performance.No matter the kidney invigorating Circulation Resolving Phlegm remembers the orientation navigation experiment of acquisition capability from the reflection space learning, or test from the space search of reflection information storage ability, all obviously is better than model group.And show as the trend of high, medium and low dosage group dose-effect relationship.Experimental result also shows, the kidney invigorating Circulation Resolving Phlegm also has a certain distance to the improvement effect of AD learning and memory in rats and normal group and sham operated rats, illustrates that this medicine can only partly improve the ability of learning and memory of AD, and can not make it recovery from illness, and this is also consistent with clinical efficacy.
Embodiment 10 medicine of the present invention is to IBO+A β 25-35induce the impact experiment of intending AD rat model pathomorphism
1 experiment material
1.1 laboratory animal is with embodiment 9.
1.2 medicine, reagent and instrument
H600 transmission electron microscope HIT other with embodiment 9.
2 experimental techniques
2.1AD model construction, animal grouping and administration are with embodiment 9.
2.2 Hippocampus Pathomorphologic observation
2.2.1 neurofibrillary tangles is observed
The conventional paraffin embedding of hippocampal tissue, slice thick 4 μ m, by silver hexamine staining method stained: section dewaxes to water, distilled water is washed section 3 times, put into hexamine silver solution (6% hexamethyl tetramine 150ml, 5% silver nitrate 715ml, 5% sodium tetraborate 9ml), hatch 2h for 56 ℃, distilled water is washed section 3 times, be placed in 4% neutral formalin solution 5min, distilled water is washed section 3 times, be placed in 5% sodium thiosulfate liquid 5min, distilled water is washed 5min, gradient ethanol (70%, 80%, 90%, 95%, dehydrated alcohol) each 5min that dewaters, dimethylbenzene is transparent, the neutral gum mounting.Nerve fiber is brownish black.Observe and take the photograph the sheet analysis under light microscopic.
2.2.2 senile plaque is observed
Section dewaxes to water, and methanol Congo red dye liquor dyes (Congo red 0.5g, methanol 80ml, glycerol 20ml) 15min, break up liquid (potassium hydroxide 0.2g, 80% ethanol 100ml) the differentiation several seconds with alkaline ethanol, washing, haematoxylin is redyed 2min, washing, dehydration, transparent, sealing.Positive findings: amyloid plaques is salmon pink.
2.2.3 the electron microscopic observation Hippocampal ultrastructure changes
Rat is through the left ventricle aortic cannulation, and 4% paraformaldehyde perfusion fixation, get brain, with the piece of tissue (1mm at the same position of Hippocampus 3), 2% glutaraldehyde is fixed, and send the Wuhan University Electron Microscopy Room to prepare ultrathin section, and transmission electron microscope (H600 of Hitachi type) is observed hippocampal neurons ultrastructure and is changed.
3 results
3.1 observe under light microscopic
Some aging performances also appear in normal group and blank group rat cortex and hippocampus cerebral tissue, show as neurocyte dyeing heterogeneity, structure and lose completely, and cell membrane, nuclear membrane are unintelligible.Also visible amyloid beta deposition speckle and neurofibrillary tangles under indivedual visuals field, may be the normal agings performance of 15 monthly ages, equal visible a large amount of vacuolar degenerations, and slight edema is arranged.Model group: hippocampal neurons obviously reduces, and after birth nuclear membrane boundary is unintelligible, karyopyknosis and vacuolar degeneration, partial nerve unit vanished cell.The model cerebral tissue shows and serious point-like interruption occurs, amyloid beta deposition speckle and neurofibrillary tangles showed increased, and visible a large amount of cavitys form, and form the change of fishnet shape.Low dose group and Western medicine group are very little to the animal pattern pathology effects, the rarely seen neuron of Western medicine group increases a little, it is intact than model that cellularity is arranged, and low dose group neuron number also is shown in and increases, and neurofibrillary tangles and amyloid beta deposition speckle reduce a little and alleviate.High dose group is apparent in view to neuronic reparation, and karyopyknosis and vacuolar degeneration also have certain improvement, and neurofibrillary tangles and amyloid beta deposition speckle obviously reduce, and the speckle body diminishes, and fiber attenuates.Middle dosage group is between low dosage and high dose.See accompanying drawing 3,4.
3.2 observe under Electronic Speculum
Normal group and blank group: hippocampus pericaryon dense arrangement, nucleus be take euchromatin as main, and kernel is obvious, the winding of the rare fiber in neuropil district, visible abundant synaptic structure.Two groups of ultrastructures do not have too many difference.Model group: neuron is arranged very disorderly, and the pericaryon pyknosis, visible high density chromatin margination under nuclear membrane, or block the distribution in core, and nuclear membrane is fuzzy.There is filamentary fibers to be wound around, the disorderly arrangement, the swelling of the high and steep property of mitochondrion.Synaptic structure obviously reduces or disappears, and the edema state of low electron density occurs.Western medicine group and low amount group: the pericaryon size is unequal, heterochromatic block the distribution is arranged, kytoplasm inner cell inner membrance structure decrease in core.The filamentary fibers disorder, the synaptolemma structure is fused, and the synaptic vesicle structure is unclear.Middle dosage group: neuronal structure still can, mitochondrion mild swelling in kytoplasm, have extremely slight chromatin margination under nuclear membrane, nuclear membrane, core week border discontinuity broadening, part of nerve fibers has swelling.High dose group: the pericaryon structure approaches normal group, and the nerve fiber structure still can.Synaptic structure still can.See accompanying drawing 5.
4 discuss
The kidney invigorating Circulation Resolving Phlegm tool benefiting essence and marrow in this experiment, the effect of waking up the patient from unconsciousness by dissipating phlegm.After the kidney invigorating Apophlegmatisant is intervened, laboratory animal neuron loss and vacuolar degeneration have certain improvement, and neurofibrillary tangles and amyloid beta deposition speckle obviously reduce, and the speckle body diminishes, and fiber attenuates.Marrow is given birth in the replenishing essence of the kidney invigorating energy, and the prosperous brains of marrow fill, and neuron loss and vacuolar degeneration just can be repaired, and reduces phlegm and can suppress and remove formation and the accumulation of retardance brain key material, and neurofibrillary tangles and amyloid-beta deposition reduce conscious.
Embodiment 11 medicine of the present invention is to IBO+A β 25-35induce the impact of intending AD rat model cholinergic system
1 experiment material
1.1 laboratory animal is with embodiment 9.
1.2 medicine, reagent and instrument
Ach, AchE and ChAT radioimmunoassay kits are purchased from Beijing North biotechnology research institute.
Other is with embodiment 9.
2 experimental techniques
2.1AD model construction, animal grouping and administration are with embodiment 9.
2.2 the preparation of brain tissue homogenate's liquid and the mensuration of total protein content
Each is organized after rat finishes water maze laboratory and breaks end, and gets rapidly Hippocampus on the ice platform and the cerebral tissue 100mg of cortical areas added normal saline by 1: 9, homogenate 30s in ice bath, and 4 ℃ of centrifugal 10min of lower 3500rpm, get supernatant frozen standby.
2.3 the Ach of rat cerebral tissue, AchE and ChAT determination of activity
Adopt radioimmunology.Separate cerebral tissue, weigh, homogenate, make 10% homogenate.Getting after supernatant is done protein quantification send Hospital Attached to Hubei Chinese Medicine College's Isotope Lab to detect.
2.4 statistical disposition is with embodiment 9.
3 results
This measuring respectively organize Ach, ChAT and the AchE activity of rat cerebral tissue, statistical result showed, model group cerebral tissue Ach compares with normal group with ChAT and all significantly reduces (P<0.01), AchE activity obviously raise (P<0.01).Ach is normal group and blank group zero difference (P>0.05) as a result.With the model group ratio, each group of Chinese medicine all has significant difference (P<0.01).Western medicine group Ach with middle low dosage than variant (P<0.01), but with high dose group than zero difference (P>0.05).From ChAT and the active testing result of AchE, both are more consistent in each group, each treatment group and model group be than all there were significant differences (P<0.01), low dosage with the Western medicine group than variant (P<0.05), with middle high dose zero difference (P>0.05).In each, high dose compares also zero difference (P>0.05).The results are shown in Table 8-1.
The active result of the table Ach of 8-1 rat cerebral tissue, ChAT and AchE
Figure BSA00000451126400241
Figure BSA00000451126400251
Annotate: with the normal group ratio △ △p<0.01 and model group ratio ▲ ▲p<0.01
With Western medicine group ratio ★p<0.05 ★ ★p<0.01
4 discuss
This measuring respectively organize Ach, ChAT and the AchE activity of rat cerebral tissue, from experimental result, model group cerebral tissue Ach compares with each group with ChAT all and significantly reduces, the AchE activity obviously raises.Each treatment group can obviously improve AD rat model Hippocampus Ach and ChAT activity, suppresses too high AchE activity, illustrates that infringement has protective effect to the kidney invigorating Circulation Resolving Phlegm to central cholinergic system.Middle high dose effect is more obvious.In addition, this experiment also shows that the ability of learning and memory of rat and cholinergic variation have dependency, illustrates that infringement has repair to the kidney invigorating Circulation Resolving Phlegm to central cholinergic system, thereby reaches the purpose of improving learning and memory.
Embodiment 12 medicine of the present invention is to IBO+A β 25-35induce the impact experiment of intending AD rat model Ubiquitin-proteasome path
1 experiment material
1.1 laboratory animal is with embodiment 9.
1.2 medicine, reagent and instrument
Ub, E1, E2-EPF antibody are all purchased from abcam company, and PGP9.5 antibody is purchased from chemicon company.ELISA auxiliary reagent (first antibody working solution, enzyme labelled antibody working solution, stop buffer, substrate working solution etc.) is purchased from Senxiong Science & Technology Industry Co., Ltd., Shanghai.Westernblot reagent (lysate, G250 Coomassie brilliant blue solution, SDS sample-loading buffer, electrophoretic buffer, transfering buffering liquid, confining liquid, TBST, TBS, eluting antibody buffer, developer solution, fixative solution, antibody, chemical illuminating reagent etc.) is purchased from Beijing Puli's lema gene technology company limited.Trizol (LifeTechnology company), dNTP, Oligo dT, RNAsin, RNA enzyme, M-MLV, taq enzyme (TaKaRa company), Mgcl2, DEPC, agarose, chloroform, isopropyl alcohol, ethanol, 4% paraformaldehyde, dimethylbenzene.Primer sequence is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.SABC SABC test kit, DAB buy in Wuhan Boster Biological Technology Co., Ltd..
Real-time fluorescence quantitative PCR instrument American AB I 5700 types.
2 experimental techniques
2.1AD model construction, animal grouping and administration are with embodiment 9.
2.2 detect index and method thereof
2.2.1 the mensuration that ubiquitin protein is expressed
2.2.1.1 the ubiquitin protein SABC detects
Rat is fixing through the 4% paraformaldehyde perfusion of left ventricle aortic cannulation, opens cranium and gets brain and do crown section, the conventional paraffin embedding of Hippocampus and cortical areas, slice thick 4 μ m.Negative control: with PBS, replace primary antibodie.The employing image processing system is analyzed.Step:
1. the conventional dimethylbenzene dewaxing of paraffin section 15min * 3 time, each 5min of gradient ethanol (dehydrated alcohol, 95%, 90%, 80%, 70%); 2. fresh 3%H 2o 2deactivation endogenous enzyme 10min, distillation washing 2min * 3 time; 3. section is placed in to the multiple antigen 5min of citrate buffer (pH6.0) hot repair * 2 times, PBS (pH7.4) washes 5min * 3 time; 4. drip the 5%BSA sealing, room temperature 20min, get rid of unnecessary liquid, do not wash; 5. drip the anti-polyclonal antibody of rabbit of dilution, 4 ℃, refrigerator spends the night, and PBS washes 2min * 3 time; 6. drip two anti-(sheep anti-mouse iggs) 37 ℃ and hatch 30min, PBS washes 2min * 3 time; 7. drip SABC reagent 30min, PBS washes 5min * 4 time; 8. drip DBA, under room temperature, act on 5-30min; 9. haematoxylin is redyed; Gradient alcohol dehydration, dimethylbenzene is transparent, the neutral gum mounting; 10. take the photograph the sheet analysis.
The positive reaction product is brown yellow granule, reacts stronger, and color is darker.Negative reaction is colourless.Every section input picture analytical system is measured the gray value of the positive cell space of expressing in selected brain district.Obtain the average gray value of positive neuron cell space in every group of section, then carry out significance test.
2.2.1.2 ubiquitin protein RT-PCR detects
All reagent of using in operating process all configure with the DEPC treated water; All containers and apparatus are all processed through past RNA enzyme.Glass toasts 4 hours under 150 ℃.Other articles for use are high pressure deactivation DEPC after the 0.1%DEPC water soaking spends the night.And overall process is carried out in fume hood.
Rat Ub primer:
Upstream 5 '-TAAGACCATCACCCTCGATT-3 ', the cDNA sequence fragment length of downstream 5 '-TGGATGTTGTAGTCAGACAGGG-3 ' amplification is 156bp
β-actin primer:
Upstream: 5 '-GAGACCTTCAAGACCCCAGCC-3 ', the cDNA sequence fragment length of downstream: 5 '-TCGGGGCATCGGAACCGCTCA-3 ' amplification is 404bp.
(1) extract total RNA
1. take out the 80mg cerebral tissue and put into rapidly the glass homogenizer that 1ml Trizol is housed from liquid nitrogen, manually homogenate, pour in 1.5ml EP pipe, vibration dissolution precipitation, standing 5min under room temperature.2. add the 0.2ml chloroform, build lid.Acutely put upside down 15s.Then standing 10min under room temperature.3. 4 ℃ of centrifugal 12000rpm of low temperature, 15min.Careful absorption upper strata colourless liquid (total RNA) approximately 500 μ l is transferred in the EP pipe of another 1.5ml.4. the isopropyl alcohol that adds equal-volume 300 μ l, vibration mixes standing 10min under rear room temperature.5. 4 ℃ of centrifugal 12000rpm of low temperature, 15min.The centrifugal rear visible RNA lamellar that is white in color is attached to tube wall bottom, towards the opposite direction of adhering to, carefully pours out supernatant liquid, and the limit bevelling notices whether RNA also is attached on wall, and remaining water sucks with suction nozzle from the opposite direction of adhering to.6. the ethanol that adds 1ml 75%DEPC to process, 4 ℃ of centrifugal 8000rpm after vertical oscillation, 10min.7. same method is removed supernatant liquid, adds the DEPC treated water of 30 μ l.-70 ℃ of preservations.(2) RT reaction
Reaction, in the EP of 0.2ml pipe, is placed on the EP pipe on ice (preventing that secondary structure from forming again), then adds other reagent.Application of sample order and dosage are
Figure BSA00000451126400261
Figure BSA00000451126400271
70 ℃ of 5min (for dissolving the secondary structure of template), put into the reaction of PCR instrument after centrifugal: 37 ℃ 1h+94 ℃ 5min+4 ℃ of 1h.
(3) the PCR reaction response is in the EP of 0.2ml pipe, and application of sample order and dosage are
Figure BSA00000451126400272
Reaction: 94 ℃ of 5min; 94 ℃ of 30s → 55 ℃ 1min → 72 ℃ of 1min 20s * 30cycles; 72 ℃ of 10min; 4 ℃ of 1h
(4) agarose gel electrophoresis
Get 10 μ l PCR product electrophoresis on 2% agarose gel, and with 0.5 μ l/ml ethidium bromide staining.Result means with band optical density Ub/ β-actin, contrast between being organized.
2.2.2 the mensuration of ubiquitin relevant enzyme (E1, E2-EPF, PGP9.5)
2.2.2.1 euzymelinked immunosorbent assay (ELISA) (ELISA) method detects
1. coated: the coated antibody diluted is added to elisa plate, and 100 μ l/ holes, place 48h for 4 ℃; 2. add successively testing sample 100 μ l in every hole.Sptting plate is put to 37 ℃ of temperature and bathe 120min.3. with cleaning mixture, Sptting plate is fully washed 4-6 time, put on filter paper and print and do.4. every hole adds first antibody working solution 100 μ l.Sptting plate is put to 37 ℃ of temperature and bathe 60min.Wash plate the same.5. every hole adds enzyme labelled antibody working solution 100 μ l.Sptting plate is put to 37 ℃ of temperature and bathe 60min.6. every hole adds substrate working solution 100 μ l, puts 37 ℃ of dark place reaction 5-10min.7. every hole adds 1 stop buffer to mix, and mixes gently.8. survey light absorption value at the 492nm place.9. the judgment experiment result is with the OD value representation as a result, and all OD values are gone and calculated again after all deducting blank value, contrast between being organized.
2.2.2.2 immunoblotting detects
(1) extraction of total protein in tissue
1. get 50mg cerebral tissue piece from liquid nitrogen, shred piece of tissue as far as possible.2. put into homogenizer, add 400 μ l buffer (containing PMSF), then be placed in and carry out homogenate on ice.3. 4 ℃ of centrifugal 5min of lower 12000rpm, get supernatant and be sub-packed in the 0.5ml centrifuge tube and be placed in-20 ℃ of preservations.
(2) mensuration of protein content
1. get the 1.5ml centrifuge tube, every pipe adds the Coomassie brilliant blue solution 1ml of 4 ℃ of storages.Room temperature is placed after 30min for surveying albumen.2. get a pipe Coomassie brilliant blue and add 0.15mol/LNaCl solution 100 μ l, mix placement 2min and do the blank detection.3. get a pipe Coomassie brilliant blue and add 95 μ l0.15mol/L NaCl solution and 5 μ l testing protein samples, mix rear standing 2min, detect sample.
(3) SDS-PAGE gel electrophoresis
1. 2. the mounting glass plate adds after TEMED (tetramethyl diethylamine) to shake up immediately and fills with 10% separation gel.Shake up immediately encapsulating after adding TEMED.Remaining space is filled to 4% concentrated glue, immediately comb is inserted in concentrated glue.3. water rinses concentrated glue, puts it in electrophoresis tank.Add the protein sample that boils degeneration, applied sample amount is consistent.4. add 1 * electrophoretic buffer in electrophoresis tank.Electrophoresis time 4-5h, voltage is 40V.Electrophoresis to bromjophenol blue has just been run out of the termination electrophoresis, carries out transferring film.
(4) transferring film
1. cut 6 equirotal filter paper, its size is the same with transfer area.Then, they being immersed in to electricity turns in buffer.Cut an onesize PVDF (Kynoar) film, be placed in gently in the ultra-pure water plate and soaked 2h.2. 3 filter paper of pad on pvdf membrane, then be laid in gel on pvdf membrane, then put 3 filter paper, with glass rod, on the filter paper face, rolls and remove each layer of bubble, then puts into electroporation, 60V transferase 12 h.3. after having turned, film is dyed to 5min with 1 * Ponceaux dye liquor.Then water rinses out the dye liquor of not catching and just can see the albumen on film.Film is dried standby.
(5) immunoreation
1. after TBS for film (tert-butyl group dimethylsilane) being soaked from bottom to top, move in the plate that contains confining liquid, shake sealing 1h under room temperature on decolorization swinging table.2. the deblocking liquid that inclines, add the primary antibodie that is diluted to debita spissitudo (in the 1.5ml centrifuge tube) with TBST (buffer); After hatching 1-2h under room temperature, with TBST, at room temperature on decolorization swinging table, wash twice, each 10min; Wash once again 10min with TBS.3. the same method is prepared two anti-diluents and is contacted with film, after hatching 1-2h under room temperature, with TBST, at room temperature on decolorization swinging table, washes twice, each 10min; With TBS, wash once, 10min, carry out chemiluminescence reaction again.(6) chemiluminescence, develop, photographic fixing
By A and two kinds of reagent of B in the first-class volume mixture of preservative film; After 1min, the memebrane protein mixed liquor therewith that faces down is fully contacted; After 1min, film is moved on another preservative film, remove most residual liquid, wrap, put into X-mating plate folder.Use X light reaching the film, developing and fixing in darkroom.
(7) film is taken pictures, by molecular weight and the clean optical density value of gel images processing system evaluating objects band.
2.3 statistical disposition is with embodiment 9.
3 results
3.1 respectively organize the variation of rat cerebral tissue's ubiquitin protein
The immunohistochemical assay result shows, the rare Ub immunity of normal rat positive expression, normal group and sham operated rats no significant difference (P>0.05), and increase to some extent at the visible brown color Ub immunity of model group positive expression, compare with normal group, there is significant difference (P<0.01).Western medicine group and model group be zero difference (P>0.05) relatively, and the positive expression of each group of Chinese medicine is showed increased also.High dose group is than the expression increase of model group comparatively obviously (P<0.01), in low amount group take second place (P<0.05).Middle high dose ratio is zero difference (P>0.05) also.The immunohistochemistry colour developing the results are shown in accompanying drawing 10.The RT-PCR result shows, the model group band has notable difference (P<0.01) than normal group, and three groups, Chinese medicine significantly strengthens (P<0.01) than the model group shading value, with the Western medicine group, notable difference (P<0.01 or P<0.05) is arranged.The results are shown in Table 9-1.Referring to accompanying drawing 6.
Table 9-1 respectively organizes rat hippocampus and the Ub of cortical areas protein level result
Figure BSA00000451126400281
Annotate: with the normal group ratio △ △p<0.01 and model group ratio ▲p<0.05 ▲ ▲p<0.01
With Western medicine group ratio ★p<0.05 ★ ★p<0.01
3.2 respectively organize the variation of rat hippocampus and the E1 of cortical areas protein expression
ELISA (elisa) method testing result shows, normal group and sham operated rats no significant difference (P>0.05), and at model group E1 content, downward trend is arranged by the numbers, but compare with normal group, significant difference (P>0.05) be there is no.Western medicine group and model group be zero difference (P>0.05) more also, and with model group, Chinese medicine is respectively organized the E1 protein content and raise comparatively obviously (P<0.01), with the Western medicine group, notable difference (P<0.01) is also arranged.Middle high dose group is than zero difference (P>0.05).Immunoblotting detection display E1 albumen is expressed at the 118kD place, consistent with expected results.OD value analysis result and ELISA result have certain same trend, and difference is, model group and normal group more variant (P<0.05), with the Western medicine group than variant (P<0.05).The results are shown in Table 9-2.Referring to accompanying drawing 7.
Table 9-2 respectively organizes rat hippocampus and the E1 of cortical areas level result
Figure BSA00000451126400292
Figure BSA00000451126400293
Annotate: with normal group △p<0.05 and model group ratio ▲p<0.05 ▲ ▲p<0.01
With Western medicine group ratio ★ ★p<0.01
3.3 respectively organize the variation of rat hippocampus and the E2-EPF of cortical areas protein level
ELISA (elisa) method testing result shows, rat hippocampus and cortex E2-EPF content, normal group and sham operated rats no significant difference (P>0.05), model group E2-EPF protein content significantly raises, compare with normal group, there is significant difference (P<0.01).With the model group ratio, each treatment group E1 protein content all has raise in various degree (P<0.01 or P<0.05).With Western medicine group ratio, high dose variant (P<0.05), between middle high dose group than zero difference (P>0.05).Immunoblotting detects that E2-EPF albumen is main between 35-40kD shows four bands, and is consistent in theory.OD value result has consistent trend with the ELISA result, and difference is, low dose group has significant difference (P<0.05) with Western medicine group ratio.The results are shown in Table 9-3.Referring to accompanying drawing 8.
Table 9-3 respectively organizes rat hippocampus and the E2-EPF of cortical areas protein level result
Annotate: with the normal group ratio △ △p<0.01 and model group ratio ▲p<0.05 ▲ ▲p<0.01
With Western medicine group ratio ★p<0.05
3.4 respectively organize the variation of rat hippocampus and the PGP9.5 of cortical areas protein level
ELISA method testing result shows, rat hippocampus and cortex PGP9.5 content, normal group and sham operated rats no significant difference (P>0.05), model group PGP9.5 protein content significantly reduces, compare with normal group, there is significant difference (P<0.05).Western medicine group and model group be zero difference (P>0.05) relatively.With the model group ratio, Chinese medicine is respectively organized PGP9.5 protein content rising comparatively obviously (P<0.01).With Western medicine group ratio, also there were significant differences for each Chinese drug-treated group (P<0.01).Middle high dose group is than variant (P<0.05).Immunoblotting detects that PGP9.5 albumen is main between 27-36kD shows three bands, and is consistent in theory.OD value testing result and ELISA result have certain same trend, and difference is, model group and normal group be zero difference (P>0.05) relatively, high dose group with middle amount group than zero difference (P>0.05).The results are shown in Table 9-4.See accompanying drawing 9.
Table 9-4 respectively organizes rat hippocampus and the PGP9.5 of cortical areas protein level result
Figure BSA00000451126400302
Annotate: with the normal group ratio △p<0.05 and model group ratio ▲ ▲p<0.01
With Western medicine group ratio ★ ★p<0.01 and middle group of amount ratio ☆p<0.05
4 discuss
Rat hippocampus and cortex Ub, E1, E2-EPF and PGP9.5 content detection result are shown, ELISA method and immunoblotting result are basically identical, normal group and sham operated rats no significant difference.Model group Ub and E2-EPF protein content significantly raise, and E1 and PGP9.5 content significantly reduce, and visible model group ubiquitin system gets muddled, and this conforms to front pathology experimental result, and amyloid beta deposition speckle and neurofibrillary tangles appear in cerebral tissue.
Experimental result shows, medicine of the present invention can play regulating action preferably to the disorder of model group ubiquitin and associated protein thereof, can effectively improve animal Hippocampus and the Ub of cortical areas, E1, E2-EPF and PGP9.5 protein content, strengthen the degradation capability of Ubiquitin-proteasome path to paraprotein, and there is an a certain amount of effect relationship, middle high dose successful is better than low dose group, except indivedual indexs, high dose not than in dose form reveal better effect.
Embodiment 13 medicine of the present invention is to IBO+A β 25-35induce the impact experiment of intending AD rat model Tau abnormal protein phosphorylation
1 experiment material
1.1 laboratory animal is with embodiment 9.
1.2 medicine, reagent and instrument
Immunoblotting chemical illuminating reagent (ECL), BCA protein concentration detection kit, the albumen Marker dyed in advance, pvdf membrane, protein extraction test kit are purchased from Beijing Puli's lema gene technology company limited.Rabbit Chinese People's Anti-Japanese Military and Political College Mus phosphorylation Tau antibody (PHF-1) is purchased from abcam company, and the goat-anti rabbit two of HRP labelling is anti-purchased from Wuhan Boster Biological Technology Co., Ltd..
CAMK-II in situ hybridization test kit is purchased from Wuhan Boster Biological Technology Co., Ltd., Trizol is purchased from Life Technology company, dNTP, Oligo dT, RNAsin, RNA enzyme, M-MLV, taq enzyme, Mgcl2, DEPC are purchased from TaKaRa company, Gsk-3 β rat Elisa detection kit is purchased from Senxiong Science & Technology Industry Co., Ltd., Shanghai, and primer is given birth to work biotechnology company purchased from Shanghai.
Acrylamide, methylene fork bisacrylamide, Ammonium persulfate., Tris (Tris), sodium lauryl sulphate (SDS), glycine, Ponceaux, dithiothreitol, DTT, 2 mercapto ethanol, N, N, N, N-tetramethylethylenediamine (TEMED), Tween20 etc. are analytical pure.
Real-time fluorescence quantitative PCR instrument (5700 types, American AB I company), microplate reader (MB-III type, Beijing Bin Daying creates scientific & technical corporation).
Other is with embodiment 9.
2 experimental techniques
2.1AD model construction, animal grouping and administration are with embodiment 9.
2.2 animal is drawn materials
Break end after the animal water maze, get rapidly brain on the ice platform, separate hippocampal tissue and put into liquid nitrogen flash freezer.
2.3 detect index and method
2.3.1Western blot detects Rat hippocampus Tau abnormal protein phosphorylation, with embodiment 12.
2.3.2 enzyme linked immunosorbent assay (ELISA) detects cerebral tissue GSK-3 beta determination
1. Criterion curve: establish 8 of gauge orifices.Add standard substance diluent 100 μ l in every hole.The first hole adds standard substance 100 μ l, mixes rear absorption 100 μ l and adds the second hole, and so two-fold dilution's to the seven apertures in the human head takes out 100 μ l and discards from seven apertures in the human head.Octal is zero hole.2. add successively testing sample 100 μ l in every hole.Sptting plate is put to 37 ℃ of temperature and bathe 120min.3. with cleaning mixture, Sptting plate is fully washed 4-6 time, put on filter paper and print and do.4. add first antibody working solution 100 μ l in every hole.Sptting plate is put to 37 ℃ of temperature and bathe 60min.Wash plate the same.5. every hole adds enzyme labelled antibody working solution 100 μ l.Sptting plate is put to 37 ℃ of temperature and bathe 60min.6. every hole adds substrate working solution 100 μ l, puts 37 ℃ of dark place reaction 5-10min.7. every hole adds 1 stop buffer to mix, and mixes gently.8. survey light absorption value at the 492nm place.
2.3.3RT-PCR method detects the mensuration of cerebral tissue cdk5 enzymatic activity:
Rat cdk5 primer:
The cDNA sequence fragment length of upstream 5 '-GAAGCGGCACTCCATCATCTCGG-3 ' downstream 5 '-ACGCCTGG ACGATGACCCGTTTGG-3 ' amplification is 317bp
β-actin primer:
Upstream 5 '-GAGACCTTCAAGACCCCAGCC-3 ' downstream 5 '-TCGGGGC ATCGGAACCGCTCA-3 '.The cDNA sequence fragment length of amplification is 404bp
Take the photograph the sheet analysis result and mean with band optical density cdk-5/ β-actin, contrast between being organized.
2.3.4 hybridization in situ detects the variation of hippocampal neuron CaMKII-alpha levels
1. paraffin section dewaxes to water through routine.30%H 2o 210 parts of mixing of 1 part+distilled water, room temperature 10min is with the deactivation endogenous enzyme.Distillation washing 3 times.2. expose the mRNA nucleic acid fragment: drip the pepsin (the 1ml3% citric acid adds 2 concentrated type pepsin, mixes) of the fresh dilution of 3% citric acid in section, 37 ℃ or the room temperature digestion 15min (digestion time that trial test is more different, 5min for example, 10min, 20min, 30min.To find best digestion time.15min the best).Wash 3 times * 5min with PBS.Distillation washing 1 time.3. fixing afterwards: fixative is 1% paraformaldehyde/0.1M PBS (pH7.2-7.6), contains 1/1000DEPC.Room temperature is 10min fixedly.Distilled water wash 3 times.4. hybridization: by every section 20 μ l, add prehybridization solution.Wet box put calorstat 38-42 ℃ 3 hours.Draw unnecessary liquid, do not wash.By every section 20 μ l hybridization solutions, be added in section.After the protecting film of in situ hybridization special cap slide is opened, cover in section.Calorstat 38-42 ℃ hybridization is spent the night.Slide, 2 * SSC washing 5min * 2 time of 37 ℃ of left and right water temperatures; 37 ℃ of 0.5 * SSC washing 15min * 1 time; 37 ℃ of 0.2 * SSC washing 15min * 1 time (if unspecific staining is arranged, repeating 0.2 * SSC washing 15min * 1-2 time).5. drip confining liquid: 37 ℃ of 30min.Get rid of unnecessary liquid, do not wash and 6. drip biotinylation mouse-anti digoxin: room temperature 120min.In situ hybridization is washed 5min * 4 time with PBS.7. drip SABC:37 ℃ of 20min or room temperature 30min.In situ hybridization is washed 5min * 3 time with PBS.8. drip the biotinylation peroxidase: 37 ℃ of 20min or room temperature 30min.In situ hybridization is washed 5min * 4 time with PBS.9. DAB colour developing: use DAB colour reagent box 1ml distilled water to add developer A, B, each of C, mix, and adds on specimen.General colour developing 20-30min.If occur continuing colour developing without background.Fully washing.10. dehydration of alcohol, dimethylbenzene is transparent, mounting.
2.4 statistical disposition is with EXPERIMENTAL EXAMPLE 6.
3 results
3.1 respectively organize the Tau of rat cerebral tissue protein content
This measuring respectively organize rat hippocampus phosphorylation Tau protein content, statistical result showed, normal group compares no significant difference (p>0.05) with blank group, model group and normal group and blank group significantly increase (P<0.01), low amount group and Western medicine group and model group be zero difference (p>0.05) relatively, high, the middle amount group of Chinese medicine and model group significantly reduce (P<0.05 or 0.01), with Western medicine group more variant (P<0.01), but zero difference (p>0.05) between the two.See accompanying drawing 10.
The impact of table 10-1 invention medicine on the rat hippocampus Phosphorylated tau
Figure BSA00000451126400322
Annotate: with the normal group ratio △p<0.05 △ △p<0.01 and model group ratio ▲p<0.05 ▲ ▲p<0.01
With Western medicine group ratio ★ ★p<0.01
3.2 respectively organize the impact of the GSK-3 of rat cerebral tissue 'beta ' activity
The results are shown in Table 10-2.The Elisa expression compared with normal group of display model group GSK-3 β as a result has the rising of significance, and the two has compared significant difference (P<0.01).After administration, the expression of GSK-3 β all decreases than model group, low amount group and Western medicine group and model group be zero difference (p>0.05) relatively, middle and high amount group has been compared significant difference (P<0.05 or 0.01) with model group, with the Western medicine group, compare, a large amount group is expressed significant difference (P<0.05).
The impact of table 10-2 invention medicine on the GSK-3 of rat cerebral tissue 'beta ' activity
Figure BSA00000451126400332
Annotate: with the normal group ratio △ △p<0.01 and model group ratio ▲p<0.05 ▲ ▲p<0.01
With Western medicine group ratio ★p<0.05
3.3 respectively organize the impact of the cdk5 of rat cerebral tissue enzyme mrna expression
The results are shown in Table 10-3.The RT-PCR expression compared with normal group of the cdk5mRNA of display model group as a result all has the raising of significance, and the two has compared significant difference (P<0.01).Low amount group and Western medicine group and model group be zero difference (p>0.05) relatively.Middle and high amount group is compared with model group, and significant difference (P<0.01) is arranged, and with Western medicine group ratio, significant difference (P<0.01) is also arranged.The a large amount group is compared with middle amount group, expresses the reduction that significance is arranged, and significant difference (P<0.01) is arranged.See accompanying drawing 11.
The impact that table 10-3 invention medicine is expressed the cdk5mRNA of rat cerebral tissue
Figure BSA00000451126400333
annotate: with the normal group ratio △ △p<0.01 and model group ratio ▲ ▲p<0.01
With Western medicine group ratio ★ ★p<0.01 and middle amount group ratio ☆ ☆p<0.01
3.4 respectively organize the variation of the CaMKII-α of rat cerebral tissue positive expression
Hybridization in situ experiment is analyzed at the cerebral hippocampal position of normal rat rare CaMKII-alpha immunization positive expression, normal group and blank group no significant difference (p>0.05), and at the visible a large amount of brown color CaMKII-alpha immunization positive expression of model group, compare with normal group, there is significant difference (P<0.01).Western medicine group and model group be zero difference (p>0.05) relatively, and in Chinese medicine, a large amount group positive expression all reduces (P<0.01) to some extent than model group.Low amount group and model group zero difference (p>0.05), but with the Western medicine group, different (P<0.05) is arranged.See accompanying drawing 21.
The impact of table 10-4 invention medicine on the CaMKII-α of rat cerebral tissue mrna expression
Figure BSA00000451126400341
Figure BSA00000451126400342
Annotate: with the normal group ratio △ △p<0.01 and model group ratio ▲ ▲p<0.01
With the matched group ratio ★p<0.05 ★ ★p<0.01
4 discuss
This laboratory observation is to A β 25-35+ IBO can induce Rat hippocampus phosphorylation Tau Tot Prot to increase, after giving the invention Drug therapy of doses, the level of Rat hippocampus phosphorylation Tau albumen obviously descends, the most obvious with high, the middle dosage effect of invention medicine, low dosage is without obvious effect, may be that the invention medicine must reach certain concentration and just can play inhibitory action to the Tau albumen of phosphorylation.The invention medicine can reduce the Tau protein level of Abnormal Phosphorylation, and its dephosphorylation is increased, and therefore likely reverses the pathological change of AD.
The horizontal compared with normal group of model group rat hippocampus GSK-3 β has significance to raise, after administration, the level of GSK-3 β all decreases than model group, wherein the effect of high, middle amount group is the most remarkable, low amount group is not improved effect to the level of GSK-3 β, prompting invention medicine can suppress the activity of GSK-3 β in range of doses, therefore, we think that the invention medicine can pass through the level of the activity decreased AD rat model Tau abnormal protein phosphorylation of inhibition GSK-3 β.
The expression compared with normal group of model group CDK5mRNA has significantly and increases, and, by after the invention pharmaceutical intervention, the expression of CDK5mRNA all descends to some extent, and wherein the effect of high, middle amount group is remarkable, and low amount group is without obvious effect.As can be seen here, the invention medicine, except by affecting GSK-3 β, also may, by the adjusting to CDK5, affect the abnormal Hyperphosphorylationof of Tau albumen.
Model group Rat hippocampus CaMK II-alpha expression compared with normal group obviously increases, after the invention pharmaceutical intervention, the expression of CaMK II-α all reduces to some extent than model group, wherein especially comparatively obvious with the expression minimizing of a large amount group, in, low amount group takes second place, prompting invention medicine also can reduce by the expression of regulating CaMK II-α AD rat model Tau abnormal protein phosphorylation level in range of doses.
Embodiment 14 medicine of the present invention is to IBO+A β 25-35induce the immune system impact experiment of intending the mediation of AD rat model microglia
1 experiment material
1.1 laboratory animal is with embodiment 9.
1.2 medicine, reagent and instrument
The Mus OX-42 of mice Chinese People's Anti-Japanese Military and Political College monoclonal antibody, SABC SABC test kit, DAB buy in Wuhan Boster Biological Technology Co., Ltd..TNF-α, IL-1 β, IL-6ELISA test kit are purchased from Senxiong Science & Technology Industry Co., Ltd., Shanghai.Enzyme-linked immunosorbent assay instrument Finland Thermol Labsystems
Other is with embodiment 9.
2 experimental techniques
2.1AD model construction, animal grouping and administration are with embodiment 9.
The same EXPERIMENTAL EXAMPLE 8 2.2 animal is drawn materials.
2.3 detect index and method
2.3.1 rat cerebral tissue's microglia method for immunohistochemical detection is with third part experiment one
With the special significant antibody OX-42 labelling microglia of microglia, the expression of microscopic examination cerebral tissue microglia.The positive reaction product is brown yellow granule, reacts stronger, and color is darker.Negative reaction is colourless.Under light microscopic, observe, and the lower counting microglia of applies image analysis system high power field (x400 doubly).
2.3.2 the detection of the TNF-α of rat cerebral tissue, IL-1 β, IL-6 activity
Enzyme linked immunosorbent assay (ELISA), with embodiment 12.
2.4 statistical procedures is with embodiment 9.
3 results
3.1 respectively organize rat cerebral tissue's microglial activation situation
Be generally quiescent condition in the normal rat brain, anti-OX-42 immunohistochemical staining is negative, and in section, difficult discovery or cellular morphology are unintelligible.After the sham operated rats wound stimulates, activate the early reaction state that is converted into, visible OX-42 understain positive cell, cellular morphology is visible, but irregular, quantity and normal group zero difference (P>0.05); Model group OX-42 engrain, OX-42 positive cell form is clear, and it is large that cell space becomes, and quantity significantly increases, and with normal group, significant difference (P<0.01) is arranged; Each treatment group reaction weakens gradually, the OX-42 engrain, and positive cell quantity reduces gradually, and cellular morphology thickens.Quantitatively middle amount group and model group relatively have significant difference (P<0.01), and Western medicine group and a large amount group and model group be zero difference (P>0.05) relatively, and a large amount group and middle amount group be more variant (P<0.01).
The table 11-1 respectively organize rat cerebral tissue's microglial activation situation (
Figure BSA00000451126400351
n)
Annotate: with the normal group ratio △ △p<0.01 and model group ratio ▲ ▲p<0.01
With Western medicine group ratio ★ ★p<0.01 and middle amount group ratio ☆ ☆p<0.01
3.2 respectively organize the level of the Nerve Cells of Rat Brain factor
This measuring respectively organize IL-1 β, IL-6 and the TNF-alpha levels of rat cerebral tissue.From table 11-2, blank each index content of group raises a little, but the upper normal group of statistics and sham operated rats IL-1 β, IL-6 and TNF-alpha content zero difference (P>0.05); The content compared with normal group of model group IL-1 β, IL-6 and TNF-α has remarkable rising (P<0.01); The more equal zero difference of Western medicine group and model group (p>0.05).The invention medicine is low, high dose group can reduce model group IL-1 β content (P<0.05), middle dose effect better (P<0.01).In, low amount group IL-6 content relatively has significant difference (P<0.01) with model group, but high dose and model group comparison zero difference (p>0.05).TNF-α result shows to only have middle dosage group and model group more variant (P<0.05), other each treatment group with model group than equal zero difference (p>0.05).
Table 11-2 respectively organize the IL-1 β of rat cerebral tissue, IL-6 and TNF-alpha levels result (
Figure BSA00000451126400361
pg/mg)
Figure BSA00000451126400362
Annotate: with the normal group ratio △ △p<0.01 and model group ratio ▲p<0.05 ▲ ▲p<0.01
With Western medicine group ratio ★p<0.05 ★ ★p<0.01
4 discuss
This experimental result shows, is generally quiescent condition in the normal rat brain, without microglial activation.After animal model, the microglia form is clear, and it is large that cell space becomes, and quantity significantly increases, and has as seen significance activation situation to occur, and consistent with senile plaque and neurofibrillary tangles performance.In the invention medicine, the dosage group can suppress the excessive activation of microglia effectively; the infringement of protection to cerebral tissue; but low, not effect of high dose; may be that low dosage is difficult to reach active drug concentration, high dose may be to body, to be a kind of activation that causes microglia that stimulates on the contrary because concentration is excessive.
Experimental result shows, the invention medicine can play regulating action preferably to the immune cytokine of model group, can effectively reduce the IL-1 β of animal brain, IL-6 and TNF-alpha levels, alleviate the infringement to cerebral tissue, and having a certain amount of effect relationship, middle dose effect is significantly better than the high and low dose group.Its mechanism may be relevant with the microglia effect that suppresses excessive activation.
Embodiment 15 medicine Contained Serum of the present invention is to A β 25-35effect PC12 induces AD cell model neuroprotective research experiment
1 experiment material
1.1 cell strain
PC12 (strain of Mus pheochromocytoma cells) is provided by the cell research of Shanghai Branch of the Chinese Academy of Sciences.
1.2 main agents
1640 culture medium, the GibcoBRL product; Hyclone (FBS), horse serum (HS) the Hangzhou four seasons clear biological engineering material institute product; Tetramethyl azo azoles blue (MTT), the Fluka product; A β 25-35, trypsin, Sigma product.Caspase-3 Mus IgG and instant SABC staining kit, Wuhan Boster Biological Technology Co., Ltd..All the other reagent are domestic analytical pure.
1.3 key instrument equipment
The CO2 incubator, U.S. Sheldon company; Superclean bench, Purifying Equipment Co., Ltd., Suzhou; Inverted phase contrast microscope, Japanese OLYMPUS (CK-TKc-3); Enzyme-linked immunosorbent assay instrument, Finland Thermol Labsystems; Flow cytometer
FACSCLibur, U.S. BD Asia company limited; The vertical electric pressure steam sterilizer, the LDZX-40 type.
1.4 laboratory animal
Clean level Wistar rat, body weight 250 ± 20g, provided by Tongji Medical College, Huazhong Science and Technology Univ.'s Experimental Animal Center.
1.5 Experimental agents
Preparation Example 3 medicines, 4 ℃ save backup.
2 methods
2.1PC12 the cultivation of cell
Cell is bought rear put into rapidly 37 ℃, 5%CO back 2in incubator, standing adaptability is cultivated 1d, and its later half amount is changed liquid, after in former culture bottle, cell reaches the standard of going down to posterity, cell is inoculated in to 25cm 2in culture bottle, cell density is 2 * 10 5/ ml, add 10% hyclone with the DMEM culture fluid and cultivate.According to the Growth of Cells situation, 1-2d changes culture fluid, and 2-3d goes down to posterity once.By observing counting under inverted microscope, by complete 1640 culture medium, by cell dilution, be 1 * 10 6the cell suspension of individual cell/ml, be inoculated in culture bottle and culture plate, and 37 ℃, 5%CO 2cultivate, cell attachment growth in 2-3 days, can be used for experiment.
2.2AD the foundation of cell model
When testing, select PC12 cell in 96 well culture plates to be paved with culture hole (every Kong Yuehan cell 1 * 10 of monolayer 6), change serum-free medium (except Normal group) into.Add respectively different final concentration A β 25-35, and be divided at random 6 groups, every group of 8 holes: 1. complete medium group (I); 2. blank PBS matched group (H); 3. 0.1 μ M group (III); 4. 1 μ M group (IV); 5. 5 μ M groups (V); 6. 10 μ M group (VI) groups.7., after 20 μ M groups (VII) are hatched 12h, 24h, 48h jointly, carry out trypan blue staining and morphological observation.
The A β prepared 25-35solution is hatched 4d prior to 37 ℃, and short it is aging, selects A β 25-35concentration in culture medium is that 5 μ mol/L are as experimental concentration.Selection enters the cell of exponential phase after going down to posterity, add the A β that final concentration is 5 μ mol/L 25-35segment, continue to cultivate after 1d to set up the AD cell model.
2.3 the preparation of pastille Mus serum and blank Mus serum:
30 of Wistar rats, minute administration group (A) and matched group (B), 15 every group.
The A group is pressed man and animal body surface area dose,equivalent ratio table scaling method [1-2]and Sero-pharmacological experiment method, the serum donor be take relevant effect dosage (as the clinical 8-10 at people's dosage doubly measures) administration of clinical application reason or block mold animal, to rat oral gavage invention medicine water decoction, 2 administrations every day (morning 8:00-9:00, afternoon 3:00-4:00), being equivalent to the crude drug amount is 25g/kgd.Continuous 7 days, 2h after last administration (before administration, 12h prohibits drinking-water), with after pentobarbital sodium (40mg/kg) intraperitoneal injection of anesthesia, blood is got from rat heart by sterile working, and centrifugal 1000rpm separation of serum, then 10 rat blood serums is mixed, 56 ℃, the 30min inactivation treatment.With 0.22 μ m filtering with microporous membrane degerming ,-20 ℃ save backup.Prepared by this method is exactly Contained Serum.When testing, with the RPMI1640 culture medium be diluted to respectively 5%, 10%, the Contained Serum of 20% 3 kind of variable concentrations.
The B group gavages 0.9% continuous normal saline 7 days, administration time, method and obtain serological method with the A group.Prepared by this kind of method is blank serum.By same method, blank serum is diluted to the serum of variable concentrations.
2.4 detection index and approach
2.4.1 cell directly is placed in its form of micro-Microscopic observation counting
Prepare cell suspension: stop cultivating, by the culture medium sucking-off, with PBS, softly clean culture once.Add the 1ml0.25% trypsin in culture bottle, under 37 ℃ of conditions, digest 3-5min, when the Microscopic observation cell rounding approaches de-wall, add the culture medium of 4.5ml, with suction pipe softly piping and druming repeatedly, make cell take off wall and make cell suspension.
Cell counting: place the special-purpose coverslip of counting in blood bead counting chamber central authorities, with glass siphon, draw 0.1ml cell suspension, allow siphon on coverslip or the counting chamber groove of downside flows out suspension, be full of by liquid to coverslip.Total cellular score under microscope in four jiaos of large grids of counting.Only count and reaching the standard grade and left line person for the cell of line ball.By following formula, count: cell density=(total cellular score/4) * 10 4(individual/ml)
2.5.2MTT detection method
4h before Contained Serum effect 12h finishes, every hole adds the MTT that final concentration is 0.5mg/ml, continue to cultivate 4h and carry out chromogenic reaction, then suck former culture medium, every hole adds 100% dimethyl sulfoxide 200 μ l, mixes and treats that first a ceremonial jade-ladle, used in libation crystalline particle dissolves, after standing 4 hours, be put on microplate reader, detect the absorbance in each hole under the light absorption value of 560nm.Contained Serum is calculated as follows the rate of increase of PC12 cell:
The rate of increase (%)=OD contained Serum-OD without medicine serum, contrast/ OD without medicine serum, contrast* 100%.
Same method, observe Contained Serum effect 24h, the impact of 48h on the PC12 cell proliferation.
2.5.2 the impact of Contained Serum on the PC12 cell proliferation
After cultivation in culture bottle is paved into monolayer, abandon former culture medium until the PC12 cell, PBS liquid swings and washes once gently.0.25% trypsin that adds 1ml, digest 2-3min under 37 ℃ of conditions, until cell monolayer is loosening when being lumps and floating, adds blood-free medium to stop digestion.With suction pipe piping and druming, making cell be single dispersion for several times, count under inverted phase contrast microscope, is 1 * 10 with serum-free medium by cell dilution 6individual cell/ml, be inoculated in 96 well culture plates, every hole 200 μ l.
The PC12 cell be inoculated in 96 well culture plates is divided into 7 groups at random, every group of 8 holes, and by following scheme administration: 1. first group adds complete culture medium group (I); 2. second group adds 5% blank serum (II); 3. the 3rd group adds 10% blank serum (III); 4. the 4th group of 20% blank serum (IV); 5. the 5th group adds 5% Contained Serum (V); 6. the 6th group adds 10% Contained Serum (VI); 7. the 7th group adds 20% Contained Serum (VII).Jointly hatch 12,24, after 48h, carry out inverted phase contrast microscope observation of cell form and carry out cell counting, MTT detects cell proliferation rate.
2.5.3 the neurovirulent protective effect of medicine of the present invention to the PC12 cell model
Select the PC12 cell to be paved with the culture hole of monolayer, be divided at random 8 groups, establish 8 holes for every group: 1. matched group (I); 2. A β model group (II); 3. the blank serum group of A β+5% (III); 4. the blank serum group of A β+10% (IV); 5. the blank serum group of A β+20% (V); 6. A β+5% Contained Serum group (VI); 7. A β+10% Contained Serum group (VII); 8. A β+20% Contained Serum group (VIII).Suck former culture medium, after PBS liquid swings gently and washes once, every hole adds serum-free medium 1ml, and after effect 30min, each group first adds A β and makes final concentration is 20 μ M effect 48h, then adds Contained Serum and the blank serum of variable concentrations.After jointly hatching 24h, carry out morphological observation, MTT, flow cytometer detection and immunohistochemical staining.
Calculate each group of blank serum to A β according to following formula 25-35cause the survival rate of PC12 cell injury:
Survival rate=OD blank serum group-ODA β/OD complete medium-OD a β* 100%
Calculate the WEIDANTANG Contained Serum to A β according to following formula 25-35cause the survival rate of PC12 cell injury: survival rate=OD a β+contain medicine serum-OD a β+blank serum group/ OD blank serum group-OD a β+blank serum group* 100%.
2.5.4 medicine of the present invention is to A β 25-35induce the apoptotic impact of NG108-15 cell model
2.5.4.1 the flow cytometer detection method is with embodiment 8.
2.5.4.2Caspase-3 the immunohistochemical staining of albumen is with embodiment 8.
2.6 statistical procedures is with embodiment 9.
3 results
3.1 the impact of Contained Serum on the PC12 cell proliferation
3.1.1 morphological observation
Under inverted phase contrast microscope, observe, complete medium group PC12 cell is the rapid adherent growth of 24h energy after inoculation, form is fusiformis mostly, minority is triangular in shape, and while cultivating 48h, cell is paved with monolayer, and cell all increases well, endochylema is even, bright, cell peripheral is the fine hair shape and short projection is arranged, and cell surface is without obviously speckle and deposit, nothing or a little fragment is only arranged some holes in.Blank serum group cell growth condition and complete medium category are seemingly.The PC12 cell is after adding the Contained Serum of variable concentrations, and cellular morphology is without significant change, and the not damaged performance, grow fine, and when Contained Serum is cultivated 48h, Growth of Cells is vigorous, is fusiformis, triangle growth, is paved with monolayer, and while cultivating 72h, the speed of growth is gradually slow.See accompanying drawing 12.
3.1.2MTT automatic fine method testing result
Result is as shown in table 12-1, and Contained Serum adds 12h in culture medium, 24h, 48h, all can promote the growth of PC12 cell, and is certain dose dependent.No significant difference between complete medium group and blank serum group (P>0.05); The Contained Serum group, apparently higher than complete medium group and blank serum group, has significant difference (P<0.01).Between the Contained Serum group of three kinds of variable concentrations, 5% Contained Serum cell inhibitory rate is minimum, and 20% Contained Serum cell proliferation rate is the highest.
Table 12-1 variable concentrations Contained Serum to the comparison of PC12 cell proliferation rate (
Figure BSA00000451126400391
n=8)
Figure BSA00000451126400392
Annotate: with complete medium, compare, △p<0.05, △ △p<0.01 is compared with blank serum matched group, ★p<0.05, ★ ★p<0.01
3.2 medicine of the present invention is to A β 25-35the effect of the PC12 cell model neurotoxicity protection of inducing
3.2.1 cellular morphology is observed
Cell is without A β 25-35when fragment is cultivated, normal rat serum group and Contained Serum are respectively organized cell and are all increased well, and endochylema is even, bright, and obvious halation is arranged, karyon is clear, the cell space refractivity is good, full, and is rich in third dimension, cell peripheral is the fine hair shape and short projection is arranged, and increase into network-likely, cell surface is without obviously speckle and deposit, but Contained Serum group cell is relatively better than normal group growth situation.Having under A β toxic action, A β group cultured cells is gathered the cluster shape, and the most neurons cell space dwindles, and cell process shortens and be few, chap, fracture or disappearance.The PC12 cell number that normal group serum control medium is processed obviously reduces, and cell edges is dim, spottiness, can see blackening, ruptured cell.And the Growth of Cells situation of Contained Serum group is compared with blank serum group and will be got well, and blackening, smudge cells are relatively less.See accompanying drawing 13.
3.2.2 the impact (trypan blue staining) of Contained Serum on cell mortality
Observe dead cell under microscope and be blue, and calculate at 20 μ M A β by blood bead counting chamber 25-35after effect 48h, 24h is to the PC12 cell mortality for the effect of variable concentrations Contained Serum.In Table 12-2.
Table 12-2 variable concentrations Contained Serum on the impact of cell mortality (n=8,
Figure BSA00000451126400401
%)
Figure BSA00000451126400402
Annotate: with complete medium group ratio △p<0.05 △ △p<0.01 and model group ratio ▲p<0.05 ▲ ▲p<0.01
With blank serum matched group ratio ★p<0.05 ★ ★p<0.01
3.2.3 the impact on the PC12 survival rate
20 μ M A β for the PC12 cell 25-35process after 48 hours, then make MTT and measure, the OD value is converted into to cell survival rate (%).Found that 20 μ M A β 25-35cell viability is descended, and Contained Serum is to A β 25-35the cytotoxicity caused has significant protective effect.
Table 12-3 variable concentrations Contained Serum on the impact (%) of cell survival rate (
Figure BSA00000451126400403
n=8)
Figure BSA00000451126400404
Annotate: with complete medium group ratio △p<0.05 △ △p<0.01
With the model group ratio ▲p<0.05 ▲ ▲p<0.01
With corresponding blank serum matched group ratio ★p<0.05 ★ ★p<0.01
3.3 medicine of the present invention is to A β 25-35induce the impact of PC12 cell AD model cell apoptosis
3.1.1 flow cytometer testing result
From table 12-4, can find out, the PC12 cell is through A β 25-35the hypodiploid peak occurs in left side, G1 peak after processing, the apoptosis rate of Contained Serum group has obvious reduction (P<0.01) than model group.And reduction (P<0.01) in various degree all arranged between the variable concentrations group.Each group of Contained Serum is compared with corresponding each group of blank serum, acts on more obviously, and significant difference (P<0.01) is arranged.See accompanying drawing 14.
Table 12-4 variable concentrations Contained Serum on the impact of apoptosis rate (n=8,
Figure BSA00000451126400411
%)
Figure BSA00000451126400412
Annotate: with complete medium group ratio △p<0.05 △ △p<0.01
With the model group ratio ▲p<0.05 ▲ ▲p<0.01 and blank serum matched group ratio ★p<0.05 ★ ★p<0.01
3.1.2Caspase-3 SABC testing result
Caspase-3 positive expression neuron is that endochylema and after birth are dyed brown color, and this laboratory observation arrives through A β 25-35the equal positive expression of PC12 cell caspase-3 of processing.The normal saline matched group also has a small amount of positive expression, but paler colour.After the Contained Serum effect, can partly reduce the expression of caspase.Under high power lens, random 5 visual field positive neuron countings as table 12-5, have statistical significance through the t check.See accompanying drawing 15.
PC12 cell Caspase-3 expression in each group of table 12-5 (n=8,
Figure BSA00000451126400413
)
Figure BSA00000451126400414
Annotate: with complete medium group ratio △p<0.05 △ △p<0.01
With the model group ratio ▲p<0.05 ▲ ▲p<0.01 and blank serum matched group ratio ★p<0.05 ★ ★p<0.01
4 discuss
At first this experiment utilizes the A β of preliminary ageing 25-35directly induced PC 12 cells apoptosis, to set up the AD cell model, utilizes the indexs such as morphology, flow cytometer to support apoptosis to participate in the viewpoint of AD morbidity.Then use ways of Chinese herbal medicine serum pharmacology to inquire into the treatment mechanism of invention medicine to AD.Result shows, Contained Serum can have proliferation function and to A β to the PC12 cell 25-35induce and cause damage AD model protective effect is arranged.We have observed the rate of increase of the PC12 cell of variable concentrations (5%, 10%, 20%) Contained Serum cultivation, and result shows, with the situation of above-mentioned AD cell model, compares, and the variable concentrations Contained Serum can resist A β to a certain extent 25-35damaging action to cell; But respectively organize differently, in the 5%-20% concentration range, its protective effect is to increase along with the increase of concentration, is certain concentration dependent.
Simultaneously, from morphology and mtt assay, we observe neuron after A β processes, and cell survival rate reduces, the visible hypodiploid of flow cytometer peak; And Contained Serum can suppress the PC12 apoptosis by the beta induced AD model of A, increase the PC12 cell survival rate that A β fragment toxicity is induced.Result confirms that Contained Serum can alleviate the neurotoxicity of cell to A β, shows that the invention medicine brings into play the therapeutical effect to AD by the pathology of antagonism AD.
Apoptosis by Flow Cytometry representation model group apoptosis rate has utmost point significance to raise, and the pathologic basis of prompting AD may come from the Neuron Apoptosis due to A β.And WEIDANTANG pastille cerebrospinal fluid and Contained Serum group cell survival rate are high than model group, apoptosis rate, than model group low (p<0.05), affirms that from the angle of serum pharmacological the invention medicament protection is by A β 25-35the effect of the neuronal apoptosis of inducing, for the clinical practice of invention medical treatment alzheimer disease provides theoretical foundation.
In addition, we have observed PC12 cell A β 25-35the protein expression of the caspase-3 induced, result shows, at AD cell model group Caspase-3 positive expression.Also find, Contained Serum can reduce the positive expression of the beta induced apoptosis of A and caspase-3, and the prompting Contained Serum may be to express by activating caspase-3, and inhibited apoptosis.This provides new foundation and new research approach for the invention medicine is used for the treatment of AD.
Embodiment 16 medicine of the present invention is to A β 25-35effect NG108 induces AD cell model neuroprotective research experiment
1 experiment material
1.1 cell strain
NG108-1 neuroma cell strain is provided by Wuhan University's Chinese Typical Representative culture collection center.
1.3 main agents and instrument
The Mus Bcl-2 of the rabbit Chinese People's Anti-Japanese Military and Political College and Bax polyclonal antibody, Wuhan doctor's moral company.All the other are same with embodiment 15.
1.4 laboratory animal
12 of healthy clean level white big ear rabbits, body weight 2.5-3kg, male and female are not limit, purchased from Tongji Medical College, Huazhong Science and Technology Univ.'s Experimental Animal Center.
1.5 Experimental agents
Preparation Example 3 medicines, 4 ℃ save backup.
1.6 main solution preparation is with embodiment 15.
2 methods
2.1NG108-15 the cultivation of cell
Cell is bought rear put into rapidly 37 ℃, 5%CO back 2in incubator, standing adaptability is cultivated 1d, and its later half amount is changed liquid, after in former culture bottle, cell reaches the standard of going down to posterity, cell is inoculated in to 25cm 2in culture bottle, cell density is 2 * 10 5/ ml, add 10% hyclone with the DMEM culture fluid and cultivate.According to the Growth of Cells situation, 1-2d changes culture fluid, and 2-3d goes down to posterity once.Cell counting under inverted microscope, adjust cell density, and cell is inoculated in to culture bottle or culture plate, the adherent situation of observation of cell under microscope, and the 8-12 cell enters exponential phase and can be used for experiment.
2.2AD the foundation of cell model is with embodiment 15.
2.3 the preparation of pastille rabbit anteserum and pastille rabbit cerebrospinal fluid
2.3.1 the preparation of pastille rabbit anteserum
2.5-3kg white big ear rabbit, adaptability is divided into two groups: blank group and Chinese drug-treated group after feeding 3d at random.Chinese medicine gavages group and gives the dense decocting liquid 4.5g/kgd of Chinese medicine gavage, every day 2 times, 3d continuously.Fasting 12h before the last administration, but can't help water, after last gavage 1h, 10% chloral hydrate (1ml/kg) anesthesia, aseptic heart blood sampling 50ml/ only, collect 4 ℃ of blood and spend the night by centrifuge tube, treats that its serum fully separates out, 3000rpms * 15min is centrifugal, collect serum, 56 ℃, 30min deactivation complement, 0.22 the packing of μ m filtering with microporous membrane ,-20 ℃ save backup.Blank group, with the normal saline gavage of equal volume, is extracted serum with same method after the identical time, and gained is blank serum.
2.3.2 the collection of pastille rabbit cerebrospinal fluid
After gavaging the intracardiac blood sampling of Chinese medicine, connect No. 7 syringe needles with Dispensable 1 ml syringe rapidly, under aseptic condition from the vertical slowly inserting needle of rabbit Foramen magnum, during puncture cerebellomedullary cistern success, syringe needle has the sense that falls through, slowly extract cerebrospinal fluid, every rabbit is collected cerebrospinal fluid 800-1000 μ l, injects rapidly sterilizing 1.5mlEP pipe, and-70 ℃ frozen standby.Blank group is collected cerebrospinal fluid with same method, and gained is blank cerebrospinal fluid.
2.4 experiment detection method
2.4.1 cell directly is placed in its form of micro-Microscopic observation counting
Prepare cell suspension: stop cultivating, by the culture medium sucking-off, with PBS, softly clean culture once.Add the 1ml0.25% trypsin in culture bottle, under 37 ℃ of conditions, digest 3-5min, when the Microscopic observation cell rounding approaches de-wall, add the culture medium of 4.5ml, with suction pipe softly piping and druming repeatedly, make cell take off wall and make cell suspension.
Cell counting: place the special-purpose coverslip of counting in blood bead counting chamber central authorities, with glass siphon, draw 0.1ml cell suspension, allow siphon on coverslip or the counting chamber groove of downside flows out suspension, be full of by liquid to coverslip.Total cellular score under microscope in four jiaos of large grids of counting.Only count and reaching the standard grade and left line person for the cell of line ball.By following formula, count: cell density=(total cellular score/4) * 10 4(individual/ml)
2.4.2MTT detection method
4h before Contained Serum effect 12h finishes, every hole adds the MTT that final concentration is 0.5mg/ml, continue to cultivate 4h and carry out chromogenic reaction, then suck former culture medium, every hole adds 100% dimethyl sulfoxide 200 μ l, mixes and treats that first a ceremonial jade-ladle, used in libation crystalline particle dissolves, after standing 4 hours, be put on microplate reader, detect the absorbance in each hole under the light absorption value of 560NM.Contained Serum is calculated as follows the rate of increase of PC12 cell:
The rate of increase (%)=OD contained Serum-OD without medicine serum, contrast/ OD without medicine serum, contrast* 100%.
Same method, observe Contained Serum effect 24h, the impact of 48h on the PC12 cell proliferation.
2.5 the protective effect of medicine of the present invention to the N6108-15 cell model
Set up 6 experimental grouies respectively: blank group, model group, blank cerebrospinal fluid group, pastille cerebrospinal fluid group, blank serum group, Contained Serum group.The cerebrospinal fluid group adds respectively blank cerebrospinal fluid, pastille cerebrospinal fluid 100 μ l, then adds complete medium to 5ml.Serum group is changed original fluid with the complete medium containing 10% blank serum, 10% Contained Serum respectively.Except the blank group, all the other each group is all set up the AD cell model in advance, and the A β segment that still adds 5 μ mol/L after pharmaceutical intervention is to maintain the AD model.After each group is carried out respective handling 24h, observation of cell growth conditions take the photograph sheet under inverted phase contrast microscope, carry out the detection of index of correlation.
2.6 medicine of the present invention is to A β 25-35induce the apoptotic impact of NG108-15 cell model
2.6.1 the flow cytometer detection method is with embodiment 15.
2.6.2Bcl-2 and the immunohistochemical staining of Bax albumen is with embodiment 15.
2.7 statistical procedures is with embodiment 9.
3 results
3.3 the invention medicine is to A β 25-35the effect of the NG108-15 cell model of inducing
3.3.1 the impact on cellular morphology
Cell is without A β 25-35blank group growth background clear, cell enlargement is good, the full refractivity of cell space is good, endochylema is even, bright, karyon is clear, the part cell peripheral has short projection, cell surface is without obviously speckle and deposit.Under aβ protein segment neurotoxic effect, it is bad that all the other respectively organize the Growth of Cells background, visible some dead cells and cell debris are floating, the NG108-15 cell number obviously reduces, and gathers the cluster shape more, and the most neurons cell space dwindles, endochylema is dimer, spottiness, have than the de-wall hydro-planing of many cells, has the neurocyte that projection is stretched out obviously to reduce.And wherein the pastille cerebrospinal fluid and Contained Serum group Growth of Cells situation better, death, blackening, fragmented cell are relatively less, have the cell of projection elongation relatively many.Counting is respectively organized the ratio that bossed cell number accounts for total cell number, the results are shown in Table 13-1.See accompanying drawing 16.
The impact of table 13-1 invention medicine on NG108-15 cell process rate (%)
Figure BSA00000451126400441
Figure BSA00000451126400442
Annotate: with the blank group, compare: ★p<0.05; With model group, compare: ▲p<0.05.
Through between group, the t assay shows, with the blank group, compares, and all the other each groups have the protrusion cell ratio to reduce, and A β is described 25-35neurotoxic effect suppressed the enation of NG108-15 cell.And Contained Serum group and pastille cerebrospinal fluid group and model group are relatively, the cell process rate all increases, its difference has significance (p<0.05), and these results show that the invention medicine can promote the growth of NG108-15 cell process, also can alleviate the inhibition of A β to cell process simultaneously.
3.3.2 the impact on cytoactive
Mtt assay detects cell survival rate, represents the cytoactive in this hole with the absorbance (OD value) in each hole, and the higher cell survival rate of OD value is higher, in Table 2-17.Result shows: Contained Serum group and pastille cerebrospinal fluid group OD value are significantly higher than model group (p<0.05), illustrate that invention medicine Contained Serum and pastille cerebrospinal fluid all can protect the NG108-15 primary cellular defect of being induced by A β segment neurotoxicity.Wherein relatively, its OD value has rising trend for pastille cerebrospinal fluid group and Contained Serum group, but not statistically significant (p>0.05).
The impact of table 13-2 invention medicine on NG108-15 cell survival rate (OD value)
Figure BSA00000451126400444
Annotate: with the blank group, compare: ★p<0.05; With model group, compare: ▲p<0.05.
3.2.1 on apoptotic impact
On the flow cytometry rectangular histogram, each group of all the other except the blank group is the apoptotic peak (Ap peak) of a visible hypodiploid of the front appearance in normal diploid cell DNA peak (G1 peak) all.Result shows A β 25-35the neurotoxicity of segment can be induced NG108-15 cellular abnormality apoptosis.Each organizes apoptosis rate in Table 13-3.With model group, compare, pastille cerebrospinal fluid and Contained Serum group apoptosis rate all reduce (p<0.01), and pastille cerebrospinal fluid group Neuron Apoptosis rate is less than Contained Serum group (p<0.05).Result supports MTT to detect the result of cytoactive.Accompanying drawing 17.
The impact of table 13-3 medicine of the present invention on NG108-15 apoptosis rate (%)
Annotate: with the blank group, compare: ★p<0.05 ★ ★p<0.01;
With model group, compare: ▲compare with the Contained Serum group p<0.05: △p<0.05
3.2.2 the impact on apoptosis-related genes Bcl-2, Bax protein expression
Cell cytoplasm is brown color, and it is the bcl-2 positive cell that brown yellow granule is arranged.Bax immunohistochemical staining mode is endochylema, and the cytoplasm of take is the positive staining that inhomogeneous brown color fine particulate or sepia lumps are Bax.Each organizes Bcl-2, Bax stained positive cell number and Bcl-2/Bax ratio in Table 13-4.Accompanying drawing 18.
The impact of table 13-4 medicine of the present invention on NG108-15 cell Bcl-2, Bax protein expression
Figure BSA00000451126400454
Annotate: with the blank group, compare: ★compare with model group p<0.05: ▲p<0.05 ▲ ▲p<0.01
Compare with the blank group, A β is arranged 25-35the Bcl-2 expression of respectively organizing of effect descends, and the expression of Bax raises (p<0.05), shows that A β causes apoptotic neurotoxic effect and the expression that reduces apoptosis suppressor gene Bcl2, and the expression of the short apoptogene Bax of rising is relevant simultaneously.With model group, compare, each serum group and cerebrospinal fluid group neuron Bcl-2 positive expression are counted there was no significant difference (p>0.05), and the expression of pastille cerebrospinal fluid group and Contained Serum group Bax reduces (p<0.05), and Bcl-2/Bax ratio raises.Compare there was no significant difference (p>0.05) between two groups.
4 discuss
From morphology and mtt assay, we observe neuron after A β processes, and cell survival rate reduces, the visible hypodiploid of flow cytometer peak; And the pastille cerebrospinal fluid can suppress the NG108-15 apoptosis by the beta induced AD model of A, increase the NG108-15 cell survival rate that A β fragment toxicity is induced.Result confirms that Contained Serum can alleviate the neurotoxicity of cell to A β, shows that the invention medicine brings into play the therapeutical effect to AD by the pathology of antagonism AD.
Compare with the blank group, A β is arranged 25-35the Bcl-2 that respectively organizes of effect expresses decline, and the expression of Bax raises, prompting A β 25-35one of possible approaches of inducing neuronal apoptosis is the regulation and control to the Bcl-2 gene family, and this is consistent with top viewpoint.Experimental result shows the rising AD model cell bcl-2/bax ratio of invention medicine energy significance, reduces apoptosis rate.In addition; with model group, compare; each serum group and cerebrospinal fluid group cell bcl-2 positive expression are counted no significant difference; the expression of pastille cerebrospinal fluid group and Contained Serum group bax has significant to reduce; the apoptotic effect of NG108-15 that prompting invention medicament protection is caused by A β, may be relevant with the expression of its downward bax gene.
Embodiment 17 medicine of the present invention is to A β 25-35effect NG108 induces the impact experiment of AD cell model signal transduction
1 experiment material
1.1 animal is with embodiment 16.
1.2 cell strain is with embodiment 16.
1.3 Experimental agents is with embodiment 16.
1.4 main agents is with embodiment 16.
1.5 key instrument is with embodiment 16.
2 experimental techniques
2.1 the reagent preparation is with embodiment 16.
2.2NG108 the cultivation of cell is with embodiment 16.
2.3 grouping
Blank group: add the blank cerebrospinal fluid that final concentration is 10% to process 24h.
Model group: the final concentration that adds preliminary ageing 4d is 5 μ mol/LA β 25-35segment is cultivated 24h, and adding afterwards final concentration is that 10% blank cerebrospinal fluid continues to cultivate 24h.
The Chinese medicine high dose group: the concentration that adds preliminary ageing 4d is 5 μ mol/LA β 25-35segment is cultivated 24h, and adding afterwards final concentration is that 20% pastille cerebrospinal fluid continues to cultivate 24h.
Dosage group in Chinese medicine: the concentration that adds preliminary ageing 4d is 5 μ mol/LA β 25-35segment is cultivated 24h, and adding afterwards final concentration is that 10% pastille cerebrospinal fluid continues to cultivate 24h.
The Chinese medicine low dose group: the concentration that adds preliminary ageing 4d is 5 μ mol/LA β 25-35segment is cultivated 24h, and adding afterwards final concentration is that 5% pastille cerebrospinal fluid continues to cultivate 24h.
SP600125 group: be that after 25 μ mol/LSP600125 process 30min, the concentration that adds preliminary ageing 4d is 5 μ mol/LA β with final concentration in advance 25-35segment is cultivated 24h, and adding afterwards final concentration is that 10% pastille cerebrospinal fluid continues to cultivate 24h.
2.4AD copying of model
The A β prepared 25-35solution is hatched 4d prior to 37 ℃, and short it is aging, selects A β 25-35concentration in culture medium is that 5 μ mol/L are as experimental concentration.Selection enters the cell of exponential phase after going down to posterity, add the A β that final concentration is 5 μ mol/L 25-35segment, after continuing to cultivate 1d, build up the AD cell model.
2.5 the extraction of medicine pastille cerebrospinal fluid of the present invention is with embodiment 16.
2.6 detection index
2.6.1 SABC detects the expression of P-JNK and P53
In akinete, JNK is positioned cytoplasm and nucleus, once just be shifted in nucleus after being phosphorylated reacting activation, promotes the expression of gene and synthesizing of new albumen by the phosphorylation to transcription factor, and promotes or cause apoptosis.Being positioned nuclear p53 is one of apoptosis-induced target gene substrate of JNK, can see that by the chromogenic reaction to antigen antibody complex the cell core of positive expression is dark brown.
2.6.2 flow cytometer detects cell cycle
Collect the NG108-15 cell that each group is cultivated, the centrifugal 5min collecting cell of 1000rpm, after PBS liquid washing 2 times, 0 ℃ of the ice ethanol with 70% is 24h fixedly, and before upper machine, the centrifugal 5min of 1000rpm is with removal ethanol, after PBS liquid washing 2 times, add 0.5ml propidium iodide dyeing liquor, lucifuge is placed 30min, 300 order nylon net filters again, upper machine is measured cell cycle distribution, 10000 cells of each pattern detection.
2.7 statistical method is with embodiment 9.
3 results
3.1 the impact of medicine pastille cerebrospinal fluid of the present invention on p-JNK and p53 expression
As show as shown in 14-1, the expression of model group p-JNK and p53 is apparently higher than other each group (P<0.01), there is utmost point significant difference between low, middle dosage group and SP600125 group, high dose group and SP600125 is the most approaching but still there were significant differences, SP600125 group and difference not statistically significant between blank group.
Table 14-1 respectively organize SABC cell dyeing positive rate analyze (
Figure BSA00000451126400471
%)
Figure BSA00000451126400472
Annotate: with model group, contrast ▲p<0.01 contrasts with the SP600125 group ★p<0.01 ☆p<0.05
4 discuss
Medicine pastille cerebrospinal fluid of the present invention has protection A β 25-35the effect of the NG108-15 damaged nerve cell wound of inducing.The JNK signal transduction pathway has been got involved A β 25-35the NG108-15 neuronal apoptosis of inducing, and activated its downstream substrate p53, thereby cell the is started program of self-regeneration, make the cell apoptosis that can't repair, cell cycle is blocked, and invention medicine pastille cerebrospinal fluid can improve the interior environment of cell, the expression of p-JNK and p53 is lowered, thereby reduced A β 25-35to the degree of injury of NG108-15 neurocyte, and this protective effect has presented dose dependent.But from high dose group and inhibitor group relatively, there is the significance difference opposite sex between them, the β through A is described 25-35nG108-15 neural cell injury process after inducing is not reversed fully.
The impact experiment of the embodiment 18 medicine of the present invention side of tearing open on AD rat model learning and memory and cholinergic system
1 material
1.1 laboratory animal
15 100 of months aged Wistar rats, male and female half and half.Body weight 400 ± 50g, provided by Tongji Medical College, Huazhong Science and Technology Univ.'s Experimental Animal Center.
1.2 medicine, reagent and instrument
The invention medicine is comprised of Radix Polygoni Multiflori Preparata, Rhizoma Acori Graminei, Rhizoma Panacis Japonici, Rhizoma Chuanxiong, Cortex et Radix Polygalae (processed) etc., and Rhizoma Panacis Japonici is purchased from the enshi city, and all the other are fine quality all purchased from medical expert's consultation section of Hubei College Of Traditional Chinese Medicine.Huperzine A-Zhulin Antun, pharmaceutical factory in Henan all living creatures Pharmacy stock Co., Ltd Henan, lot number: 060321.The deep propylhomoserin (Ibotenic acid, IBO) of goose cream and A β 25-35all from Sigma company, order.The automatic experimental record instrument of treated rats in Morris water maze performance provides for the Chinese Academy of Medical Sciences.Ach, AchE and ChAT radioimmunological kit are purchased from Military Medical Science Institute.Brain solid positioner is provided by U.S. TPI company.
2 methods
2.1AD model construction is with embodiment 9.
2.2 animal grouping and administration
15 100 of months aged Wistar rats.Be divided at random 10 groups, it is normal group, sham operated rats, model group, the Huperzine A-Zhulin Antun group, the former side's group of brain-care (Radix Polygoni Multiflori Preparata, Rhizoma Acori Graminei, Rhizoma Panacis Japonici, Rhizoma Chuanxiong, Cortex et Radix Polygalae (processed)), the kidney invigorating group (Radix Polygoni Multiflori Preparata, Rhizoma Panacis Japonici), the group of reducing phlegm (Rhizoma Acori Graminei, Radix Polygalae), the group of invigorating blood circulation (Rhizoma Panacis Japonici, Rhizoma Chuanxiong), the kidney invigorating reduce phlegm group (Radix Polygoni Multiflori Preparata, Rhizoma Panacis Japonici, Rhizoma Acori Graminei, Radix Polygalae) and invigorating kidney, promoting blood circulation group (Radix Polygoni Multiflori Preparata, Rhizoma Panacis Japonici, Rhizoma Chuanxiong), 10 every group.Each is organized the equal decocting of medicine and makes concentrated solution (being equivalent to containing crude drug 1g/ml).Intracerebral injection administration after 2 weeks.6 groups, Chinese medicine is respectively to this group medicine decocting liquid of rat oral gavage; And Huperzine A-Zhulin Antun suspension (0.036mg/ml) for Huperzine A-Zhulin Antun group gavage; Normal group, sham operated rats, model group all give isometric normal saline gavage.Dose is definite by people and rat body surface area coefficient ratio, and each organizes rat all by the 1ml/100gd gavage.Continuous 28 days.
2.3 detect index and method thereof
2.3.1Morris water maze test is with EXPERIMENTAL EXAMPLE 6.
2.3.2 rat hippocampus district Ach, AchE and ChAT determination of activity
Adopt radioimmunology.Separate hippocampal tissue, weigh, homogenate, make 10% homogenate.Getting supernatant detects.
2.4 statistical disposition is with embodiment 9.
3 results
3.1 the impact of the medicine of the present invention side of tearing open on the learning and memory in rats level
In experiment a few days ago, each treated animal is all substantially around pool wall swimming, and less trip is near platform, and its movement locus is and is randomly distributed among all quadrants.But along with the propelling of experiment, each is organized rat and relies on spatial cues to find the time of position of platform to shorten gradually, and its movement locus also slowly is positioned at the platform quadrant, or adjacent left and right sides quadrant is found at the platform quadrant, and descend its incubation period rapidly.But the model group rat is except only a few can find platform, and the situation while substantially keeping a few days ago, slightly be improved.For the analytical information acquisition capability, measured total incubation period and the training end rear incubation period of 2 days of training in 4 days.Statistical result showed, normal group and sham operated rats no significant difference, and model group incubation period all higher than other each group, tool significant difference (P<0.05).The group of invigorating blood circulation, higher than the Huperzine A-Zhulin Antun group, relatively has notable difference (p<0.05).The kidney invigorating group, the group of reducing phlegm and invigorating kidney, promoting blood circulation group with Huperzine A-Zhulin Antun group no significant difference (p>0.05), and reduce phlegm group and former side's group and Huperzine A-Zhulin Antun group of the kidney invigorating has significant difference (P<0.05), but comparison zero difference (p>0.05) between two groups.
In orientation navigation test, observed experimental rat and always accounted for apart from percentage ratio in the swimming distance of test platform quadrant, pool wall 20% and 40% zone swimming is apart from percentage ratio.From testing, normal group can rely on spatial cues to find position of platform, and its movement locus is positioned at most the platform quadrant, and the model group animal is seldom swum near platform, and its movement locus is and is randomly distributed among all quadrants.Each medication group mainly concentrates on the left and right sides quadrant that the platform quadrant is adjacent and finds, and major part can find platform, but between each group, different is arranged.Statistical result showed, each group show with on the table incubation period essentially identical trend.Difference be 20% when zone the kidney invigorating group and the Huperzine A-Zhulin Antun group significant difference (P<0.05) is arranged, but be worse than former side's group and the kidney invigorating group (P<0.05) of reducing phlegm.And invigorate blood circulation group and model group zero difference (p>0.05).
Table 15-1 rat orientation navigation test ability of learning and memory result (
Figure BSA00000451126400491
s)
Figure BSA00000451126400492
In the space search experiment, the swimming distance of platform quadrant accounts for always apart from percentage ratio, pool wall 20% is tested each group with 40% zone swimming apart from percentage result and orientation navigation and is shown consistent trend, and each group of model and other is than notable difference (P<0.05) is arranged.The kidney invigorating group, the group of reducing phlegm, invigorating kidney, promoting blood circulation group and Huperzine A-Zhulin Antun group comparing difference not obvious (p>0.05).Each is organized rat and strides across original platform position number of times and compare in orientation navigation experiment obviously and descend.The model group rat only has only a few to pass, and each medication group is at platform circuit that quadrant is swum and to stride across the platform number of times obviously many than model, shows than model group memory ability preferably.
Table 15-2 rat space search test ability of learning and memory result
Figure BSA00000451126400493
Figure BSA00000451126400494
3.2 the impact of the invention medicine side of tearing open on the hippocampus cholinergic system
This measuring respectively organize Ach, ChAT and the AchE activity of rat hippocampus, statistical result showed, model group hippocampus Ach compares with each group with ChAT and all significantly reduces (P<0.05), AchE activity obviously raise (P<0.05).Normal group and sham operated rats no significant difference (p>0.05).Chinese medicine each group and Huperzine A-Zhulin Antun group comparing difference not obvious (p>0.05).And the kidney invigorating is reduced phlegm group and former side's group and sham operated rats without significant difference (p>0.05), be close to sham operated rats level (p>0.05), but do not reach yet normal group level (P<0.05).But compare zero difference (p>0.05) between two groups.
Table 15-3 rat hippocampus district Ach, ChAT and the active result of AchE
Figure BSA00000451126400501
4 discuss
This experimental result shows, medicine of the present invention is respectively torn Fang Junneng open and improved to a certain extent AD rat model action learning level, improving aspect the cholinergic system damage, and each group of Chinese medicine also shows effect preferably.But with the former side of invention medicine and invention effect of drugs the best, between the two without significant difference.The Comprehensive Experiment result, we think that the effective ingredient that the party acts on is the kidney invigorating apophlegmatisant Radix Polygoni Multiflori Preparata, Rhizoma Panacis Japonici, Rhizoma Acori Graminei, Radix Polygalae, can be used as the best prescription of next step research.
Embodiment 19
Medicine of the present invention is to the old dementia patients lanqin oral solutions
Alzheimer disease refers to that the geratic period take a kind of chronic progressive external encephalopathy that cognitive dysfunction is main manifestations.
1 research standard
1.1 diagnostic criteria
Western medicine diagnose standard: adopt the 4th edition diagnostic criteria about Alzheimer ' s Disease (AD) and Vaseular dementia (VD) of the CDSM-IV of American Psychiatric Association's " diagnostic & statistical manual of mental disorder " [, 1. both ways, dysmnesia and Cognitive function damage at least possess following one (aphasia, apraxia, agnosia, abstract thinking or judgment infringement) in the cognitive dysfunction performance.2. above-mentioned two class cognitive dysfunction have obviously disturbed occupation and social activity, or compare obviously and go down in the past with the individual.3. just do not occur among the course of disease of delirium.4. above-mentioned infringement can not be explained (as depression, schizophrenia etc.) by other spirit and thymopathy.
The tcm diagnosis standard: " Research of Senile Dementia Treated diagnosis, differentiation of symptoms and signs for classification of syndrome and the curative effect determinate standard " that adopt Gerontological Society of 1990-05 All-China Association of Traditional Chinese Medicine to formulate meets
1.2 deciding degree standard
Adopt the clinical dementia evaluation form, CDR=0 is that CDR=0.5 is suspicious dementia without dull-witted, and CDR=1 is mild dementia, and CDR=2 is moderate dementia, and CDR=3 is severe dementia.
1.3 judging standard
1. adopt Hachinski IS scale (HIS) difference vascular dementia (VD) and Alzheimer (AD) disease, the 7 minutes persons that mark are vascular dementia; 5,6 are divided into Mixed dementia; 4 are divided into Alzheimer.2. adopt depression except Cornell depression scale (CSDD), >=8 are divided into depression.
1.4 inclusive criteria
The all cases of clinical data all meet the DSM-IV-R diagnostic criteria and NINCDS-ADRDA is light, moderate AD, and MMSE marks between 15～25, HIS (Hachinsk) ischemia scale≤4; CSDD depression scale scoring<8 minutes; In Syndrome Differentiation of Traditional Chinese Medicine, deficiency of marrow-reservoir, turbid phlegm blocking the clear orifices, people who gets stagnation of vital energy and blood stasis are all as observing treatment target.
1.5 exclusion standard
1. dull-witted severe (CDR=3) or the patient of serious neurologic impairment is arranged, as various aphasias, agnosia etc.; 2. merge intentionally, serious primary disease, the psychotics such as brain, liver, kidney and hemopoietic system; 3. include case generation drug anaphylaxis or other severely adverse events in, develop complications and should not continue the reception test case; 4. other reasons has caused the whole course for the treatment of and has affected the treatment or the case of safety judgement.But surpassed 1/2 the course for the treatment of person add up curative effect.
2 clinical datas
All cases are from the attached institute of the Hubei traditional Chinese medical science, mental hospital, Shashi, Hubei, the central hospital of autonomous prefecture that bestows favour, each relevant section office in-patient department of Wuhan City No.1 Hospital.Choose senile dementia patients totally 56 examples that meet above-mentioned standard, by random method fully, patient is divided into to two groups, medicine instant treatment group 30 examples of the present invention, male's 18 examples wherein, women's 12 examples; 72 ± 8.3 years mean aves, average course of disease 1.26 ± 0.13 years, schooling 9.36 ± 4.73 years, dull-witted slight 10 examples, moderate 20 examples.Piracetam matched group 26 examples, male's 15 examples wherein, women's 11 examples; 71 ± 11.5 years mean aves, average course of disease 1.31 ± 0.28 years, schooling 9.8 ± 4.85 years; Slight 9 examples, moderate 17 examples.Epidemiological Analysis by statistics, two groups of no significant differences on age, sex, average course of disease, schooling, severity extent (P>0.05), have comparability.
3 research methoies
3.1 Therapeutic Method
Treatment group is taken the medicine of the present invention (product of Preparation Example 1.The Drug Manufacturing Room of Hubei Province institute of traditional Chinese medicine produce, and supervise Hubei College Of Traditional Chinese Medicine's Gerontological Research Center chamber, lot number: 2001006), and every day 2 times, each 6g; Matched group is taken Piracetam Tablet (produced lot number by east doctor group Dongbei Pharmaceutical General Factory: 200006066, every containing piracetam 0.4g), and each 0.4～0.8g, every day 3 times, 3 months be a course for the treatment of.With diabetes, hypertension patient, can add with blood sugar lowering, antihypertensive drugs stop using during treatment other cerebral vasodilators and nootropics.All patients all coordinate psychological counseling and intelligent training; As set up warm home care, require family numbers of patients often with it, to exchange emotion, comfort and care patient; And according to patient's schooling, hobby etc., encourage to listen to the music, to see TV, do housework, read, read newspaper etc.
3.2 observation index is measured
3.2.1 safety is observed
General procuratorial work project: blood, urine, just routine test; The heart, liver, kidney function test.
3.2.2 health giving quality is observed
3.2.2.1 intelligence and viability detect
Before and after treatment respectively according to 20, simple and easy Mental status schedule (MMSE) and daily life self-care ability table (ADL) [86]check scoring, formulate form, be responsible for carrying out by the special messenger.
3.2.2.2 check the blood Main Biochemical
(serum cholesterol-employing CHOD-PAP method, triglyceride-employing GPO-PAP method, high density lipoprotein-employing phosphotungstic acid magnesium precipitate method detect to adopt the enzymatic assays blood fat, medicine box is provided by Beijing Zhongsheng Biological Engineering High Technology Company), adopt cone-plate method mensuration whole blood viscosity, plasma viscosity packed cell volume to adopt.Measure lipid peroxide (LPO) content by the TBA method, adopt the purine oxidase method to measure SOD in serum, medicine box is purchased from attached Concord Hospital of the Central China University of Science and Technology, and by specification operates.
4 criterions of therapeutical effect and therapeutic outcome
4.1 criterion of therapeutical effect
4.1.1 clinical efficacy standard
" efficacy assessment standard of Research of Senile Dementia Treated " according to Chinese Chinese medicine Gerontological Society of association and Society of Internal Medicine's revision in May nineteen ninety carried out.Produce effects: cardinal symptom is recovered substantially, conscious, directed sound, answers a question correct, is quick on the draw, and takes care of oneself, and can carry out the mass society activity; Effectively: the main spirits symptom alleviates to some extent or part disappears, and life is taken care of oneself substantially, answering a question is in the main true, but reaction is still blunt, and intelligence and personality still have partial impairment; Invalid: cardinal symptom has development without change or the state of an illness, can't take care of oneself, answer a question correct not, and mind moronism.
4.1.2 simple and easy Mental status schedule scoring
Score value is treated front increase >=5 and is divided into produce effects, increases by 2～4 and is divided into effectively, and score value increase<2 or minimizing person are invalid.
4.1.3 daily life self-care ability Table A DL20 item rating
After treatment, total points descends and >=6 to be divided into produce effects, descends to >=3 being divided into effectively, descends<3 minutes or ascensionist is invalid.
5 statistical methods
Mean ± standard deviation for measurement data
Figure BSA00000451126400521
mean, relatively reach cross-reference between group, with the t check, enumeration data adopts X 2check.
6 therapeutic outcomes
6.1 two groups of curative effects are relatively in Table 16-1
Two groups of curative effects of table 16-1 are [n (%)] relatively
Figure BSA00000451126400522
Annotate: two groups relatively ※p<0.05
6.2 before and after two groups of treatments, MMSE, ADL integral contrast are in Table 16-2
Before and after two groups of treatments of table 16-2, MMSE, ADL integral contrast (divide
Figure BSA00000451126400523
)
Figure BSA00000451126400524
Annotate: with this group before treatment ※ ※p<0.01
With comparison after treatment of control group △p<0.05 △ △p<0.01
6.3 before and after two groups of patient treatments, the blood Main Biochemical compares, in Table 16-3
Before and after two groups of treatments of table 16-3, the blood Main Biochemical relatively
Figure BSA00000451126400531
Annotate: with this group before treatment ※p<0.05 ※ ※p<0.01
With comparison after treatment of control group △p<0.05 △ △p<0.01
6.4 before and after two groups of treatments, hemorheology index changes relatively in Table 16-4
Before and after two groups of treatments of table 16-4, hemorheology index changes relatively
Figure BSA00000451126400532
Figure BSA00000451126400533
Annotate: with this group before treatment ※p<0.05 ※ ※p<0.01
With comparison after treatment of control group △p<0.05 △ △p<0.01
6.5 after two groups of treatments, clinical cardinal symptom changes situation in Table 16-5
After two groups of treatments of table 16-5, clinical cardinal symptom change situation relatively
Annotate: with this group before treatment ※p<0.05 ※ ※p<0.01
With comparison after treatment of control group △ △p<0.01
7 conclusions
With the kidney invigorating and essence nourishing, the Chinese medicine compound of phlegm reduction of blood circulation promoting is that the master suffers from and just carries out rehabilitation 30 routine senile dementias.Matched group 26 examples, coordinate intelligent training to carry out rehabilitation with piracetam.The clinical observation discovery, the Chinese traditional treatment group is for patient's dementia symptom, and there is effect preferably the aspects such as activity of daily living, and general curative effect is better than matched group.And, to before and after every patient treatment, having done the inspection items such as Main Blood Biochemical Index, hemorheology, result shows to treat rear serum cholesterol, triglyceride and significantly descends, high density lipoprotein, the active significantly rising of erythrocyte sod, blood plasma LP descend.Can inference according to check result and modern pharmacological research, the mechanism of action of traditional Chinese medicine composition for treating senile dementia of the present invention is as follows: (1) blood fat reducing, prevent and treat arteriosclerosis, vessel softening is conducive to blood circulation; (2) remove free radical, suppress formation and the accumulation of lipofuscin in brain, improve and delay the aging of brain; (3) expansion of cerebral vascular, increase cerebral blood flow, anti-hypoxia, thrombolytic, inhibition thrombosis; (4) strengthen the energy metabolism of brain, improve the cell viability of brain neuron, strengthen citing sb. for meritorious service of brain, improve the intelligence of brain.
Embodiment 20 medicine of the present invention is preferably studied
1 experiment material
1.1 medicine
Medicine, all purchased from Chinese Medicinal Material In Hubei Province company, send professor Chen Keli of Hubei Chinese medicine institute check.Calculate to obtain the mice dose,equivalent by man and animal body surface area coefficient (0.0026).
Positive control drug: Piracetam Tablet.Piracetam Tablet is pulverized, with normal saline, be made into the suspension of every ml containing medicine 24mg, 0～4 ℃ of cold preservation is standby.
1.2 equipment
The Morris water maze, medicine institute of the Chinese Academy of Sciences.
2 experimental techniques
2.1 medicine, animal and grouping
3 the monthly age mice 200, body weight 18～22g, be divided into 20 groups at random, 10 every group.
With Rhizoma Pinelliae (processed with Rhizoma Zingiberis Recens), Caulis Bambusae In Taenia, Fructus Aurantii Immaturus, as substantially square, fix tentatively as A0 side, and separately by the kidney invigorating (Radix Rehmanniae Preparata, Rhizoma Gastrodiae), reduce phlegm (Radix Polygalae, Rhizoma Acori Graminei, Semen Sinapis Albae), invigorate blood circulation (Rhizoma Chuanxiong), the prescription research respectively of QI invigorating (Radix Ginseng) rule for the treatment of medicine.Compile respectively as A0, A1, A2, A3, A4.According to combinatorial principle, tentatively determine and have 16 kinds of different compatibility modes, and establish the former side's group of model control group, positive drug matched group, Normal group and WEIDANTANG.Show after experiment that best prescription fixes tentatively as B side.
2.2 learning and memory horizontal detection
Each organizes administration 10 days, trains once continuous 4 days in the 11st day each treated animal water maze every day.Test in the 5th day, each organizes 40min after mouse stomach, the lumbar injection scopolamine, dosage is 3mg/kg (except normal group), after 30min, with the Morris water maze, is tested, and records its time that searches out platform in 1min (escape latency).6d removes platform, and optional 1 place of entry of rat is put into, and records the number of times of crossing over the original platform position in its 1min.Two experiments are also recorded rat and are always accounted for apart from percentage ratio in the swimming of platform quadrant distance, and pool wall 20% and 40% zone swimming are apart from percentage ratio.
2.3 statistical method
Each is organized data and all adopts
Figure BSA00000451126400541
mean that the analysis of SPSS 17.0 statistical softwares adopts single factor ANOVA to analyze.
3. experimental result
From following table, with the model group ratio, normal group, piracetam group have significant differences (p<0.01) incubation period.7,14,15,17,18,20 groups, Chinese medicine has significant differences (p<0.01) with the model group ratio, and 4,5,6,8,9,16 groups, Chinese medicine has significant difference (p<0.05).Chosen the significant differences Chinese drug-treated group organize in twos between relatively, 7,14,15,17,18 mutual not statistically significants (p>0.05), 20 groups and other group have significant difference (p<0.05).
Figure BSA00000451126400551
4. conclusion
Through screening experiment research, show that the most effective prescription is: 10 parts of Rhizoma Pinelliae (processed with Rhizoma Zingiberis Recens)s, 12 parts of Caulis Bambusae In Taenia, 10 parts of Fructus Aurantii Immaturuss, 12 parts, Radix Rehmanniae Preparata, 10 parts of Radix Polygalaes, 10 parts of Rhizoma Acori Graminei, 10 parts of Rhizoma Chuanxiongs, 6 parts of Radix Ginsengs.

Claims (19)

1. a Chinese medicine composition for the treatment of senile dementia, it is by effective ingredient or also have pharmaceutically acceptable carrier to form, it is characterized in that, its contained effective ingredient is mainly made by the raw material of Chinese medicine of following weight proportioning: Rhizoma Pinelliae 1-15 part, Caulis Bambusae In Taenia 1-15 part, Fructus Aurantii Immaturus 1-15 part, Rhizoma Acori Graminei 1-15 part, Radix Polygalae 1-15 part, Radix Ginseng 1-10 part, Rhizoma Chuanxiong 1-15 part, Radix Rehmanniae Preparata 1-20 part.
2. a kind of Chinese medicine composition for the treatment of senile dementia as claimed in claim 1, it is characterized in that, its contained effective ingredient is mainly made by the raw material of Chinese medicine of following weight proportioning: 10 parts of the Rhizoma Pinelliaes, 12 parts of Caulis Bambusae In Taenia, 10 parts of Fructus Aurantii Immaturuss, 10 parts of Rhizoma Acori Graminei, 10 parts of Radix Polygalaes, 6 parts of Radix Ginsengs, 10 parts of Rhizoma Chuanxiongs, 12 parts, Radix Rehmanniae Preparata.
3. a kind of Chinese medicine composition for the treatment of senile dementia as claimed in claim 1, it is characterized in that, raw material of Chinese medicine also comprises one or more combinations of raw material of Chinese medicine that are selected from following weight proportion: Rhizoma Panacis Japonici 1-15 part, Poria 1-20 part, Pericarpium Citri Reticulatae 1-15 part, Semen Sinapis Albae 1-10 part, Hirudo 1-10 part, Radix Polygoni Multiflori Preparata 1-25 part.
4. a kind of Chinese medicine composition for the treatment of senile dementia as claimed in claim 2, it is characterized in that, raw material of Chinese medicine also comprises one or more combinations of raw material of Chinese medicine that are selected from following weight proportion: 10 parts of Rhizoma Panacis Japonicis, 15 parts, Poria, 10 parts of Pericarpium Citri Reticulataes, 6 parts of Semen Sinapis Albaes, 6 parts of Hirudos, 20 parts of Radix Polygoni Multiflori Preparata.
5. a kind of Chinese medicine composition for the treatment of senile dementia as described as claim 1-4 any one, is characterized in that, described raw material of Chinese medicine replaces with its extract.
6. a kind of Chinese medicine composition for the treatment of senile dementia as described as claim 1-4 any one, it is characterized in that, Chinese medicine composition is by the raw material of Chinese medicine of described weight proportion or also has said decoction on the pharmaceutics that pharmaceutically the acceptable carrier is made, powder, tablet, capsule, dispersible tablet, micropill, injection, oral liquid, granule.
7. a kind of Chinese medicine composition for the treatment of senile dementia as described as claim 1-4 any one, it is characterized in that, Chinese medicine composition is by the raw material of Chinese medicine of described weight proportion or also has said tablet, capsule, granule, pill or decoction on the pharmaceutics that pharmaceutically the acceptable carrier is made.
8. a kind of Chinese medicine composition for the treatment of senile dementia as claimed in claim 6, is characterized in that, on described pharmaceutics, the acceptable carrier is: excipient starch and derivant thereof, dextrin, calcium hydrogen phosphate, magnesium stearate, micropowder silica gel; Disintegrating agent carboxymethyl base sodium cellulosate, hydroxypropyl cellulose; Magnesium stearate lubricant; Sugar coating material: sucrose, Pulvis Talci, gelatin, pigment, river wax; Thin film coating material stomach dissolution type water, pure coating material.
9. a kind of preparation method for the treatment of the Chinese medicine composition of senile dementia as described as claim 1-4 any one, it is characterized in that, described raw material of Chinese medicine directly is cut into small pieces or is ground into coarse powder, water or ethanol extraction obtain each drug extract again, or continue to be condensed into dry extract, or with acceptable carrier on pharmaceutics, mix again.
10. a kind of preparation method for the treatment of the Chinese medicine composition of senile dementia as described as claim 1-4 any one, it is characterized in that, described raw material of Chinese medicine directly is cut into small pieces or is ground into coarse powder, raw material of Chinese medicine is divided into the extract of several different components after difference water or ethanol extraction and mixes, or continue to be condensed into dry extract, or with acceptable carrier on pharmaceutics, mix again.
11. a kind of preparation method for the treatment of the Chinese medicine composition of senile dementia as described as claim 1-4 any one, it is characterized in that, described raw material of Chinese medicine directly is cut into small pieces or is ground into coarse powder, water or total ethanol extract to obtain extract again, or continue to be condensed into dry extract, or with acceptable carrier on pharmaceutics, mix again.
12. a kind of preparation method for the treatment of the Chinese medicine composition of senile dementia as claimed in claim 1 or 2, it is characterized in that, comprise the Rhizoma Pinelliae, Caulis Bambusae In Taenia, Fructus Aurantii Immaturus, Rhizoma Acori Graminei, Radix Polygalae, Radix Ginseng, Rhizoma Chuanxiong and Radix Rehmanniae Preparata, water decocts 3 times, each 20 minutes, filter merging filtrate, be evaporated to every 1 milliliter and be equivalent to raw medicinal herbs amount 1 gram, obtain decoction; Through sterilizing, filling bottle, obtain oral liquid again.
13. a kind of preparation method for the treatment of the Chinese medicine composition of senile dementia as claimed in claim 1 or 2, is characterized in that, comprises the Rhizoma Pinelliae, Caulis Bambusae In Taenia, Fructus Aurantii Immaturus, Rhizoma Acori Graminei, Radix Polygalae, Radix Ginseng, Rhizoma Chuanxiong and Radix Rehmanniae Preparata, water decocts 3 times, each 20 minutes, filters, merging filtrate, be evaporated to thick paste, continues drying under reduced pressure to dry cream, add starch, pulverize, mix, cross 80 mesh sieves, with 95% ethanol wet granulation, dry below 80 ℃, granulate, the granulation agent.
14. a kind of preparation method for the treatment of the Chinese medicine composition of senile dementia as claimed in claim 1 or 2, is characterized in that, comprises the Rhizoma Pinelliae, Caulis Bambusae In Taenia, Fructus Aurantii Immaturus, Rhizoma Acori Graminei, Radix Polygalae, Radix Ginseng, Rhizoma Chuanxiong and Radix Rehmanniae Preparata, water decocts 3 times, each 20 minutes, filter merging filtrate, be evaporated to thick paste, continue drying under reduced pressure to dry cream, add starch, pulverize, mix, cross 80 mesh sieves, add magnesium stearate, mix, with 95% ethanol moistening, tabletting, coating, obtain tablet.
15. a kind of preparation method for the treatment of the Chinese medicine composition of senile dementia as claimed in claim 1 or 2, it is characterized in that, comprise the Rhizoma Pinelliae, Caulis Bambusae In Taenia, Fructus Aurantii Immaturus, Rhizoma Acori Graminei, Radix Polygalae, Radix Ginseng, Rhizoma Chuanxiong and Radix Rehmanniae Preparata, water decocts 3 times, each 20 minutes, filter, merging filtrate, be evaporated to thick paste, continue drying under reduced pressure to dry cream, add excipient, incapsulate, obtain capsule.
16. a kind of preparation method for the treatment of the Chinese medicine composition of senile dementia as claimed in claim 1 or 2, it is characterized in that, comprise and use soak with ethanol after 12 hours Fructus Aurantii Immaturus, Rhizoma Acori Graminei and Radix Polygalae, with the 75%-85% ethanol of 20-25 times of quality, reflux at twice, each micro-rear backflow 1～1.5 hour of boiling, filter, reclaim ethanol to without the alcohol flavor, obtain extract; Get the Rhizoma Pinelliae, Caulis Bambusae In Taenia, Radix Ginseng, Rhizoma Chuanxiong and Radix Rehmanniae Preparata and decoct 40-50 minute, filter, filtrate is concentrated into the 1/6-1/2 volume, with described extract, merges, and amalgamation liquid is evaporated to every 1 milliliter and is equivalent to raw medicinal herbs amount 1 gram, obtains decoction; Through sterilizing, filling bottle, obtain oral liquid.
17. a kind of preparation method for the treatment of the Chinese medicine composition of senile dementia as claimed in claim 1 or 2, it is characterized in that, comprise and use soak with ethanol after 12 hours Fructus Aurantii Immaturus, Rhizoma Acori Graminei and Radix Polygalae, with the 75%-85% ethanol of 20-25 times of quality, reflux at twice, each micro-rear backflow 1～1.5 hour of boiling, filter, reclaim ethanol to without the alcohol flavor, obtain extract; Get the Rhizoma Pinelliae, Caulis Bambusae In Taenia, Radix Ginseng, Rhizoma Chuanxiong and Radix Rehmanniae Preparata and decoct 40-50 minute, filter, filtrate is concentrated into the 1/6-1/2 volume, with described extract, merge, amalgamation liquid is evaporated to thick paste, continues drying under reduced pressure to dry cream, add excipient, incapsulate, obtain capsule.
18. a kind of preparation method for the treatment of the Chinese medicine composition of senile dementia as claimed in claim 1 or 2, it is characterized in that, comprise and use soak with ethanol after 12 hours Fructus Aurantii Immaturus, Rhizoma Acori Graminei and Radix Polygalae, with the 75%-85% ethanol of 20-25 times of quality, reflux at twice, each micro-rear backflow 1～1.5 hour of boiling, filter, reclaim ethanol to without the alcohol flavor, obtain extract; Get the Rhizoma Pinelliae, Caulis Bambusae In Taenia, Radix Ginseng, Rhizoma Chuanxiong and Radix Rehmanniae Preparata and decoct 40-50 minute, filter, filtrate is concentrated into the 1/6-1/2 volume, with extract, merge, amalgamation liquid is evaporated to thick paste, continues drying under reduced pressure to dry cream, add starch, pulverize, mix, cross 80 mesh sieves, with 95% ethanol wet granulation, dry below 80 ℃, granulate, granulation agent.
19. a kind of preparation method for the treatment of the Chinese medicine composition of senile dementia as claimed in claim 1 or 2, it is characterized in that, comprise and use soak with ethanol after 12 hours Fructus Aurantii Immaturus, Rhizoma Acori Graminei and Radix Polygalae, doubly measuring 75%-85% ethanol with 20-25 refluxes at twice, each micro-rear backflow 1～1.5 hour of boiling, filter, reclaim ethanol to without the alcohol flavor, obtain extract; Get the Rhizoma Pinelliae, Caulis Bambusae In Taenia, Radix Ginseng, Rhizoma Chuanxiong and Radix Rehmanniae Preparata and decoct 40-50 minute, filter, filtrate is concentrated into the 1/6-1/2 volume, with extract, merge, amalgamation liquid is evaporated to thick paste, continues drying under reduced pressure to dry cream, add starch, pulverize, mix, cross 80 mesh sieves, add magnesium stearate, mix, with 95% ethanol moistening, tabletting, coating, obtain tablet.
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