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

Abstract

A medicine for treating senile dementia and a preparation method thereof. The medicine is composed of active ingredients or pharmaceutically acceptable carriers, and the active ingredients are mainly made of the following raw materials in the weight ratio: 1-15 parts of pinellia, 1-15 parts of Zhuru, and 1-15 parts of citrus aurantium . Or it also includes one or more combinations selected from the following raw materials: 1-15 parts of Panax ginseng, 1-15 parts of Poria cocos, 1-15 parts of tangerine peel, 1-10 parts of white mustard seed, 1-10 parts of leech, and Shouwu 1-20 servings. The dosage forms made of the traditional Chinese medicine include decoction, powder, tablet, capsule, dispersible tablet, pellet, injection, oral liquid and granule. The preparation method is to cut the raw materials into small pieces or pulverize them into coarse powder, then extract them with water or ethanol, mix them, or continue to concentrate them into dry extracts, or mix them with carriers. Clinical application proves that this drug is an effective drug for treating Alzheimer's disease and improving patients' memory, cognitive impairment and living ability.

Description

A kind of medicine for treating senile dementia and preparation method thereof

technical field

The invention relates to a medicine for treating senile dementia and a preparation method thereof, more specifically, a medicine for treating senile dementia mainly caused by Alzheimer's disease prepared from Chinese herbal medicine and a preparation method thereof.

Background technique

Senile dementia includes Alzheimer's disease (Alzheimer's disease, AD) and vascular dementia (vascuIardementia, VaD) and other types. With the acceleration of population aging in my country, the incidence of senile dementia continues to increase, seriously affecting the health of the elderly population. Due to the insufficient understanding of the pathogenesis, so far, there is no ideal treatment. FDA-approved drugs include three cholinesterase inhibitors, such as Donepezil, Galantamine, and Rivastigmine, and an excitatory amino acid receptor antagonist, such as memantine (Memantine), which can improve the symptoms of dementia patients in the short term but cannot delay the progression of the disease.

AD belongs to the categories of "dementia" and "dementia" in traditional Chinese medicine. The prevention and treatment of AD with traditional Chinese medicine has been recognized by more and more doctors for its good curative effect and low toxic and side effects. In recent years, it has become a research hotspot at home and abroad. , and achieved great success.

The disease location of senile dementia is in the brain, and its pathogenesis always belongs to deficiency in origin and excess in superficiality, and mostly clinical deficiency and excess are interlaced, and the disease and syndrome are complicated. Its root deficiency lies in the deficiency of the vital energy of the kidney and the deficiency of the sea of marrow, and its symptom is that phlegm turbidity and blood stasis blind the brain orifices and block the brain collaterals. Kidney deficiency, essence deficiency, and phlegm and stasis blockage are two different pathological changes in the development of senile dementia. Multi-terminal.

Among them, phlegm turbidity plays an important role in the pathogenesis of AD. Most of the patients are people with visceral deficiency and damage for a long time. Their qi gradually declines, and their body fluid metabolism is abnormal, resulting in water and dampness stop gathering, and internal turbidity of phlegm. Phlegm turbidity blocks the brain orifice, resulting in turbidity of the marrow sea, poor brain function, mental disorder, and dementia. "Syndrome Differentiation" clearly pointed out that "phlegm accumulates in the brain and entrenches outside the heart, making the mind unclear and causing dementia." Chen Shiduo further explained the relationship between the degree of dementia and phlegm turbidity in "Stone Chamber Secret Record", "phlegm The momentum is the most prosperous, and the dullness is the deepest." In the prevention and treatment of this disease in Chinese medicine, expectoration is also placed in an important position. Domestic physicians analyzed 40 prescriptions that were reported to be effective against senile dementia from 1992 to 1997, and found that all effective prescriptions had the medicinal flavor of eliminating phlegm and resuscitation. Japan's Kitasato Institute of Oriental Medicine has confirmed that Wendan Decoction, a classic prescription for resolving phlegm, has achieved a positive effect in the treatment of senile dementia.

Contents of the invention

The object of the present invention is to provide a medicine for treating senile dementia and a preparation method thereof. Said senile dementia includes Alzheimer's disease, especially Alzheimer's disease of kidney essence deficiency, phlegm and blood stasis obstructing orifices syndrome, memory and cognitive impairment, and low living ability.

In order to achieve the above object, the technical solution of the present invention is based on traditional Chinese medicines, and the effective components of Chinese medicines are combined. According to the theory of TCM syndrome differentiation and holistic medication, the treatment principle of "dissolving phlegm and kidney, nourishing qi and blood circulation, and improving wisdom" is adopted. If it is sufficient, the brain will be nourished, the qi and blood will be unobstructed, and the phlegm and blood stasis will be dissolved to open the orifices and achieve the effect of improving the learning and memory ability.

Based on clinical research, in vitro experiments and in vivo experiments, the present invention adds and subtracts the classic Huatan prescription Wendan Decoction to form a new compound prescription. The sources of the compound Chinese medicinal materials of the present invention mainly include: pinellia, bamboo rutabaga, citrus aurantium, calamus aurantium, polygala, ginseng, chuanxiong and rehmannia glutinosa.

Among them, Pinellia is pungent and warm, dries dampness and resolves phlegm, and is the monarch drug.

Zhuru is sweet and slightly cold, clearing away heat and resolving phlegm. Pinellia and Bamboo Ru, one warm and one cool, resolving phlegm and stomach upset, complement each other; Shichangpu rejuvenates the mind, clears dampness and harmonizes the stomach, calms the mind and boosts the will, and Polygala calms the mind and calms the nerves; The effect of phlegm resuscitating is that Shichangpu tends to be pungent and loose to release its phlegm dampness, while polygala tends to be bitter to reduce phlegm dampness. The two are used at the same time, and they work together to make the qi flow naturally and block it open, the turbid phlegm dissipates, the orifices are cleared, and the mind is clear. Rehmannia glutinosa nourishes the blood, nourishes the essence and fills the marrow; ginseng nourishes vitality, nourishes the spleen and lungs, calms the nerves and strengthens the mind. "New Compendium of Materia Medica": Shichangpu, the happy orifice must be supplemented with ginseng; Citrus aurantium lowers qi and guides stagnation, eliminates phlegm and ruffian, smoothes qi and eliminates phlegm, and increases the power of drugs for monarchs and ministers to reduce phlegm. Ligusticum chuanxiong promotes qi and blood circulation, is a medicine for qi in the blood, good at promoting blood qi up to the head, clearing the orifices and strengthening the brain.

A traditional Chinese medicine composition for treating senile dementia of the present invention, which is composed of active ingredients or a pharmaceutically acceptable carrier, is characterized in that the active ingredients it contains are mainly made of the following traditional Chinese medicine raw materials in weight ratio : Pinellia 1-15 parts, Zhuru 1-15 parts, Citrus aurantium 1-15 parts, Shichangpu 1-15 parts, Polygala 1-15 parts, Ginseng 1-10 parts, Chuanxiong 1-15 parts, Rehmannia glutinosa 1-15 parts 20 servings.

Preferably: 10 parts of pinellia, 12 parts of Zhuru, 10 parts of Citrus aurantii, 10 parts of Shichangpu, 10 parts of polygala, 6 parts of ginseng, 10 parts of Chuanxiong, and 12 parts of Rehmannia glutinosa.

The Chinese medicine composition of the present invention, in addition to the above-mentioned Chinese medicine raw materials, also includes one or more combinations of Chinese medicine raw materials selected from the following weight ratios: 1-15 parts of Panax ginseng, 1-20 parts of Poria cocos, 1-20 parts of tangerine peel 15 parts, 1-10 parts of white mustard seeds, 1-10 parts of leeches, and 1-25 parts of Shouwu. Preferred: 10 parts of Bamboo Ginseng, 15 parts of Poria cocos, 10 parts of tangerine peel, 6 parts of white mustard seed, 6 parts of leech, and 20 parts of Shouwu.

The medicines described in the present invention can be replaced by their extracts.

The traditional Chinese medicine composition of the present invention is any pharmaceutical dosage form made from the above-mentioned traditional Chinese medicine raw materials in proportion by weight or a pharmaceutically acceptable carrier, including decoction, powder, tablet, capsule, Dispersible tablets, pellets, injections, oral liquids and granules.

The pharmaceutically acceptable carrier of the present invention includes but not limited to: excipients, such as starch and its derivatives, dextrin, calcium hydrogen phosphate, magnesium stearate, micro silica gel, etc.; Sodium cellulose, propyl cellulose, etc.; lubricants, such as magnesium stearate, etc.; sugar coating materials, such as sucrose, talcum powder, gelatin, pigments, Sichuan wax, etc.; film coating materials, such as gastric soluble water, Alcohol coating materials, etc.

The dosage form of the medicine of the present invention is preferably an oral preparation, such as tablet, capsule, granule, pill or decoction, wherein the method for preparing the preparation and the pharmaceutically acceptable carrier and/or excipient used are in the technical field conventional technology.

The preparation method of the traditional Chinese medicine composition of the present invention is a mixture of various drug extracts obtained by using different processes and different extraction methods from the raw materials of the traditional Chinese medicine, or a mixture of extracts obtained by dividing the drug into several different components and extracting them separately, Or the total extract, made of preparations.

The preparation method of the traditional Chinese medicine composition of the present invention comprises directly cutting the said traditional Chinese medicine raw materials into small pieces or pulverizing them into coarse powder, and then mixing the medicinal extracts obtained by extracting them with water or ethanol, or continuing to concentrate them into Dry extract, or mixed with pharmaceutically acceptable carrier.

The preparation method of the traditional Chinese medicine composition of the present invention comprises decocting Pinellia, Zhuru, Citrus aurantium, Shichangpu, Polygala, Ginseng, Rhizoma Chuanxiong, Rehmannia glutinosa with water for 3 times, each time for 20 minutes, filtering, combining the filtrates, reducing Concentrate under pressure until every 1 ml is equivalent to 1 gram of raw medicinal material to obtain a decoction; or sterilize and fill a bottle to obtain an oral liquid; or concentrate to a thick paste, continue to dry under reduced pressure to a dry paste, add starch, Crush, mix, pass through 80-mesh sieve, wet granulate with 95% ethanol, dry below 80°C, granulate, and make granules; or add magnesium stearate, mix, compress, and coat to obtain tablets agent; or add excipients and pack into capsules to obtain capsules.

The preparation method of the traditional Chinese medicine composition of the present invention; comprising soaking Fructus Citrus Fructus Fructus Aurantii, Shichangpu and Polygala with ethanol for 12 hours, then refluxing twice with 75-85% ethanol with 20-25 times the mass, and refluxing for 1 hour after slightly boiling each time. ~1.5 hours, filter, recover ethanol until there is no alcohol smell, and get the extract; take Pinellia, Bamboo Ru, Ginseng, Chuanxiong, and Shudi and decoct for 40-50 minutes, filter, and concentrate the filtrate to 1/6-1/2 volume, combined with the extract, and the combined solution is concentrated under reduced pressure to 1 gram of raw medicinal material per 1 milliliter to obtain a decoction; or made into oral liquid, granules, tablets, and capsules according to the above method.

The administration method of the medicine of the present invention is conventional, and the dosage is determined by the attending physician according to factors such as the nature of the disease, the patient's age, body weight, condition, and administration method. The medicine of the present invention is composed of representative traditional Chinese medicines in the methods of invigorating the kidney, replenishing qi, resolving phlegm and promoting blood circulation. The effect of cognition and memory impairment is significantly better than that of chemical drugs, and has the advantages of clear effective parts, definite curative effect, less medicinal smell and low cost.

The technical solution of the present invention can realize one or more objects of the present invention.

In the present invention, the drug effect and pharmacology research on the medicine of the present invention is carried out from three levels of cell experiment, animal experiment and clinical experiment respectively, and good effect has been obtained. The improvement effect of the medicament of the present invention on the learning and memory of senile dementia patients has been clinically observed. In terms of experimental research, the Meynert nucleus-like AD rat model was damaged by Ibotenic acid (IBO), the Meynert nucleus-like AD rat model was damaged by IBO+Aβ25-35, and PC12 cells and NG108 cells were induced by Aβ25-35. Cells and the AD cell model of the nerve cell damage model induced by glutamate in the primary culture of the rat brain hippocampus were taken as the research object, and the protective effect of the drug of the present invention on the neurotoxicity of model animals and cells was observed, and at the same time, it was multi-targeted from multiple links. researched more systematically.

Note: the ethanol % used in the present invention all refers to volume percent concentration.

Description of drawings

Fig. 1 is the track diagram of the invention drug on AD model rat positioning navigation experiment, wherein: a1 is the normal group, b1 is the blank group, c1 is the model group, d1 is the western medicine group, e1 is the low dose group, f1 is the middle dose group, g1 is the high dose group.

Fig. 2 is the track diagram of the space search experiment of the invented drug on AD model rats, wherein: a2 is the normal group, b2 is the blank group, c2 is the model group, d2 is the western medicine group, e2 is the low-dose group, f2 is the middle-dose group, g2 is the high dose group.

Figure 3 is the effect of the invented drug on the senile plaques in the brain tissue of AD model rats, wherein: a3 is the normal group, b3 is the blank group, c3 is the model group, d3 is the western medicine group, e3 is the low-dose group, f3 is the middle-dose group, g3 is the high dose group.

Figure 4 is the effect of the invented drug on the neurofibrillary tangles in the brain tissue of AD model rats, wherein: a4 is the normal group, b4 is the blank group, c4 is the model group, d4 is the western medicine group, e4 is the low-dose group, and f4 is the middle-dose group. Dosage group, g4 is the high-dose group.

Figure 5 is the effect of the invented drug on the ultramicropathology of the brain tissue of AD model rats, wherein: a5 is the normal group, b5 is the blank group, c5 is the model group, d5 is the western medicine group, e5 is the low dose group, and f5 is the middle dose group, g5 is the high-dose group.

Fig. 6 is the RT-PCR result of the invention drug on rat hippocampus Ub protein level.

Figure 7 is the western blot results of the inventive drug on the E1 protein level in rat hippocampus.

Fig. 8 is the western blot result of the inventive drug on rat hippocampal E2-EPF protein level.

Figure 9 is the western blot results of the inventive drug on the PGP9.5 protein level in rat hippocampus.

Figure 10 is the western blot results of the invented drug on rat phosphorylated tau protein.

Fig. 11 is the result of RT-PCR detection of the invented drug on rat cdk5.

Figure 12 is the status of PC12 cells under the action of the invented drug on drug-containing serum and blank serum, wherein: a12 is 5% blank serum, b12 is 10% blank serum, c12 is 20% blank serum, d12 is 5% drug-containing serum, e12 It is 10% medicated serum, and f12 is 20% medicated serum.

Figure 13 is a diagram showing the protection status of the invented drug against Aβ25-35 damaged PC12 cells and drug-containing serum, wherein: a13 is PC12+20μMAβ+5% blank serum, b13 is PC12+20μMAβ+5% drug-containing serum, and c13 is PC12+20μMAβ+10% blank serum, d14 was PC12+20μMAβ+10% drug-containing serum, e13 was PC12+20μMAβ+20% blank serum, and f13 was PC12+20μMAβ+20% drug-containing serum.

Figure 14 is the flow cytometric result of the inventive drug on the apoptosis rate of AD model cells, wherein: a14 is the normal control, b14 is the model group, c14 is 5% blank serum, d14 is 10% blank serum, e14 is 20% blank serum, f14 is 5% drug-containing serum, g14 is 10% drug-containing serum h14 is 20% drug-containing serum.

Fig. 15 Invention medicine is to AD model cell caspase-3 immunohistochemical result (10X10), wherein: a15 is the complete medium group, b15 is Aβ _25-35 group, c15 is Aβ _25-35 +5% blank serum, d15 is Aβ _25-35 + 10% blank serum, e15 is Aβ _25-35 + 20% blank serum, f15 is Aβ _25-35 + 5% drug-containing serum, g15 is Aβ _25-35 + 10% drug-containing serum, h15 is Aβ _25-35 + 20% medicated serum.

Fig. 16 Invention drug on AD model cell morphology results (10x20), wherein: a16 is the blank group, b16 is the model group, c16 is the blank serum group, d16 is the drug-containing serum group, e16 is the blank cerebrospinal fluid group, f16 is the drug-containing cerebrospinal fluid Group.

Fig. 17 Influence of the inventive drug-containing cerebrospinal fluid on the apoptosis rate of AD model cells, wherein: a17 is the blank group, b17 is the model group, c17 is the blank serum group, d17 is the drug-containing serum group, e17 is the blank cerebrospinal fluid group, f17 is the Drug-containing cerebrospinal fluid group.

The influence of Fig. 18 invention medicine on Bcl-2, Bax protein expression, wherein: a18 is the blank group bax (40×10), b18 is the model group bcl-2 (40×10), c18 is the blank serum group bax (40×10) 10), d18 is the bcl-2 (40×10) of the blank cerebrospinal fluid group, e18 is the bax (40×10) of the drug-containing serum group, and f18 is the bcl-2 (40×10) of the drug-containing cerebrospinal fluid group.

Detailed ways

The following experimental examples are used to illustrate the preparation of the medicament of the present invention and its properties, but in any case they cannot constitute a limitation of the present invention.

Preparation Example

The raw materials of traditional Chinese medicine were purchased from the expert outpatient department of Hubei University of Traditional Chinese Medicine.

Example 1

1. Take 10 parts of pinellia, 12 parts of Zhuru, 10 parts of citrus aurantium, 20 parts of Polygonum multiflorum, 6 parts of red ginseng, 10 parts of Chuanxiong, 6 parts of leech, 10 parts of Shichangpu and 10 parts of bamboo ginseng;

2. Decoct Pinellia, Bamboo Ru, Citrus Citrus, Polygonum multiflorum, Red Ginseng, Ligusticum Chuanxiong, Leeches, and Shichangpu with water for 3 times, each time for 20 minutes, to extract the medicinal liquid as much as possible, combine the water extracts, and concentrate under reduced pressure to the density It is a thick paste of 1.0-3.0 (50°C), continue drying under reduced pressure to a dry paste, add starch, pulverize, mix evenly, pass through a 80-mesh sieve, wet granulate with 95% ethanol, dry below 80°C, granulate, and prepare into granules.

Example 2

1. Take 10 parts of pinellia, 12 parts of Zhuru, 10 parts of aurantium, 20 parts of Polygonum multiflorum, 6 parts of red ginseng, 10 parts of Chuanxiong, 6 parts of leeches and 10 parts of Shichangpu by weight;

2. Decoct Pinellia, Bamboo Ru, Citrus Citrus, Polygonum multiflorum, Red Ginseng, Ligusticum Chuanxiong, Leeches, and Shichangpu with water for 3 times, each time for 20 minutes, to extract the medicinal liquid as much as possible, combine the water extracts, and concentrate under reduced pressure to the density It is a thick paste of 1.0-3.0 (50°C).

Example 3

1. Prepare by weight 20 parts of Shouwu, 10 parts of Shichangpu, 10 parts of Bamboo Ginseng, 15 parts of Poria cocos, 10 parts of tangerine peel, 10 parts of pinellia, and 6 parts of white mustard seed;

2. Decoct Radix Polygoni Multiflori, Shichangpu, Panax Ginseng, Poria, Tangerine Peel, Pinellia, and White Mustard Seed with water for 3 times, each time for 20 minutes, to extract the medicinal liquid as much as possible, combine the water extracts, and concentrate under reduced pressure to the density It is a thick paste of 1.0-3.0 (50°C).

Example 4

1. Take 10 parts of Jiang Pinellia, 12 parts of Zhuru, and 10 parts of Citrus aurantium by weight; decoct Jiang Pinellia, Zhuru, and Citrus aurantium in water for 3 times, each time for 20 minutes, try to extract the medicinal liquid, combine the water extracts, and reduce Concentrate under pressure to a thick paste with a density of 1.0-3.0 (50°C).

2. Take 10 parts of ginger pinellia, 12 parts of Zhuru, 10 parts of citrus aurantium, 10 parts of tangerine peel, 15 parts of tuckahoe, and 6 parts of licorice by weight; decoct ginger pinellia, zhuru, citrus aurantium, tangerine peel, tuckahoe, and licorice for 3 times , 20 minutes each time, try to leaching the medicinal solution, combine the water extracts, concentrate under reduced pressure to a thick paste with a density of 1.0-3.0 (50°C).

3. Take 10 parts of Jiang Pinellia, 12 parts of Zhuru, 10 parts of Zhishi, 12 parts of Shudi, and 20 parts of Zhishouwu by weight; decoct Jiang Pinellia, Zhuru, Zhishi, Shudi and Zhishouwu with water for 3 times. Every 20 minutes, try to make the medicinal solution leaching, combine the water extracts, and concentrate under reduced pressure to a thick paste with a density of 1.0-3.0 (50°C).

4. Take 10 parts of ginger pinellia, 12 parts of Zhuru, 10 parts of citrus aurantium, 10 parts of polygala, 10 parts of Shichangpu, and 6 parts of white mustard seeds by weight; put ginger pinellia, bamboo Ru, citrus aurantium, polygala, Shichangpu, and white mustard seeds in water Decoct 3 times, 20 minutes each time, try to leaching the medicinal liquid, combine the water extracts, concentrate under reduced pressure to a thick paste with a density of 1.0-3.0 (50°C).

5. Take 10 parts of Jiang Pinellia, 12 parts of Zhuru, 10 parts of Zhishi, and 10 parts of Chuanxiong according to weight; decoct Jiangpinia, Zhuru, Zhishi, and Chuanxiong with water for 3 times, each time for 20 minutes, to make the liquid leached as much as possible. The combined water extracts were concentrated under reduced pressure to a thick paste with a density of 1.0-3.0 (50°C).

6. Take 10 parts of ginger pinellia, 12 parts of Zhuru, 10 parts of citrus aurantium, and 6 parts of ginseng by weight; decoct ginger pinellia, bamboo rut, citrus aurantium, and ginseng in water for 3 times, each time for 20 minutes, to make the liquid leached as much as possible. The combined water extracts were concentrated under reduced pressure to a thick paste with a density of 1.0-3.0 (50°C).

7. Take 10 parts of ginger pinellia, 12 parts of Zhuru, 10 parts of Zhishi, 12 parts of Rehmannia glutinosa, 20 parts of Zhishouwu, 10 parts of polygala, 10 parts of Shichangpu, and 6 parts of white mustard seed by weight; Stew, Rehmannia glutinosa, Zhishouwu, Polygala, Shichangpu, and white mustard seeds, decoct 3 times with water, 20 minutes each time, try to extract the medicinal liquid, combine the water extracts, and concentrate under reduced pressure to a density of 1.0-3.0 (50°C) thick paste.

8. Take 10 parts of Jiang Pinellia, 12 parts of Zhuru, 10 parts of Zhishi, 12 parts of Shudi, 20 parts of Zhishouwu, and 10 parts of Chuanxiong by weight; Decoct 3 times, 20 minutes each time, try to leaching the medicinal liquid, combine the water extracts, concentrate under reduced pressure to a thick paste with a density of 1.0-3.0 (50°C).

9. Take 10 parts of Jiang Pinellia, 12 parts of Zhuru, 10 parts of Zhishi, 12 parts of Rehmannia glutinosa, 20 parts of Zhishouwu, and 6 parts of ginseng by weight; Decoct 3 times, 20 minutes each time, try to leaching the medicinal liquid, combine the water extracts, concentrate under reduced pressure to a thick paste with a density of 1.0-3.0 (50°C).

10. Take 10 parts of ginger pinellia, 12 parts of Zhuru, 10 parts of citrus aurantium, 10 parts of polygala, 10 parts of Shichangpu, 6 parts of white mustard seed, and 10 parts of Chuanxiong by weight; , white mustard seed, and Chuanxiong were decocted with water for 3 times, each time for 20 minutes, to extract the medicinal liquid as much as possible, combined the water extracts, and concentrated under reduced pressure to a thick paste with a density of 1.0-3.0 (50°C).

11. Take 10 parts of ginger pinellia, 12 parts of Zhuru, 10 parts of citrus aurantium, 10 parts of Polygala, 10 parts of Shichangpu, 6 parts of white mustard seed, and 6 parts of ginseng by weight; , white mustard seed, and ginseng were decocted with water for 3 times, each time for 20 minutes, to extract the medicinal liquid as much as possible, combined the water extract, and concentrated under reduced pressure to a thick paste with a density of 1.0-3.0 (50°C).

12. Take 10 parts of ginger pinellia, 12 parts of Zhuru, 10 parts of Citrus aurantium, 10 parts of Chuanxiong, and 6 parts of ginseng by weight; decoct ginger Pinellia, Zhuru, Zhishi, Chuanxiong and ginseng in water for 3 times, each time for 20 minutes, Make the medicinal liquid leaching out as much as possible, combine the water extracts, and concentrate under reduced pressure to a thick paste with a density of 1.0-3.0 (50°C).

13. Take 10 parts of ginger pinellia, 12 parts of Zhuru, 10 parts of Citrus aurantium, 12 parts of Rehmannia glutinosa, 20 parts of Zhishouwu, 10 parts of polygala, 10 parts of Shichangpu, 6 parts of white mustard seed, and 10 parts of Chuanxiong; Zhuru, Citrus aurantium, Rehmannia glutinosa, Zhishouwu, Polygala, Shichangpu, white mustard seeds, and Chuanxiong are decocted in water for 3 times, 20 minutes each time, to extract the medicinal liquid as much as possible, combine the water extracts, and concentrate under reduced pressure to a density of 1.0 -3.0 (50°C) thick paste.

14. Take 10 parts of ginger pinellia, 12 parts of Zhuru, 10 parts of citrus aurantium, 12 parts of Rehmannia glutinosa, 20 parts of Zhishouwu, 10 parts of polygala, 10 parts of Shichangpu, 6 parts of white mustard seed, and 6 parts of ginseng; Zhuru, Citrus aurantium, Rehmannia glutinosa, Zhishouwu, Polygala, Shichangpu, white mustard seeds, and ginseng are decocted in water for 3 times, 20 minutes each time, to extract the medicinal liquid as much as possible, combine the water extracts, and concentrate under reduced pressure to a density of 1.0 -3.0 (50°C) thick paste.

15. Take 10 parts of Jiang Pinellia, 12 parts of Zhuru, 10 parts of Zhishi, 12 parts of Shudi, 20 parts of Zhishouwu, 10 parts of Chuanxiong, and 6 parts of Ginseng by weight; Aconite, Chuanxiong, and ginseng were decocted in water for 3 times, 20 minutes each time, to extract the medicinal liquid as much as possible, combined the water extracts, and concentrated under reduced pressure to a thick paste with a density of 1.0-3.0 (50°C).

16. Take 10 parts of ginger pinellia, 12 parts of Zhuru, 10 parts of citrus aurantium, 10 parts of polygala, 10 parts of Shichangpu, 6 parts of white mustard seed, 10 parts of Chuanxiong, and 6 parts of ginseng by weight; Polygala, Shichangpu, white mustard seeds, Chuanxiong, and ginseng were decocted with water for 3 times, 20 minutes each time, to extract the medicinal liquid as much as possible, combined the water extracts, and concentrated under reduced pressure to a thick paste with a density of 1.0-3.0 (50°C).

17. Take 10 parts of ginger pinellia, 12 parts of Zhuru, 10 parts of citrus aurantium, 12 parts of Rehmannia glutinosa, 20 parts of Zhishouwu, 10 parts of polygala, 10 parts of Shichangpu, 6 parts of white mustard seed, 10 parts of Chuanxiong, and 6 parts of ginseng ; decoct ginger Pinellia, Zhuru, Citrus aurantium, Rehmannia glutinosa, Zhishouwu, Polygala, Shichangpu, white mustard seed, Chuanxiong, and ginseng in water for 3 times, each time for 20 minutes, try to extract the medicinal liquid, combine the water extract, reduce Concentrate under pressure to a thick paste with a density of 1.0-3.0 (50°C).

Experimental example

Embodiment 5 Effect experiment of the medicine of the present invention on the learning and memory level of the pseudo AD model rats induced by IBO injection

1 Experimental materials

1.1 Animals

Sixty Wistar rats, half male and half female, weighing 300±50 g, were provided by the Experimental Animal Center of Tongji Medical College, Huazhong University of Science and Technology. After one week of feeding under stable conditions in the laboratory, the Y-type water maze method was used to coarsely screen, and those whose correct times reached more than 12 times in 15 tests were qualified.

1.2 Drugs and preparation

Prepare the medicament of Example 2. Naofukang (piracetam) tablet, 400mg/tablet (produced by Northeast Pharmaceutical Factory, batch number: 990618), crushed with water and added double distilled water to make a 10mg/ml suspension. IBO (Sigma Corporation).

1.3 Main reagents and instruments

The rat Morris water maze automatic experiment recorder and the rat program-controlled shuttle box were provided by the Chinese Academy of Medical Sciences.

2 Experimental methods

2.1 AD model making

After the animals were anesthetized by intraperitoneal injection of pentobarbital sodium (40mg/kg), they were fixed on the brain stereotaxic apparatus, the scalp was prepared, the skin in the surgical area was routinely disinfected, and the operation was performed under aseptic conditions. The ball separates the periosteum and the skull is drilled with a dental drill. Referring to the stereotaxic coordinates of the brain, ear rod +0.2, outside the midline 0.3, and below the skull 7.0, advance the injection needle of the micro-injector, and inject 0.5 μl (1ug/1ul) of amanita (IBO) at each point, and the injection time lasts for 2 days. -3min to prevent diffusion. After the needle was slowly withdrawn, the wound was cleaned with hydrogen peroxide, the skull hole was sealed with sponge glue, the scalp was sutured, and 100,000 u of penicillin was injected intramuscularly to prevent infection. Contralateral lesion injections were performed at the same coordinates after the animal survived for one week. The blank group took the same coordinates and injected the same amount of normal saline after needle insertion.

2.2 Grouping and administration

The rats were randomly divided into 6 groups: normal group, sham operation group (blank group), model group, Naofukang group (abbreviated as control group), low-dose group of invented drug (abbreviated as low-dose group), high-dose group of invented drug (abbreviated as low-dose group). high volume group). 10 in each group. Three days after the model was made, another screening was performed. The model was successful if the correct number of tests decreased by 1/3 compared with that before the model was made. Start administration after success, each administration group all adopts intragastric method administration, and high-dose group is given the decoction of invented medicine by 20g/kg.d. The low-dose group was given the diluent of the invented drug at 10 g/kg d, the control group was given the Nafukang suspension at 100 mg/kg d, and the normal group, the blank group and the model group were all given the same volume (1ml/100g d) Oral administration of normal saline; 28 consecutive days. After all the animals were processed until the brain was taken, 10, 9, 7, 8, 9 and 9 rats survived in the normal group, blank group, model group, control group, low-volume group, and high-volume group, respectively.

2.3 Morris water maze test

a. The water temperature of the positioning navigation test was controlled at about 25°C. Four times a day in the morning and afternoon, the rats were put into the water facing the pool wall and divided into four quadrants, and the time it took to find the platform within 2 minutes (escape latency period) was recorded. If the rat does not find the platform within 2 minutes, it is pulled onto the platform by hand, and the rat is allowed to stay for 10 seconds before being put back into the cage. The latency period is calculated as 120 seconds. It lasted 5 days.

b The platform was removed on the 6th day of the space exploration test, and the rat was put into any water entry point, and the number of times the rat crossed the original platform position within 2 minutes was recorded. The two experiments also recorded the swimming distance of the rats in the quadrant of the platform as a percentage of the total distance, and the swimming distance percentages of the 10% and 30% areas of the pool wall.

2.4 Rat shuttle box experiment

After dosing was complete, a shuttle box test was performed. The experiment was divided into two parts: training and formal testing. The training period was 4 days and 5 days for the formal test. The electric stimulation time was set to 15s, the beeping time was 5s, the interval time was 2 minutes, the current was 20mA, and each training cycle was 20 cycles, once a day.

表示。采用SPSS 10.0统计软件包统计分析。包括t检验。2.5 Statistical processing All results are expressed as mean ± standard deviation

express. SPSS 10.0 statistical software package was used for statistical analysis. Include t-test.

3 results

3.1 General behavior of animals

Compared with the control group, the animals in the model group had less glossy coat, less food and less activity, and improved after gavage treatment with traditional Chinese and western medicine. The wounds of the dead rats all showed signs of infection such as suppuration.

3.2 Morris water maze test

3.2.1 The effect on the incubation period of the positioning navigation test in rats

It can be seen from the influence of the incubation period that the incubation period of the two groups of rats decreased rapidly two days before training, and tended to be stable from the third day, basically maintaining a constant level. In order to analyze the information acquisition ability, the total latency of the 5-day training and the post-latency of the last 3 days of training were measured. The results are shown in Table 2-1.

3.2.2 Effects on the learning and memory of rat positioning navigation test and space search test

The swimming distance of the quadrant of the rat positioning navigation test and the space search test platform accounted for the percentage of the total distance, and the swimming distance percentages of the 10% and 30% areas of the pool wall. The results are shown in Tables 2-2 and 3.

3.2.3 The effect on the number of rats crossing the original platform position

See Table 2-4 for the comparison of the number of times the rats in each group crossed the original platform in the space search test. The above results all show that the rats in the normal group, sham operation group, low-volume group, and high-volume group can find the location of the platform by relying on spatial cues, and their movement trajectories are located in the quadrant of the original platform at most, while the rats in the control group are mostly located in the quadrant adjacent to the original platform. Look in the left and right quadrants. But the rats in the model group basically swam around the pool wall, and seldom swam to the vicinity of the original platform, and their movement trajectories were randomly distributed in each quadrant. No matter from the positioning navigation test reflecting the ability of spatial learning and memory acquisition, or from the space search experiment reflecting the ability of information storage, there are statistical differences between the control group, low volume group and high volume group and the other three groups (P<0.01) . The high amount group showed significantly better ability than the control group (P<0.05). However, each index did not reach the level of the normal group and the sham operation group.

Table 2-1 The influence of medicine of the present invention on the average incubation period of rat positioning navigation test

Note: Compared with the normal group, ^△△ P<0.01 Compared with the model group, ^▲▲ P<0.01

Compared with the control group ^★★ P＜0.01

Table 2-2 The influence of medicine of the present invention on the learning and memory of rat positioning navigation test

Note: Compared with the normal group ^△ P<0.05 ^△△ P<0.01 Compared with the model group ^▲▲ P<0.01

Compared with the control group ^★ P＜0.05 ^★★ P＜0.01

Table 2-3 The influence of medicine of the present invention on rat space search test learning and memory

Note: Compared with the normal group, ^△△ P<0.01 Compared with the model group, ^▲▲ P<0.01

Compared with the control group ^★ P＜0.05 ^★★ P＜0.01

Table 2-4 The influence of medicine of the present invention on rat space search test passing through former platform position number of times

Note: Compared with the normal group, ^△△ P<0.01 Compared with the model group, ^▲▲ P<0.01

Compared with the control group ^★ P＜0.05

3.3 Rat shuttle box experiment

3.3.1 Active avoidance response rate

In the first 3 days of training, the treatment groups received fewer electric shocks, but there was no significant difference in the rate of active avoidance among the three groups. From the 4th day, the model group still basically maintained the original level, showing obvious behavioral disorder. The active avoidance response rate of the rats in other groups increased significantly, compared with the model group (P<0.01), but it still did not reach the level of the normal group. There was no significant difference between the low amount group and the control group (P>0.05). On the 5th day, the official test results were consistent with the above, but the difference was more obvious. The results are shown in Table 2-5.

Table 2-5 The influence of medicine of the present invention on the correct rate of active avoidance in rats

Note: Compared with the normal group ^△ P<0.05 ^△△ P<0.01 Compared with the model group ^▲▲ P<0.01

Compared with the control group ^★★ P＜0.01

3.3.2 Passive escape time From the 4th day, the passive escape time of the control group, low dose group, and high dose group was significantly shorter than that of the model group (P<0.01), and continued to shorten on the 5th day (P<0.01). The model group continued to maintain a longer passive escape time. The results of each group were consistent with the active avoidance response rate. See Table 2-6

The impact of table 2-6 medicine of the present invention on rat passive escape time

Note: Compared with the normal group, ^△△ P<0.01 Compared with the model group, ^▲▲ P<0.01

Compared with the control group ^★★ P＜0.01

4 discussions

Experimental result shows, the above-mentioned detection index of the Morris water maze of rats of high and low dose group of medicine of the present invention is all obviously better than model group, and shows the trend that high dose group will be slightly better than low dose group; Show that medicine of the present invention is to IBO-induced The learning and memory function of AD model rats with Meynert nuclear damage can be significantly improved. Experimental result also shows: there is still a certain gap between the high and low dose treatment groups of the present invention on the learning and memory of AD rats compared with the normal group and the sham operation group, indicating that the inventive medicine can only partially improve the learning and memory ability of AD, while Can not make it fully recovered, and this also is similar with embodiment 4 clinical manifestations.

Embodiment 6 The influence experiment of the drug of the present invention on the cholinergic system of IBO-induced AD rats

1 material

1.1 Animals, medicine and preparation are the same as in Example 5.

1.2 Detection reagents and instruments

ChAT and Ach radioimmunoassay kits were purchased from Beijing Zhongshan Institute of Biotechnology.

2 methods

2.1 Modeling, grouping and administration are the same as in Example 5.

2.2 Determination of ChAT and Ach activity in rat hippocampus

The hippocampal tissue was separated, weighed, and homogenized to make a 10% homogenate. Carried out by radioimmunoassay, ChAT activity is represented by the ρmol number of Ach catalyzed by ChAT per minute per mg of wet brain tissue (ρmol Ach/min·mg brain), and Ach activity is represented by Ach produced per mg of wet brain tissue ( ρmol/mg brain). See the product manual for specific operations.

2.3 Statistical processing

表示。采用SPSS 10.0统计软件包统计分析。All results are presented as mean ± standard deviation

express. SPSS 10.0 statistical software package was used for statistical analysis.

3 results

3.1 The effect of the drug of the present invention on the activity of ChAT in the hippocampus.

This experiment measured the ChAT activity of the hippocampus of rats in each group. It can be seen from Table 7 that the ChAT in the hippocampus of the model group was significantly reduced compared with each group (P<0.01). Compared with the model group, the ChAT activity in the hippocampus increased in varying degrees (all P<0.01). The activities of the high-quantity group and low-quantity group were 1.5 and 1.3 times that of the model group, respectively. The ChAT in the hippocampus of rats in the high amount group was close to the level of the blank group, but not yet reached the level of the normal group. The results are shown in Table 3-1.

Table 3-1 The influence of the medicine of the present invention on the activity of ChAT in the hippocampus

Note: Compared with the normal group ^△ P<0.05 ^△△ P<0.01 Compared with the model group ^▲▲ P<0.01

Compared with the control group ^★★ P＜0.01

3.2 The influence of the medicine of the present invention on the activity of Ach in rat hippocampus

In this experiment, the Ach activity in the hippocampus of the rats in each group was measured, and the results showed that Ach in the hippocampus of the model group was significantly reduced compared with the other groups (P<0.01). The Ach activity in the hippocampus of the rats in the low and high dosage groups and the control group increased to varying degrees (P<0.01). The activities of the high amount group and the high amount group were 1.6 and 1.9 times that of the model group, respectively. Compared with the control group, the increase of Ach in the sea area of the low and high amount groups was significantly different (P<0.05-0.01), and the high amount group was close to the level of the normal group (P<0.01). The results are shown in Table 3-2

The influence of table 3-2 medicine of the present invention on rat hippocampus Ach activity

Note: Compared with the normal group, ^△△ P<0.01 Compared with the model group, ^▲▲ P<0.01

Compared with the control group ^★ P＜0.05 ^★★ P＜0.01

4 discussions

The medicine of the invention can increase the activity of CHAT in the hippocampus of AD model rats, thereby increasing the activity of Ach in the brain tissue and achieving the purpose of improving learning and memory, indicating that the medicine of the invention has a repairing effect on central cholinergic damage. Analyzing its mechanism, the invented drug may increase the number of memory synapses in the necrotic marginal zone of rats due to modeling, that is, the number of cholinergic synapses, and increase its density, so that the remaining neurons can play a greater role make the memory synaptic gap narrow, so as to accelerate the conduction function between synapses; meanwhile, there are some nerve conduction bypasses in the brain, and the medicine of the present invention may cause nerve impulses, which are transmitted to the central nervous system (brain) through peripheral nerves, The bypass pathway that usually does little work stimulates the cerebral cortex, thereby causing changes in the number and structure of synapses in the necrotic marginal zone; thickening the dense material of the post-synaptic membrane, which may be caused by the phosphorylation process of certain enzymes and their substrate proteins It is related to the change of its molecular configuration, which may be the morphological basis of the functional change.

Experimental results show that the drug of the present invention can increase the activity of ChAT in the hippocampus of AD model rats, inhibit the excessively high activity of AchE, thereby increasing the activity of Ach in the brain tissue, and achieve the purpose of improving learning and memory. It shows that the invented drug has a protective effect on central cholinergic damage.

Example 7 The protective effect and mechanism of the drug of the present invention on the neurons of IBO-induced AD rats

1 material

1.1 Animals, medicine and preparation are the same as in Example 5.

1.2 Detection reagents and instruments

iNOS, NGF and nNOS monoclonal antibodies, P38 in situ hybridization kit, immunohistochemistry kit SABC, DAB color development kit were purchased from Wuhan Boster Bioengineering Co., Ltd. .Flow Cytometry (BD, USA, FACS) test.

2 methods

2.1 Modeling, grouping and administration are the same as in Example 5.

2.2 Detection of nerve cell apoptosis

Flow cytometry—propidium iodide staining method, collect and process hippocampal cells in a centrifuge tube, count 1×10 ⁶ cells, centrifuge at 800-1000 r/min for 5 min, resuspend cells with blank, centrifuge, discard supernatant , fix with 1ml of 70% ethanol overnight at 4°C, centrifuge at 800r/min for 8min, discard the supernatant, wash with PBS, centrifuge again, keep 0.2ml of liquid, add RNase 10μl (final concentration is 50g/l) and shake well, 37°C 45min. After adding 50 μl of Promideiodine PI (final concentration: 50 g/l), add PBS to 0.5 ml, and shake well. Store at 4°C in the dark for 1 hour. Filter through a 300-mesh nylon mesh, and test with a flow cytometer (BD Company, FACS, USA).

2.3 Detection of P38 in rat hippocampus

The in situ hybridization technique was used, and the specific experimental operation was carried out according to the instructions.

2.4 Detection of NOS and NGF in the hippocampus

After the administration, the rats in each group were perfused with 4% paraformaldehyde and internally fixed. The brain tissue was routinely embedded in paraffin. The cortex and hippocampus were sectioned 20 times with a thickness of 4 μm, and one piece was taken every 4 pieces. SABC immunohistochemical staining method was used for detection, and the specific operation was found in the product manual.

2.5 Image analysis and processing

Five slices were randomly taken from each rat, and the number of n NOS positive cells was randomly selected in four visual fields of the right cortex and hippocampus under the light microscope, and the average number of each visual field was calculated.

表示。采用SPSS 10.0统计软件包统计分析。2.6 Statistical processing All test results are expressed as mean ± standard deviation

express. SPSS 10.0 statistical software package was used for statistical analysis.

3 results

3.1 The influence of the medicine of the present invention on the expression of nNOS in rat cortex and hippocampus

Under the light microscope, it can be seen that the positive substances accumulate in the cytoplasm and processes. The colored cells are of different sizes and shades. Most of them are oval, and a few are round. The number of nNOS-positive cells in the model group was significantly lower than that in the normal group and the blank group (P<0.01), and the cells were very lightly stained. The expression of nNOS cells in the cortex and hippocampus of rats with high amount was close to normal, and the cells stained darker. The number of nNOS-positive cells in the low-dose group and the control group also increased to varying degrees, and the results are shown in Table 4-1.

The influence of table 4-1 medicine of the present invention on cortex and hippocampus area nNOS positive cell number

Note: ^△△ P<0.01 compared with normal group ^▲ P<0.05 compared with model group ^▲▲ P<0.01

Compared with the control group ^★ P＜0.05

3.2 Effect of the drug of the present invention on the expression of iNOS in rat cortex and hippocampus

Under the microscope, iNOS-positive substances accumulated in the cytoplasm and processes, stained brownish yellow, and the colored cells were of different sizes and irregular shapes. The results are shown in Table 4-2, iNOS was rarely expressed in the normal group and the blank group; the number of iNOS positive cells in the model group was significantly higher than that in the normal group and the blank group (P<0.01). The expression of iNOS cells in the high amount group was significantly lower than that in the model group (P<0.01). The number of iNOS-positive cells in the low amount group and the control group also increased to varying degrees (P<0.01), but there was no difference between the two groups (P>0.05). The reduction in the high amount group was the most significant (P<0.01).

The influence of table 4-2 medicine of the present invention on iNOS expression

Note: Compared with the normal group ^△ P<0.05 ^△△ P<0.01 Compared with the model group ^▲▲ P<0.01

Compared with the control group ^★★ P＜0.01

3.3 The influence of the medicine of the present invention on the expression of P38 in rat cortex and hippocampus

This experimental study showed that P38 in the hippocampus and cortex of the model group was significantly reduced. In the normal group, the hippocampus and cortex were widely expressed, and diffuse synaptophysin protein distribution was seen, and the staining was darker. The difference was statistically significant (P<0.01). Synaptophysin protein in Meynert nucleus and its periphery in each treatment group decreased compared with the normal group, but increased significantly compared with the model group (P<0.01), especially in the high amount group (P<0.01). The results are shown in Table 4-3.

The influence of table 4-3 medicine of the present invention on AD model rat cortex and hippocampus P38 expression

Note: Compared with the normal group, ^△△ P<0.01 Compared with the model group, ^▲▲ P<0.01

Compared with the control group ^★★ P＜0.01

3.4 Effects of the medicine of the present invention on the expression of NGF in rat cortex and hippocampus

As shown in Table 4-4, the distribution of NGF-positive neurons in the cortex and hippocampus of rats in the normal group and blank group was more, and the number of NGF-positive neurons in the model group was significantly reduced compared with each group (P<0.01). The expression of NGF increased in different degrees in rats with low amount, high amount and control group (P<0.01). The number of positive neurons in the high-volume and low-volume groups was 6.5 and 4.5 times that of the model group, respectively. The level of NGF-positive neurons in the high-dose group was close to that of the normal group (P<0.01), and its effect on increasing the expression of NGF in the rat cortex and hippocampus was the most obvious.

The influence of table 4-4 medicine of the present invention on rat NGF

Note: Compared with the normal group, ^△△ P<0.01 Compared with the model group, ^▲▲ P<0.01

Compared with the control group ^★★ P＜0.01

3.5 Effect of the medicine of the present invention on the apoptosis rate of rat cortex and hippocampus neurons

Flow cytometry detection found that the peak type of hypodiploid cell population appeared on the left side of the G1 peak of nerve cells in the model group. This characteristic is consistent with the characteristics of apoptosis, indicating that IBO can induce apoptosis of nerve cells. There was no hypodiploid peak in the neurons of rats in the normal group and the sham operation group in the pre-G1 phase, indicating that there was no or very small amount of apoptosis. There was a small hypodiploid peak in the pre-G1 phase of nerve cells in the high-dose group, showing only a small amount of apoptosis, indicating that the high-dose invented drug had a protective effect on IBO injury. The nerve cells in the low dose group and the control group also had a small hypodiploid peak in the pre-G1 phase, but it was higher than that in the high group, showing that there was also a small amount of apoptosis, indicating that the low dose of the invented drug also had a protective effect on IBO injury . Compared with the normal group, the apoptosis rate of the model group significantly increased (P<0.01); compared with the model group, the apoptosis rate of the control group, the high amount group, and the low amount group also had a significant decrease (P<0.01), illustrating that the present invention The drug can partly antagonize the apoptosis induced by IBO, and the effect is best with high dose. The results are shown in Table 4-5.

Table 4-5 The influence of medicine of the present invention on nerve cell apoptosis

Note: Compared with the normal group ^△ P<0.05 ^△△ P<0.01 Compared with the model group ^▲▲ P<0.01

Compared with the control group ^★★ P＜0.01

4 discussions

This experiment proves that the intervention effect of the drug of the present invention on the cholinergic damage of experimental rats is realized by adjusting the level of cholinergic neurotransmitters, increasing the content of synaptophysin protein, inhibiting nerve cell apoptosis and other factors. The following is its specific mechanism of action:

4.1 Increase rat brain nNOS neurons and reduce iNOS content and activity

The results of this experiment show that the drug of the present invention can increase the number of nNOS cells in the rat cerebral cortex and hippocampus, reduce the expression of iNOS, and cause less damage to local neurons. The medicine of the present invention can inhibit damage to NOS neurons in the cerebral cortex and hippocampus of model rats, and the effect of the high dosage group is better than that of the low dosage group. It is thus suggested that the drug of the present invention can improve the learning and memory of rats, which is achieved by increasing the activity of nNOS neurons in the rat brain, inhibiting the expression of iNOS, and protecting local neurons.

4.2 Promote the formation of new synapses and increase the number of synapses in rats

Experiments show that the drug of the present invention can effectively promote the formation of new synapses, increase the number of synapses in model rats, and improve the level of learning and memory, which indicates that its effect on the plasticity mechanism of hippocampal synapses may be the neurobiological basis for improving learning and memory one. Previous studies have confirmed that the increase of cholinergic fibers can provide a material basis for synaptogenesis. In addition, it may be related to improving the protective effect on neurons and promoting the synthesis of synaptic auxin, which needs further study.

4.3 Inhibition of brain nerve cell apoptosis

The results of this experiment show that the apoptosis of brain nerve cells in model rats is significantly increased, and the drug of the present invention can significantly reduce the apoptosis of brain nerve cells, indicating that Naofukang and the drug of the present invention have the effect of inhibiting the apoptosis of brain nerve cells, and the effect of high dose most. The anti-apoptosis effect of the medicine of the present invention may be to protect the nerve cells by eliminating free radicals in the nerve cells, thereby achieving the effect of inhibiting cell apoptosis.

4.4 Increase the expression of NGF in brain tissue

From the foregoing experimental results, the medicine of the present invention has a significant improvement effect on the information acquisition ability and information storage ability of experimental rats, and the expression of endogenous NGF in each group of rats is consistent with its behavioral learning level, indicating that the model is large. There is a close relationship between the increase of endogenous NGF content in rats and the learning and memory level of experimental rats. It is speculated that this effect may be due to the fact that the drug of the present invention increases the content of endogenous NGF, and NGF promotes the increase in the number of cholinergic fibers in the innervation area, and promotes the formation of more synapses at the end of a single axon, which contributes to neuron damage. rapid recovery of function. Its specific mechanism of action needs to be further explored. Embodiment 8 The effect of the medicine of the present invention on apoptosis and calcium ion of hippocampal nerve cells cultured in vitro

1 material

1.1 Reagents

Poly-L-lysine (poly-l-lysine), Sigma product; calf serum, product of Hangzhou Sijiqing Institute of Bioengineering Materials; complete DMEM medium, product of Gibco, 1L DMEM medium containing 3.7g of NaHCO ₃ , penicillin G sodium 100U/ml, streptomycin 100 μg/ml and 10% calf serum, pH 7.4; Trypsin, Sigma product; L-glutamic acid, Shanghai Boao Biotechnology Company product; MTT (dimethylthiazole di phenyltetrazolium bromide), a product from Fluka; nimodipine, a product from Tianjin Central Pharmaceutical Factory; other reagents were commercially available analytically pure. Annexin-V-FITC apoptosis detection kit was purchased from Beijing Baosai Biotechnology Co., Ltd.

1.2 Prescription and preparation of the medicine of the present invention

With embodiment 5.

1.3 Preparation of rat serum and blank serum containing the drug of the present invention

Ten clean-grade Wistar rats, weighing 190-220 g, were provided by the Animal Center of Tongji Medical College, Huazhong University of Science and Technology. They were randomly divided into two groups, A and B, with 5 rats in each group.

Group A feeds and takes medicine water decoction of the present invention, calculates the daily dosage of rats according to the conversion method of human and rat body surface area, and presses serum pharmacology experiment method again, serum donor is clinical dosage or the effective dose of whole model animal. Dosage (8-10 times of clinical dose in people) administration, so the daily dose of rats is 2g/100g, i.e. 1ml/100g, and the water decoction of the medicine of the present invention is administered 2 times a day (8:00- 9:00, 3:00-4:00 pm) for 7 consecutive days, 2 hours after the last administration (drinking water was prohibited 12 hours before the administration), after being anesthetized with pentobarbital, blood was collected from the heart of the rat by aseptic operation, centrifuged Serum was obtained, mixed with sera from 5 rats, and inactivated at 56°C for 30 minutes. Sterilize by filtration with a 0.22 μm microporous membrane, and store at -20°C for later use. What is prepared by this method is the rat serum containing the drug of the present invention. The serum was diluted with 20% FBS DMEM/F12 into two different concentrations of 10% and 20% drug-containing serum.

Group B was fed with 0.9% normal saline for 7 consecutive days, and the administration time, method and method of obtaining serum were the same as group A. This method prepares the blank serum. Dilute the blank serum into 10% drug-containing serum in the same way.

1.4 Main Instruments

2003 type _CO2 incubator (Sheldon Company, USA), aseptic operating table (Suzhou Purification Equipment Factory), LG-10 centrifuge (Beijing Medical Centrifuge Factory), constant temperature water tank, enzyme-linked immunoassay instrument (Huadong Electronic Tube Factory) ), FACSCaliber flow cytometer (BD company in the United States), CK-2 inverted phase contrast microscope (Japan), BX-60 fluorescence microscope (Japan), RF-5000 fluorescence spectrophotometer (Japan), electronic balance, sterile centrifuge Tubes, 96-well culture plates, 24-well culture plates.

1.5 Preparation of main solution

PBS solution: NaCL 8g/L, KCL 0.20g/L, NaHPO ₄ ·H ₂ O 1.56g/L, KH ₂ PO ₄ 0.20g/L Dissolve the bagged powder in an appropriate amount of distilled water, stir to make it fully Melt, put the liquid into saline bottles, sterilize and sterilize with high-pressure steam, adjust the pH value of the PBS solution to 7.2-7.4 with sterile NaHCO ₃ solution, and store in a 4°C refrigerator for later use.

DMEM/F12 culture medium: Warm the prepared triple distilled water to 16-30°C; dissolve the bagged DMEM/F12 dry powder in water, stir to make it dissolve; add appropriate amount of NaHCO ₃ ; adjust with sterile NaHCO ₃ solution The pH value of the culture solution is 7.2-7.4; filter, because the pH may be affected during filtration, filter with a microporous membrane of 0.22 μm or smaller, subpackage, and store in a refrigerator at 4°C for later use.

MTT solution: Weigh 250mg of MTT, put it into a small beaker, add 50ml of PBS solution (0.01mol/L, pH7.4), stir on a magnetic stirrer for 30 minutes, filter through a 0.22μm microporous membrane, and store in 4°C. Backup, valid for two weeks.

Preparation of Annexin V-FITC/PI: Prepare according to the instruction manual.

2 methods

2.1 Primary culture of rat brain hippocampus neurons

Put the newborn rats into 75% alcohol for disinfection, put them in the ultra-clean workbench, move them into a beaker with PBS, soak for a while, take them out and place them in a plate filled with D-Hanks liquid, cut off the skin of the head, Peel off the cerebral cortex and quickly place it in ice-cold D-Hanks solution, remove the meninges and blood vessels, take the rat hippocampal tissue and move it to a small bottle containing a small amount of D-Hanks solution, cut the tissue into minced shape with ophthalmic scissors, and add 0.25% Cover the tissue fragments with trypsin, digest at 37°C for 30 minutes, blow and beat repeatedly to disperse the brain tissue, add DMEM medium containing 10% calf serum to stop the digestion, let it stand, suck out the upper cell suspension and put it into a sterilized graduated centrifuge tube , centrifuge at 800rpm for 10min, discard the supernatant, add DMEM containing 10% calf serum to the pellet to make a cell suspension, inoculate in a 24-well plate pre-coated with poly-l-lysine, place at 37°C in 5% CO ₂ Culture in an incubator, change the medium once after 24 hours, and then change the medium every 3 days, and replace it with a culture medium containing cytarabine (final concentration: 5 μg/ml) on the 7th day to inhibit the growth of glial cells and other non-neural cells. Proliferate, change to normal medium after 48h, and prepare for drug experiment.

2.2 The effect of the drug-containing serum of the present invention on the growth and proliferation of hippocampal nerve cells.

Culture wells with similar cell density and consistent growth state were selected for the experiment. The cells are divided into: 1. culture medium group, 2. 5% drug-containing serum group of the present invention, 3. low-dose group of drug-containing serum of the present invention, 4. high-dose group of inventive drug-containing serum, 5. blank serum Group. Suck off the original culture solution, gently wash twice with sugar-free Earle's solution, add 1ml of sugar-free Earle's solution to each well, and let it act for 30 minutes. Add the drug-containing serum group of the present invention with a final concentration of 5% respectively Serum; the blank serum group was added with a final concentration of 10% drug-free serum. Cultivate at 37°C, 5% CO ₂ for 24h. Add MTT (final concentration: 0.5 mg/ml), continue to culture for 4 hours, suck off the culture medium, add 200 μl of 100% dimethyl sulfoxide to each well, and after the blue particles in the wells are completely dissolved, use an enzyme-linked immunosorbent assay Optical density values (OD) were measured at 490 nm.

2.3 Effects of the drug-containing serum of the present invention on the apoptosis of nerve cell damage induced by glutamate

Select culture wells with similar cell density and consistent growth status for the experiment. The cells were then divided into: 1. model group, 2. nimodipine group, 3. 10% blank serum group, 4. 10% drug-containing serum group of the present invention, and 5. 20% drug-containing serum group of the present invention. Aspirate the original culture solution, gently shake and wash twice with sugar-free Earle's solution, add 1ml of sugar-free Earle's solution to each well, and let it act for 30min, add nimodipine with a final concentration of 5×10-6mol/L in the nimodipine group, The low-dose and high-volume groups of the medicine-containing serum of the present invention add the medicine-containing serum of the invention with a final concentration of 5% respectively, and act for 20 minutes, then add glutamic acid (Glu) with a final concentration of 500 μmol/L, and act for 30 minutes; the normal control group Only Glu with a final concentration of 500 μmol/L was added; the blank serum group was added with a final concentration of 5% drug-free serum, while the normal control group was treated with D-Hanks for 30 minutes without Glu. Finally, each group sucked off the liquid, washed gently with D-Hanks solution twice, added 1ml of DMEM/F12 culture solution to each well, and cultured at 37°C, 5% CO2 for 24h. The apoptosis rate was detected on a flow cytometer with the Annexin V-FITC/PI method.

2.4 Effects of the drug-containing serum of the present invention on intracellular calcium ions induced by glutamic acid

Cell grouping, treatment, and culture were the same as above. After culturing for 24 hours, take the cultured nerve cells, suspend the cells with D-Hanks solution, adjust the cell concentration to 105/ml, add Fura-2 to the cell suspension, incubate at 37°C, 5% _CO2 incubator for 50min, take out the cell suspension Wash the cells twice with D-Hanks solution to remove Fura-2 in the extracellular fluid. Then use D-Hanks solution to suspend the cells, adjust the cell concentration to 105/ml, and observe the survival of the cells. Use a fluorescence spectrophotometer to excite at dual wavelengths of 340 and 380nm, and emit at a wavelength of 505nm to measure intracellular free calcium under different experimental conditions. The fluorescence intensity was corrected for cellular autofluorescence. According to the formula: Calcium ion (n mol/L)=Kd(FF _min )/(F _max -F) to calculate the calcium ion concentration (F is the fluorescence intensity when Triton-100 is not added, F _max is the maximum fluorescence intensity, F _min minimum fluorescence intensity, Kd=224nmol/L).

3 results

3.1 MTT test results

The results of the influence of the invented drug on the primary cultured sea nerve cells are shown in Table 5-1. The high dose group of the invented drug-containing serum has obvious effects on the growth and proliferation of the sea nerve cells, and there is a significant difference compared with the control group (p<0.05 ). There was no significant difference between the blank serum group and the low-dose serum group containing the invented drug compared with the control group (p>0.05).

Table 5-1 OD value and cell growth and growth rate of each group

Note: Compared with culture medium group, △p<0.05

3.2 Containing the change of drug rat serum of the present invention to apoptosis

As shown in Table 5-2, the apoptosis of nerve cells is the highest in the model group, followed by the blank serum group, and the drug serum group of the present invention is the lowest. Compared with the model group, the Xingnaoyizhi low and high dose groups have significant statistical differences ( P<0.05), there was no significant difference between the blank serum group and the model group (P>0.05).

The influence of table 5-2 drug rat serum of the present invention on nerve cell apoptosis

Note: △Compared with culture medium group, p<0.05

3.3 Effects of rat serum containing the drug of the present invention on calcium ions in nerve cells

As shown in Table 5-3, the apoptosis of nerve cells is the highest in the model group, followed by the blank serum group, and the drug serum group of the present invention is the lowest. Compared with the model group, the Xingnao Yizhi low and high dose group has a significant statistical difference ( P<0.05), there was no significant difference between the blank serum group and the model group (P>0.05).

Table 5-3 The influence of drug rat serum of the present invention on calcium ion in nerve cells

Note: Compared with culture medium group, △p<0.05, △△p<0.01

4 discussions

4.1 Drugs of the present invention can inhibit nerve cell apoptosis

The experimental results show that the drug-containing serum of the present invention can obviously promote the growth of nerve cells and inhibit nerve apoptosis. A large number of research results have shown that blood-activating Chinese medicines have the effect of removing free radicals in the body, so the drug of the present invention has the effect of inhibiting cell apoptosis by removing free radicals in nerve cells, thereby protecting nerve cells and achieving the effect of inhibiting cell apoptosis. In addition, the drug of the present invention can reduce the calcium ion overload in nerve cells, and can also inhibit cell apoptosis. 4.2 The medicine of the present invention can alleviate the calcium ion overload of nerve cells

In this experiment, the addition of glutamic acid to the cultured nerve cells caused intracellular calcium ion overload, and the calcium fluorescent probe was used to detect the effect of the invented drug on the calcium ion overload of nerve cells. have obvious inhibitory effect. This shows that the effect of the drug of the present invention on inhibiting nerve cell apoptosis may be related to the inhibition of calcium ion overload in nerve cells. The drug of the present invention can antagonize the entry of calcium ions into nerve cells, reduce the damage of calcium ion overload to cells, and achieve the purpose of inhibiting cell apoptosis. role.

Example 9 Effect of the drug of the present invention on the learning and memory level of IBO+Aβ _25-35 induced pseudo-AD model rats

1 Experimental materials

1.1 Experimental animals

Seventy 15-month-old Wistar rats, half male and half male. Body weight 450±50g, provided by Hubei Experimental Animal Research Center.

1.2 Drugs, reagents and instruments

The medicine of Example 3 was prepared, and the high, middle and low dose groups were equivalent to containing crude drug 2g/ml, 1g/ml and 0.5g/ml respectively. Hubin, Henan Zhongsheng Pharmaceutical Co., Ltd. Yuzhong Pharmaceutical Factory. Amanita acid (Ibotenicacid, IBO) and Aβ _25-35 were ordered from Sigma.

Rat Morris water maze automatic experiment recorder, Chinese Academy of Medical Sciences. Brain Stereotaxic Instrument Beijing Shuolinyuan Technology Co., Ltd.

2 Experimental methods

2.1 AD model construction

1 mg of Aβ _25-35 peptide was dissolved in 100 μl of sterilized physiological saline at a concentration of 10 μg/μl. After sealing the parafilm, incubate at 37°C for 96 hours to allow aggregation and aging. Take 1 mg of IBO and dissolve it in it to make a mixed solution of IBO and Aβ _25-35 , which is prepared immediately for use.

Model group: same as the composite model group of Experimental Example 5.

Sham operation group: a one-time injection of 1 μl sterilized physiological saline was given to the rats under localization as a negative control.

Normal group: no treatment.

2.2 Animal grouping

70 15-month-old Wistar rats were randomly divided into 7 groups, namely normal group, sham operation group (abbreviated as blank group), model group, Haberin treatment group (abbreviated as Western medicine group), high, medium and low doses of invented drugs Treatment groups (referred to as high, medium and low dose groups), 10 rats in each group.

2.3 Administration

Two weeks after the intracerebral injection, the dosage was determined according to the ratio of the body surface area coefficients of humans and rats, and the clinical human equivalent dosage was used as the middle dosage, and the high, middle and low dosages were determined according to 4:2:1. The rats in the three groups of traditional Chinese medicine were given the decoction of the medicine in the stomach, while the Western medicine group was given the Haberine suspension; the normal group, the blank group and the model group were given the same volume of normal saline. Rats in each group were intragastrically administered at 1ml/100g·d. 28 consecutive days.

2.4 Morris water maze experiment is the same as in Example 5.

2.5 Statistical processing

Cit.

3 results

3.1 Morris water maze behavior test results

3.1.1 Average escape latency of rats

Two days before the experiment, the animals in each group basically swam around the pool wall, seldom swam to the vicinity of the platform, and their movement trajectories were randomly distributed in each quadrant. However, as the experiment progressed, the time for the rats in the normal group and the sham operation group to rely on spatial cues to find the platform position gradually shortened, and their movement tracks also gradually located in the platform quadrant, or searched in the left and right quadrants adjacent to the platform quadrant. The latency period drops rapidly. But the rats in the model group basically maintained the situation of the previous two days, with a slight improvement, except for a very small number of rats who could find the platform. To analyze information acquisition ability, the total latency of the 4-day training and the post-latency of the last 2 days of training were measured. It can be seen from Table 6-1 that the average incubation period of the model group was significantly longer than that of the normal group and the blank group, and there was a significant difference (P<0.01). Compared with the model group, the average incubation period of the western medicine group and the high, middle and low dose groups was significantly shortened, and there was a statistical difference (P<0.01) compared with the model group. Further inter-group comparison found that there was a significant difference between the high-dose group and the western medicine group (P<0.01). There is also a significant difference in the ratio of middle and high dose groups (P<0.01). See Figure 1.

Table 6-1 The average incubation period results of the first 4 days and the last 2 days of the rat positioning navigation experiment

Note: Compared with the normal group, ^△△ P<0.01 Compared with the model group, ^▲▲ P<0.01

Compared with the western medicine group ^★ P＜0.05 ^★★ P＜0.01Compared with the moderate group ^☆☆ P＜0.01

3.1.2 Average swimming distance of rats

The quadrant percentage of rats in model group was significantly shorter than that of rats in normal group and blank group. Compared with the model group, the percentage of quadrants in the high-dose and middle-dose groups was significantly increased (P<0.01). The quadrant percentage of the low dose group was not different from that of the model group (P>0.05). Further inter-group comparison found that the difference between the low-dose group and the western medicine group was significant (P<0.01). The 20% and 40% regions of the high, middle and low dose groups were significantly reduced compared with the model group (P<0.01), and their values decreased correspondingly with the increase of the dose. However, there was no difference between the middle and high dose groups except for the 20% area (P>0.05). Compared with the western medicine group, there was no difference (P>0.05). The 20% and 40% regions of the low-dose group were significantly different from those of the western medicine group (P<0.01). The results are shown in Table 6-2.

Table 6-2 The learning and memory results of the positioning and navigation experiment of rats

Note: Compared with the normal group, ^△ P<0.05 ^△△ P<0.01 Compared with the model group, ^▲▲ P<0.01

Compared with the western medicine group ^★★ P＜0.01 Compared with the moderate group ^☆ P＜0.05

3.1.3 Change Trend of Rat Search Platform

It can be seen from Table 6-3 that the spatial search ability of rats in the model group decreased significantly, and its quadrant percentage was significantly shorter than that of the normal group and the blank group (P<0.01), and the 20% and 40% areas were significantly higher than those of the normal group and the blank group. increase (P<0.01). There was no difference between normal group and blank group (P>0.05). The quadrant percentages of the high, middle and low dose groups were significantly increased compared with the model group (P<0.01). The quadrant percentage and 20% area of the high-dose group were significantly different from those of the western medicine group (P<0.01 or P<0.05). There was a significant difference between the high-dose groups in the 20% area (P<0.01). There were differences in 40% of regions between low and middle dose groups (P<0.05). See Figure 2.

Table 6-3 Learning and memory results of rat space search experiment

Note: ^△△ P<0.01 compared with normal group and ^▲▲ P<0.01 compared with model group

Compared with the western medicine group ^★ P＜0.05 ^★★ P＜0.01 Compared with the moderate group ^☆ P＜0.05 ^☆☆ P＜0.01

3.1.4 Times of Rats Crossing the Platform

The number of times the rats in each group crossed the original platform position was compared. It can be seen from Table 6-4 that the number of times the rats in the model group crossed the original platform position was significantly lower than that of the other groups of rats, and compared with the other two groups. There was a significant difference (P<0.01). There were statistically significant differences between the Chinese medicine groups and the model group (P<0.01). There was a difference (P<0.01 or P<0.05) between the western medicine group and the middle-dose group, but no difference (P>0.05) between the low-dose group and a difference between the high-dose group and the middle-dose group (P<0.05).

次)Table 6-4 The results of the number of times rats passed through the original platform position (

Second-rate)

Note: ^△△ P<0.01 compared with normal group and ^▲▲ P<0.01 compared with model group

Compared with the western medicine group ^★ P＜0.05 ^★★ P＜0.01Compared with the moderate group ^☆ P＜0.05

4 discussions

The results of this experiment show that the rats in the model group cannot remember the location of the platform due to the loss of vision-based spatial positioning memory, and they still blindly look for escape routes, so their swimming tracks are mainly distributed in the outer ring without platform (pool wall 20 %area). Its spatial positioning memory ability was severely damaged, suggesting that memory cannot be established through acquisition training, indicating that the animal model's intelligence caused by this method decreased significantly, and the animal had a certain "dementia" performance. Kidney-tonifying and phlegm-resolving method is obviously superior to the model group in terms of the positioning and navigation experiment reflecting the ability of spatial learning and memory acquisition, and the spatial search experiment reflecting the ability of information storage. And it showed the trend of dose-effect relationship in high, medium and low dose groups. The experimental results also showed that there is still a certain gap between the effect of tonifying the kidney and resolving phlegm on the learning and memory of AD rats compared with the normal group and the sham operation group, indicating that the drug can only partially improve the learning and memory ability of AD, but cannot make it recover. This is also consistent with clinical efficacy.

Example 10 Effect experiment of the medicine of the present invention on the pathological morphology of the induced AD model rats induced by IBO+Aβ _25-35

1 Experimental materials

1.1 Experimental animals are the same as in Example 9.

1.2 Drugs, reagents and instruments

H600 Transmission Electron Microscope Hitachi Japan Co., Ltd. Others are the same as in Embodiment 9.

2 Experimental methods

2.1 AD model construction, animal grouping and administration are the same as in Example 9.

2.2 Observation of pathological morphology of hippocampus

2.2.1 Observation of neurofibrillary tangles

The hippocampus tissue was routinely embedded in paraffin, the section thickness was 4 μm, and the section was stained according to the hexamine silver staining method: the section was dewaxed to water, washed with double distilled water for 3 times, and put into hexamine silver solution (6% hexamethyltetramine 150ml, 5% silver nitrate 715ml, 5% sodium tetraborate 9ml), incubate at 56°C for 2h, wash the slices with double distilled water for 3 times, place them in 4% neutral formalin solution for 5min, wash the slices with double distilled water for 3 times, place in In 5% sodium thiosulfate solution for 5 minutes, washed in double distilled water for 5 minutes, dehydrated in gradient alcohol (70%, 80%, 90%, 95%, absolute ethanol) for 5 minutes each, transparent in xylene, and sealed with neutral gum. Nerve fibers are brownish black. Observed under a light microscope and photographed for analysis.

2.2.2 Senile plaque observation

The sections were dewaxed to water, stained with methanol Congo red staining solution (Congo red 0.5g, methanol 80ml, glycerol 20ml) for 15min, differentiated with alkaline ethanol differentiation solution (potassium hydroxide 0.2g, 80% ethanol 100ml) for a few seconds, washed with water, hematoxylin Counterstained for 2 minutes, washed with water, dehydrated, transparent, and mounted. Positive result: Amyloid plaques are orange-red.

2.2.3 Observation of ultrastructural changes of hippocampus by electron microscope

Rats were intubated through the left ventricle and aorta, fixed with 4% paraformaldehyde perfusion, and the brain was taken out, and the tissue block (1mm ³ ) from the same part of the hippocampus was fixed with 2% glutaraldehyde, and sent to the electron microscope room of Wuhan University School of Medicine to prepare ultra-thin Slice and observe ultrastructural changes of hippocampal neurons with a transmission electron microscope (Hitachi H600).

3 results

3.1 Observation under light microscope

The cortex and hippocampus of rats in the normal group and the blank group also showed some signs of aging, manifested as uneven staining of nerve cells, loss of complete structure, and unclear cell membranes and nuclear membranes. Amyloid-deposited plaques and neurofibrillary tangles can also be seen in individual fields of view, which may be the normal aging performance of 15 months old. A large number of vacuolar degeneration and slight edema can be seen in both. Model group: hippocampal neurons were significantly reduced, the boundary of cell membrane and nuclear membrane was not clear, nucleus pyknosis and vacuolar degeneration, and some neuron cells disappeared. The brain tissue of the model showed severe punctate disruption, significantly increased amyloid plaques and neurofibrillary tangles, and a large number of vacuoles formed, forming fishnet-like changes. The low-dose group and the western medicine group had little effect on the pathology of the model animals. The western medicine group only saw a slight increase in neurons, and the arrangement of the cell structure was better than that of the model. The number of neurons in the low-dose group also increased, and neurofibrillary tangles and amyloid plaques Slightly reduce and lighten. In the high-dose group, the repair of neurons was more obvious, and the nuclear pyknosis and vacuolar degeneration also improved to a certain extent. The neurofibrillary tangles and amyloid deposition plaques were significantly reduced, the plaques became smaller, and the fibers became thinner. The medium-dose group was between low-dose and high-dose. See attached drawings 3 and 4.

3.2 Observation under electron microscope

Normal group and blank group: the neuron cell bodies in the hippocampal area were densely arranged, the nuclei were mainly composed of euchromatin, the nucleoli were obvious, and the winding of fibers was rarely seen in the neuropil area, and abundant synaptic structures could be seen. There was not much difference in ultrastructure between the two groups. Model group: the arrangement of neurons is very disordered, and the cell bodies of neurons are condensed, and high-density chromatin borders can be seen under the nuclear membrane, or scattered in the nucleus in blocks, and the nuclear membrane is blurred. There are filamentous filament winding, disorderly arrangement, and acute swelling of mitochondria. Synaptic structures were significantly reduced or disappeared, and a state of edema with low electron density appeared. Western medicine group and low dose group: the size of neuron cell body is not uniform, there is blocky distribution of heterochromatin in the nucleus, and the structure of intracellular membrane in the cytoplasm is reduced. The filamentous fibers were disordered, the structure of synaptic membrane was fused, and the structure of synaptic vesicles was unclear. Medium-dose group: the neuron structure is acceptable, the mitochondria in the cytoplasm are slightly swollen, there is a very slight border of chromatin under the nuclear membrane, the nuclear membrane and the nucleus are intermittently widened, and some nerve fibers are swollen. High-dose group: the neuron cell body structure was close to the normal group, and the nerve fiber structure was acceptable. The synaptic structure is acceptable. See attached drawing 5.

4 discussions

In this experiment, the method of tonifying the kidney and resolving phlegm has the functions of replenishing essence and marrow, resolving phlegm and resuscitation. After the intervention of kidney-reinforcing and phlegm-resolving drugs, the neuron loss and vacuolar degeneration of the experimental animals improved to a certain extent, the neurofibrillary tangles and amyloid deposition plaques were significantly reduced, the plaques became smaller, and the fibers became thinner. Tonifying the kidney can fill the essence and produce the marrow, the marrow is prosperous, the brain marrow is filled, the loss of neurons and vacuolar degeneration can be repaired, and the phlegm can be inhibited and cleared. If the protein deposition is reduced, the mind is clear.

Example 11 Effects of Drugs of the Present Invention on the Cholinergic System of IBO+Aβ _25-35 Induced Pseudo-AD Model Rats

1 Experimental materials

1.1 Experimental animals are the same as in Example 9.

1.2 Drugs, reagents and instruments

Ach, AchE and ChAT radioimmunoassay kits were purchased from Beijing North Institute of Biotechnology.

Others are the same as embodiment 9.

2 Experimental methods

2.1 AD model construction, animal grouping and administration are the same as in Example 9.

2.2 Preparation of brain tissue homogenate and determination of total protein content

After the water maze test, the rats in each group were decapitated, and 100 mg of brain tissue in the hippocampus and cortex was quickly taken on the ice platform and added to normal saline at a ratio of 1:9. The homogenate was homogenized in an ice bath for 30 seconds, centrifuged at 3500 rpm for 10 minutes at 4°C, and the supernatant was taken. Freeze for later use.

2.3 Determination of Ach, AchE and ChAT activity in rat brain tissue

Using radioimmunoassay. Brain tissue was separated, weighed, and homogenized to make a 10% homogenate. The supernatant was taken for protein quantification and then sent to the Isotope Laboratory of the Affiliated Hospital of Hubei University of Traditional Chinese Medicine for testing.

2.4 Statistical processing is the same as in Example 9.

3 results

In this experiment, the activities of Ach, ChAT and AchE in the brain tissue of the rats in each group were measured. The statistical results showed that compared with the normal group, Ach and ChAT in the brain tissue of the model group were significantly reduced (P<0.01), while the activity of AchE was significantly increased ( P<0.01). There was no difference in Ach results between the normal group and the blank group (P>0.05). Compared with the model group, there were significant differences in each group of traditional Chinese medicine (P<0.01). There was a difference (P<0.01) between Ach in the western medicine group and the low-dose group (P<0.01), but no difference (P>0.05) compared with the high-dose group. From the detection results of ChAT and AchE activity, the two are consistent in each group, and there are significant differences between the treatment groups and the model group (P<0.01), and there are differences between the low dose and the western medicine group (P<0.05), and the middle and high doses. There was no difference in dose (P>0.05). There was no difference in the ratio of middle and high doses (P>0.05). The results are shown in Table 8-1.

Table 8-1 Results of Ach, ChAT and AchE activities in rat brain tissue

Note: ^△△ P<0.01 compared with normal group and ^▲▲ P<0.01 compared with model group

Compared with western medicine group ^★ P＜0.05 ^★★ P＜0.01

4 discussions

In this experiment, the activities of Ach, ChAT and AchE in the brain tissue of rats in each group were measured. From the experimental results, the Ach and ChAT in the brain tissue of the model group were significantly reduced compared with those of the other groups, while the activity of AchE was significantly increased. Each treatment group can significantly increase the activities of Ach and ChAT in the hippocampus of AD model rats, and inhibit the excessively high AchE activity, indicating that the method of tonifying the kidney and resolving phlegm has a protective effect on central cholinergic damage. The effect of medium and high doses is more obvious. In addition, this experiment also shows that the learning and memory ability of rats is correlated with the change of cholinergic, indicating that the method of tonifying the kidney and resolving phlegm has a repairing effect on the central cholinergic damage, so as to achieve the purpose of improving learning and memory.

Example 12 Effect experiment of the drug of the present invention on the ubiquitin-proteasome pathway induced by IBO+Aβ _25-35 in pseudo-AD model rats

1 Experimental materials

1.1 Experimental animals are the same as in Example 9.

1.2 Drugs, reagents and instruments

Ub, E1, E2-EPF antibodies were purchased from abcam company, and PGP9.5 antibody was purchased from chemicon company. ELISA auxiliary reagents (primary antibody working solution, enzyme-labeled antibody working solution, stop solution, substrate working solution, etc.) were purchased from Shanghai Senxiong Technology Industrial Co., Ltd. Westernblot reagents (lysate, G250 Coomassie brilliant blue solution, SDS loading buffer, electrophoresis buffer, transfer buffer, blocking solution, TBST, TBS, elution antibody buffer, developer, fixer, antibody, chemiluminescent reagent etc.) were purchased from Beijing Pulilai Gene Technology Co., Ltd. Trizol (Life Technology Company), dNTP, OligodT, RNAsin, RNase, M-MLV, taq enzyme (TaKaRa Company), Mgcl2, DEPC, agarose, chloroform, isopropanol, ethanol, 4% paraformaldehyde, xylene. The primer sequences were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd. SABC immunohistochemistry kit and DAB were purchased from Wuhan Boster Bioengineering Co., Ltd.

The real-time fluorescent quantitative PCR instrument is American ABI 5700 type.

2 Experimental methods

2.1 AD model construction, animal grouping and administration are the same as in Example 9.

2.2 Detection indicators and methods

2.2.1 Determination of ubiquitin protein expression

2.2.1.1 Immunohistochemical detection of ubiquitin protein

Rats were perfused and fixed with 4% paraformaldehyde through left ventricle aorta cannulation, and the brain was taken out for coronal slices. Negative control: replace the primary antibody with PBS. Analyzed by image processing system. step:

①Paraffin sections were routinely dewaxed with xylene for 15 min×3 times, and graded alcohol (absolute ethanol, 95%, 90%, 80%, 70%) for 5 min each; ②Fresh 3% H ₂ O ₂ inactivated endogenous enzymes for 10 min , washed with distilled water for 2 min×3 times; ③put the slices in citrate buffer (pH6.0) for 5min×2 times for heat-recovered antigen, and washed with PBS (pH7.4) for 5min×3 times; ④drop 5% Block with BSA, room temperature for 20min, shake off excess liquid, do not wash; ⑤ add diluted rabbit anti-polyclonal antibody dropwise, freeze overnight at 4°C, wash with PBS for 2min×3 times; ⑥ add secondary antibody (goat anti-mouse IgG) dropwise at 37°C Incubate for 30 min, wash with PBS for 2 min x 3 times; ⑦ add SABC reagent dropwise for 30 min, wash with PBS for 5 min x 4 times; ⑧ add DBA dropwise, react at room temperature for 5-30 min; Seal the slides with sexual gum; ⑩ take the slides and analyze them.

Positive reaction products are brownish-yellow particles, and the stronger the reaction, the darker the color. Negative reactions are colorless. Each slice is input to the image analysis system to measure the gray value of the positive cell body expressed in the selected brain area. Calculate the average gray value of the positive neuron cell bodies in each group of slices, and then conduct a significance test.

2.2.1.2 RT-PCR detection of ubiquitin protein

All reagents used in the operation were prepared with DEPC-treated water; all containers and utensils were treated to remove RNase. The glassware was baked at 150°C for 4 hours. For other supplies, DEPC was inactivated by high pressure after soaking overnight in 0.1% DEPC water. And the whole process is carried out in the fume hood.

Rat Ub primers:

Upstream 5'-TAAGACCATCACCCTCGATT-3', downstream 5'-TGGATGTTGTAGTCAGACAGGG-3' amplified cDNA sequence fragment length is 156bp

β-actin primer:

Upstream: 5'-GAGACCTTCAAGACCCCAGCC-3', Downstream: 5'-TCGGGGCATCGGAACCGCTCA-3' The length of the amplified cDNA sequence fragment is 404bp.

(1) Extraction of total RNA

①Take out 80 mg of brain tissue from liquid nitrogen and quickly put it into a glass homogenizer containing 1 ml Trizol, homogenize it manually, pour it into a 1.5 ml EP tube, oscillate to dissolve the precipitate, and let it stand at room temperature for 5 minutes. ② Add 0.2ml of chloroform and cover the lid. Vigorously upside down for 15s. Then stand at room temperature for 10 min. ③ Centrifuge at 12000rpm at low temperature for 15min at 4°C. Carefully draw about 500 μl of the upper layer of colorless liquid (total RNA) and transfer it to another 1.5ml EP tube. ④ Add an equal volume of 300 μl of isopropanol, vortex and mix well, and let stand at room temperature for 10 minutes. ⑤Centrifuge at 12000rpm at low temperature for 15min at 4°C. After centrifugation, it can be seen that the RNA is attached to the bottom of the tube wall in the form of white flakes. Carefully pour out the upper layer of liquid towards the opposite direction of attachment. While pouring, pay attention to whether the RNA is still attached to the wall, and absorb the remaining water from the opposite direction of attachment. go. ⑥ Add 1ml of 75% DEPC-treated ethanol, shake vertically and centrifuge at 8000rpm at 4°C for 10min. ⑦ Remove the upper liquid in the same way, and add 30 μl of DEPC-treated water. Store at -70°C. (2) RT reaction

The reaction was in a 0.2ml EP tube, and the EP tube was placed on ice (to prevent the secondary structure from re-forming), and then other reagents were added. The order of adding samples and the dosage are

70°C for 5min (for dissolving the secondary structure of the template), after centrifugation, put it into a PCR instrument for reaction: 37°C for 1h+94°C for 5min+4°C for 1h.

(3) The PCR reaction is carried out in a 0.2ml EP tube, and the sequence and dosage of the samples are as follows:

Reaction: 94°C for 5min; 94°C for 30s→55°C for 1min→72°C for 1min 20s×30cycles; 72°C for 10min; 4°C for 1h

(4) Agarose gel electrophoresis

Take 10 μl of PCR reaction product and electrophoresis on 2% agarose gel, and stain with 0.5 μl/ml ethidium bromide. The results were expressed in band optical density Ub/β-actin for comparison between groups.

2.2.2 Determination of ubiquitin-related enzymes (E1, E2-EPF, PGP9.5)

2.2.2.1 Enzyme-linked immunoassay (ELISA) detection

① Coating: Add the diluted coated antibody to the ELISA plate, 100 μl/well, and place at 4°C for 48 hours; ② Add 100 μl of the sample to be tested in turn to each well. Place the reaction plate in a warm bath at 37°C for 120min. ③ Wash the reaction plate 4-6 times with washing solution, put it on filter paper and print it dry. ④ Add 100 μl of primary antibody working solution to each well. Place the reaction plate in a warm bath at 37 °C for 60 min. Wash the plate as before. ⑤ Add 100 μl of enzyme-labeled antibody working solution to each well. Place the reaction plate in a warm bath at 37 °C for 60 min. ⑥Add 100 μl of substrate working solution to each well, and react in the dark at 37°C for 5-10 minutes. ⑦ Add 1 drop of stop solution to each well and mix gently. ⑧ Measure the absorbance at 492nm. ⑨Result Judgment The experimental results are expressed by OD value, and all OD values are calculated after subtracting the blank value for comparison between groups.

2.2.2.2 Western blot detection

(1) Extraction of total protein in tissue

① Take 50 mg brain tissue block from liquid nitrogen, and cut the tissue block as much as possible. ②Put it into a homogenizer, add 400 μl of buffer solution (containing PMSF), and then place it on ice for homogenization. ③ Centrifuge at 12,000 rpm for 5 min at 4°C, take the supernatant and put it in a 0.5ml centrifuge tube and store at -20°C.

(2) Determination of protein content

①Take a 1.5ml centrifuge tube and add 1ml of Coomassie Brilliant Blue solution stored at 4°C to each tube. After standing at room temperature for 30 minutes, it was used to measure protein. ②Take a tube of Coomassie Brilliant Blue plus 0.15mol/L NaCl solution 100μl, mix well and let it stand for 2min for blank control detection. ③Take a tube of Coomassie Brilliant Blue plus 95μl 0.15mol/L NaCl solution and 5μl protein sample to be tested, mix well and let stand for 2min to test the sample.

(3) SDS-PAGE gel electrophoresis

①Install the glass plate. ②Add TEMED (tetramethyldiethylamine) and shake well immediately, then pour 10% separating gel. Shake the glue well immediately after adding TEMED. Fill the remaining space with 4% stacking gel and immediately insert the comb into the stacking gel. ③ Rinse the stacking gel with water and put it into the electrophoresis tank. Add the protein sample that has been boiled and denatured, and the sample volume is consistent. ④ Add 1× electrophoresis buffer to the electrophoresis tank. The electrophoresis time is 4-5h, and the voltage is 40V. Electrophoresis until the bromophenol blue just ran out to stop the electrophoresis and transfer to the membrane.

(4) transfer film

①Cut 6 pieces of filter paper of the same size, which is the same size as the transfer area. Then, soak them in electroporation buffer. Cut a piece of PVDF (polyvinylidene fluoride) film of the same size, and gently place it in an ultrapure horizontal dish for 2 hours. ②Put 3 pieces of filter paper on the PVDF membrane, spread the gel on the PVDF membrane, put 3 pieces of filter paper on it, roll the glass rod on the surface of the filter paper to remove the air bubbles in each layer, then put it into the electrotransfer instrument, transfer at 60V 2h. ③ After the transfer, the membrane was stained with 1× Ponceau staining solution for 5 min. Then wash off the unstained dye solution with water to see the protein on the membrane. Allow the film to dry for later use.

(5) immune response

① After soaking the membrane with TBS (tert-butyldimethylsilane) from bottom to top, move it to a plate containing blocking solution, and shake it on a decolorizing shaker at room temperature for 1 hour. ② Pour off the blocking solution, add the primary antibody diluted to an appropriate concentration (in a 1.5ml centrifuge tube) with TBST (buffer solution); after incubating at room temperature for 1-2 hours, wash twice with TBST on a decolorizing shaker at room temperature, each 10min each time; wash again with TBS for 10min. ③ Prepare the secondary antibody dilution as above and contact with the membrane. After incubating at room temperature for 1-2 hours, wash twice with TBST on a decolorizing shaker at room temperature, each time for 10 minutes; then wash once with TBS for 10 minutes, and perform chemiluminescent reaction. (6) Chemiluminescence, developing, fixing

Mix the two reagents A and B in equal volumes on the plastic wrap; after 1 minute, fully contact the mixture with the membrane protein side down; after 1 minute, move the membrane to another plastic wrap, remove the residual liquid, wrap OK, put it in the x-ray holder. Sensitize, develop and fix with X film in the darkroom.

(7) Take pictures on film, and use a gel image processing system to analyze the molecular weight and net optical density of the target band.

2.3 Statistical processing is the same as in Example 9.

3 results

3.1 Changes of ubiquitin protein in brain tissue of rats in each group

The results of immunohistochemical experiments showed that Ub immunopositive expression was rare in normal rats, and there was no significant difference between the normal group and the sham operation group (P>0.05), while the brownish yellow Ub immunopositive expression in the model group increased, which was comparable to that in the normal group. There was a statistical difference (P<0.01). There was no difference between the Western medicine group and the model group (P>0.05), and the positive expressions of the Chinese medicine groups also increased significantly. Compared with the model group, the expression in the high-dose group increased significantly (P<0.01), followed by that in the low-dose group (P<0.05). There was no difference in the ratio of medium and high doses (P>0.05). See Figure 10 for the results of immunohistochemical color development. The results of RT-PCR showed that the bands of the model group were significantly different from those of the normal group (P<0.01), and the luminosity value of the three groups of traditional Chinese medicine was significantly enhanced compared with that of the model group (P<0.01), which was significantly different from that of the western medicine group (P<0.01 or P<0.01). <0.05). The results are shown in Table 9-1. See accompanying drawing 6.

Table 9-1 The results of Ub protein levels in the hippocampus and cortex of each group of rats

Note: Compared with the normal group, ^△△ P<0.01 Compared with the model group, ^▲ P<0.05 ^▲▲ P<0.01

Compared with western medicine group ^★ P＜0.05 ^★★ P＜0.01

3.2 Changes of E1 protein expression in hippocampus and cortex of rats in each group

The results of ELISA (enzyme-linked immunosorbent assay) method showed that there was no significant difference between the normal group and the sham operation group (P>0.05), while the E1 content in the model group had a downward trend in numbers, but compared with the normal group, there was no Statistical difference (P>0.05). There was no difference between the Western medicine group and the model group (P>0.05). Compared with the model group, the E1 protein content of each Chinese medicine group increased significantly (P<0.01), and there was also a significant difference with the Western medicine group (P<0.01). There was no difference between the middle and high dose groups (P>0.05). Western blotting showed that the E1 protein was expressed at 118kD, which was consistent with the expected results. The OD value analysis results had the same trend as the ELISA results. The difference was that the model group was different from the normal group (P<0.05), and compared with the western medicine group (P<0.05). The results are shown in Table 9-2. See attached drawing 7.

Table 9-2 Results of E1 levels in hippocampus and cortex of rats in each group

Note: Compared with the normal group ^△ P<0.05 and the model group ^▲ P<0.05 ^▲▲ P<0.01

Compared with western medicine group ^★★ P＜0.01

3.3 Changes of E2-EPF protein levels in the hippocampus and cortex of rats in each group

The results of ELISA (enzyme-linked immunosorbent assay) method showed that there was no significant difference between the normal group and the sham operation group in the content of E2-EPF in the rat hippocampus and cortex (P>0.05), while the E2-EPF protein content in the model group was significantly increased. Compared with the normal group, there was a statistical difference (P<0.01). Compared with the model group, the E1 protein content in each treatment group was increased to varying degrees (P<0.01 or P<0.05). Compared with the Western medicine group, there was a difference in the high dose (P<0.05), but there was no difference in the ratio between the middle and high dose groups (P>0.05). Western blotting detected E2-EPF protein mainly showed four bands between 35-40kD, which was consistent with theory. The OD value results have a consistent trend with the ELISA results, the difference is that there is a significant difference between the low dose group and the western medicine group (P<0.05). The results are shown in Table 9-3. See attached drawing 8.

Table 9-3 The results of E2-EPF protein levels in the hippocampus and cortex of each group of rats

Note: Compared with the normal group, ^△△ P<0.01 Compared with the model group, ^▲ P<0.05 ^▲▲ P<0.01

Compared with western medicine group ^★ P＜0.05

3.4 Changes of PGP9.5 protein levels in hippocampus and cortex of rats in each group

The results of ELISA detection showed that the content of PGP9.5 in rat hippocampus and cortex had no significant difference between the normal group and the sham operation group (P>0.05), while the PGP9.5 protein content in the model group was significantly reduced. There were no statistical differences (P<0.05). There was no difference between the western medicine group and the model group (P>0.05). Compared with the model group, the protein content of PGP9.5 in each group of traditional Chinese medicine increased significantly (P<0.01). Compared with the Western medicine group, each Chinese medicine group also had a significant difference (P<0.01). There was a difference in the ratio of middle and high dose groups (P<0.05). Western blotting detected PGP9.5 protein mainly showed three bands between 27-36kD, which was consistent with the theory. The OD value test results had the same trend as the ELISA results, the difference was that there was no difference between the model group and the normal group (P>0.05), and there was no difference between the high-dose group and the middle-dose group (P>0.05). The results are shown in Table 9-4. See attached drawing 9.

Table 9-4 Results of PGP9.5 protein levels in hippocampus and cortex of rats in each group

Note: ^△ P<0.05 compared with normal group and ^▲▲ P<0.01 compared with model group

Compared with the western medicine group ^★★ P＜0.01 Compared with the middle group ^☆ P＜0.05

4 discussions

The detection results of Ub, E1, E2-EPF and PGP9.5 in rat hippocampus and cortex showed that the results of ELISA and Western blot were basically the same, and there was no significant difference between the normal group and the sham operation group. In the model group, the protein content of Ub and E2-EPF was significantly increased, and the content of E1 and PGP9.5 was significantly decreased. It can be seen that the ubiquitination system in the model group was disordered, which was consistent with the previous pathological experiment results, that is, amyloid plaques appeared in the brain tissue and neurofibrillary tangles.

Experimental results show that the drug of the present invention can better regulate the disorder of ubiquitin and its related proteins in the model group, and can effectively improve the protein content of Ub, E1, E2-EPF and PGP9.5 in the hippocampus and cortex of animals, Enhance the ability of the ubiquitin-proteasome pathway to degrade abnormal proteins, and there is a certain dose-effect relationship. The effect of medium and high doses is significantly better than that of the low dose group. Except for some indicators, the high dose does not show a better effect than the medium dose.

Example 13 Effect of the drug of the present invention on the abnormal phosphorylation of Tau protein induced by IBO+Aβ _25-35 in pseudo-AD model rats

1 Experimental materials

1.1 Experimental animals are the same as in Example 9.

1.2 Drugs, reagents and instruments

Western blotting chemiluminescent reagent (ECL), BCA protein concentration detection kit, pre-stained protein marker, PVDF membrane, protein extraction kit were purchased from Beijing Pulilai Gene Technology Co., Ltd. Rabbit anti-rat phosphorylated Tau antibody (PHF-1) was purchased from abcam company, and HRP-labeled goat anti-rabbit secondary antibody was purchased from Wuhan Boster Bioengineering Co., Ltd.

CAMK-II in situ hybridization kit was purchased from Wuhan Boster Bioengineering Co., Ltd., Trizol was purchased from LifeTechnology Company, dNTP, Oligo dT, RNAsin, RNase, M-MLV, taq enzyme, Mgcl2, DEPC were purchased from TaKaRa Company, Gsk -3β rat Elisa detection kit was purchased from Shanghai Senxiong Technology Industrial Co., Ltd., and primers were purchased from Shanghai Sangon Bioengineering Technology Company.

Acrylamide, Methylenebisacrylamide, Ammonium Persulfate, Tris (Tris), Sodium Lauryl Sulfate (SDS), Glycine, Ponceau, Dithiothreitol, 2-Mercaptoethanol , N, N, N, N-tetramethylethylenediamine (TEMED), Tween20, etc. are analytically pure.

Real-time fluorescent quantitative PCR instrument (type 5700, ABI Company of the United States), microplate reader (type MB-III, Beijing Binda Yingchuang Technology Co., Ltd.).

Others are the same as embodiment 9.

2 Experimental methods

2.1 AD model construction, animal grouping and administration are the same as in Example 9.

2.2 Animal materials

After the animal was decapitated in the water maze, the brain was quickly removed on the ice platform, and the hippocampal tissue was separated and frozen in liquid nitrogen.

2.3 Detection indicators and methods

2.3.1 Western blot detection of abnormal phosphorylation of Tau protein in rat hippocampal tissue, same as in Example 12.

2.3.2 Detection of GSK-3β in brain tissue by enzyme-linked immunosorbent assay (ELISA)

① Establish a standard curve: set 8 standard wells. Add 100 μl of standard dilution solution to each well. Add 100 μl of the standard to the first well, mix well, pipette 100 μl into the second well, and then dilute to the seventh well, take out 100 μl from the seventh well and discard. The eighth hole is the zero hole. ② Add 100 μl of the sample to be tested in turn to each well. Place the reaction plate in a warm bath at 37°C for 120min. ③ Wash the reaction plate 4-6 times with washing solution, put it on filter paper and print it dry. ④ Add 100 μl of primary antibody working solution to each well. Place the reaction plate in a warm bath at 37 °C for 60 min. Wash the plate as before. ⑤ Add 100 μl of enzyme-labeled antibody working solution to each well. Place the reaction plate in a warm bath at 37 °C for 60 min. ⑥Add 100 μl of substrate working solution to each well, and react in the dark at 37°C for 5-10 minutes. ⑦ Add 1 drop of stop solution to each well and mix gently. ⑧ Measure the absorbance at 492nm.

2.3.3 Detection of cdk5 enzyme activity in brain tissue by RT-PCR:

Rat cdk5 primers:

Upstream 5'-GAAGCGGCACTCCATCATCTCGG-3'downstream 5'-ACGCCTGG ACGATGACCCGTTTGG-3'The length of the amplified cDNA sequence fragment is 317bp

β-actin primer:

Upstream 5'-GAGACCTTCAAGACCCCAGCC-3' Downstream 5'-TCGGGGC ATCGGAACCGCTCA-3'. The length of the amplified cDNA sequence fragment is 404bp

The results of radiographic analysis were expressed as band optical density cdk-5/β-actin for inter-group comparison.

2.3.4 In situ hybridization to detect changes in CaMKII-α levels in hippocampal neurons

① Paraffin sections were routinely dewaxed to water. Mix 1 part of 30% H ₂ O ₂ + 10 parts of distilled water, room temperature for 10 minutes to inactivate endogenous enzymes. Wash with distilled water 3 times. ② Exposure of mRNA nucleic acid fragments: add freshly diluted pepsin with 3% citric acid dropwise on the slice (1ml of 3% citric acid plus 2 drops of concentrated pepsin, mix well), digest at 37°C or room temperature for 15min (pre-test to compare different digestion times , such as 5min, 10min, 20min, 30min. To find the best digestion time. 15min is the best). Wash with PBS 3 times × 5min. Wash with distilled water once. ③ Post-fixation: The fixative solution is 1% paraformaldehyde/0.1M PBS (pH7.2-7.6), containing 1/1000 DEPC. Fix at room temperature for 10 min. Wash with distilled water 3 times. ④ Hybridization: Add 20 μl of pre-hybridization solution to each slice. The wet box is placed in a thermostat at 38-42°C for 3 hours. Absorb excess liquid without washing. Add 20 μl of hybridization solution to each slice and add it to the slice. After peeling off the protective film of the special cover slip for in situ hybridization, cover it on the section. Hybridize overnight at 38-42°C in an incubator. Slides were washed with 2×SSC at about 37°C for 5 min×2 times; at 37°C with 0.5×SSC for 15 min×1 time; at 37°C with 0.2×SSC for 15 min×1 time (if there is non-specific staining, repeat the 0.2×SSC wash 15min×1-2 times). ⑤ Add blocking solution dropwise: 37°C for 30min. Shake off excess liquid without washing ⑥ Add biotinylated mouse anti-digoxin dropwise: room temperature for 120 minutes. In situ hybridization was washed with PBS for 5min×4 times. ⑦ Add SABC dropwise: 20 minutes at 37°C or 30 minutes at room temperature. Wash in situ hybridization with PBS for 5min×3 times. ⑧ Add biotinylated peroxidase dropwise: 20 minutes at 37°C or 30 minutes at room temperature. In situ hybridization was washed with PBS for 5min×4 times. ⑨DAB color development: use 1ml distilled water of DAB color development kit, add one drop each of color developers A, B, and C, mix well, and add to the specimen. Generally, the color develops for 20-30 minutes. Color development can continue if no background appears. Rinse well. ⑩Alcohol dehydration, xylene transparent, cover slides.

2.4 Statistical processing is the same as Experimental Example 6.

3 results

3.1 Tau protein content in brain tissue of rats in each group

In this experiment, the content of phosphorylated Tau protein in the hippocampus of rats in each group was measured. The statistical results showed that there was no significant difference between the normal group and the blank group (p>0.05), and the model group increased significantly compared with the normal group and the blank group (P<0.01). , there was no difference between the low dosage group and the western medicine group and the model group (p>0.05), the high and middle dosage groups of traditional Chinese medicine were significantly lower than the model group (P<0.05 or 0.01), and there was also a difference compared with the western medicine group (P<0.01) , but there was no difference between the two (p>0.05). See attached drawing 10.

Table 10-1 Influence of the invented drug on phosphorylated tau protein in rat hippocampus

Note: Compared with normal group ^△ P<0.05 ^△△ P<0.01 Compared with model group ^▲ P<0.05 ^▲▲ P<0.01

Compared with western medicine group ^★★ P＜0.01

3.2 Effects of GSK-3β activity on brain tissue of rats in each group

The results are shown in Table 10-2. The results of Elisa showed that the expression of GSK-3β in the model group was significantly higher than that in the normal group, and there was a statistical difference between them (P<0.01). After administration, the expression of GSK-3β was lower than that of the model group, and there was no difference between the low-dose group and the Western medicine group and the model group (p>0.05), and there was a significant difference between the middle and high-dose groups and the model group (P <0.05 or 0.01), compared with the Western medicine group, the expression of the high-volume group had a significant difference (P<0.05).

Table 10-2 The effect of the invented drug on the activity of GSK-3β in rat brain tissue

Note: Compared with the normal group, ^△△ P<0.01 Compared with the model group, ^▲ P<0.05 ^▲▲ P<0.01

Compared with western medicine group ^★ P＜0.05

3.3 Effects of cdk5 enzyme mRNA expression in brain tissue of rats in each group

The results are shown in Table 10-3. RT-PCR results showed that the expression of cdk5mRNA in the model group was significantly higher than that in the normal group, and there was a statistical difference between the two (P<0.01). There was no difference between the low dose group, the western medicine group and the model group (p>0.05). There is a significant difference (P<0.01) between the medium and high dosage groups and the model group, and a significant difference (P<0.01) compared with the western medicine group. Compared with the medium-dose group, the expression of the high-dose group was significantly reduced, and there was a statistical difference (P<0.01). See attached drawing 11.

Table 10-3 Influence of the invented drug on cdk5mRNA expression in rat brain tissue

Note: ^△△ P<0.01 compared with normal group and ^▲▲ P<0.01 compared with model group

Compared with the western medicine group ^★★ P＜0.01 Compared with the moderate group ^☆☆ P＜0.01

3.4 Changes in the positive expression of CaMKII-α in the brain tissue of rats in each group

In situ hybridization analysis showed that CaMKII-α immunopositive expression was rare in the hippocampus of normal rats, and there was no significant difference between the normal group and the blank group (p>0.05), while a large number of brown-yellow CaMKII-α immunopositive expressions were seen in the model group , compared with the normal group, there was a statistical difference (P<0.01). There was no difference between the Western medicine group and the model group (p>0.05), and the positive expression in the high-dose Chinese medicine group was lower than that in the model group (P<0.01). There was no difference between the low dose group and the model group (p>0.05), but there was a certain difference with the western medicine group (P<0.05). See attached drawing 21.

Table 10-4 Influence of the invented drug on the expression of CaMKII-αmRNA in rat brain tissue

Note: ^△△ P<0.01 compared with normal group and ^▲▲ P<0.01 compared with model group

Compared with the control group ^★ P＜0.05 ^★★ P＜0.01

4 discussions

In this experiment, it was observed that Aβ _25-35 + IBO could induce an increase in the total amount of phosphorylated Tau protein in rat hippocampus tissue. 1. The middle dose has the most obvious effect, while the low dose has no obvious effect. It may be that the invented drug must reach a certain concentration before it can inhibit the phosphorylated Tau protein. The invented drug can reduce the level of abnormally phosphorylated Tau protein and increase its dephosphorylation, so it is possible to reverse the pathological changes of AD.

The level of GSK-3β in the hippocampus of rats in the model group was significantly higher than that in the normal group, and the level of GSK-3β after administration was lower than that in the model group. The level of GSK-3β has no obvious improvement effect, suggesting that the invented drug can inhibit the activity of GSK-3β within a certain dose range. Therefore, we believe that the invented drug can reduce the abnormal phosphorylation of Tau protein in AD model rats by inhibiting the activity of GSK-3β the level of

The expression of CDK5mRNA in the model group was significantly higher than that in the normal group, and after the intervention of the invented drug, the expression of CDK5mRNA decreased, among which the high and medium dose groups had significant effects, while the low dose group had no obvious effect. It can be seen that, in addition to affecting GSK-3β, the invented drug may also affect the abnormal hyperphosphorylation of Tau protein through the regulation of CDK5.

The expression of CaMK II-α in the hippocampus of rats in the model group was significantly higher than that of the normal group. After the invention drug intervention, the expression of CaMK II-α was reduced compared with the model group, especially in the high-level group. The low-dose group took second place, suggesting that the invented drug can also reduce the abnormal phosphorylation level of Tau protein in AD model rats by regulating the expression of CaMK II-α within a certain dose range.

Example 14 Effect of the drug of the present invention on the immune system mediated by IBO+Aβ _25-35 -induced AD rat model microglia

1 Experimental materials

1.1 Experimental animals are the same as in Example 9.

1.2 Drugs, reagents and instruments

Mouse anti-rat OX-42 monoclonal antibody, SABC immunohistochemistry kit, and DAB were purchased from Wuhan Boster Bioengineering Co., Ltd. TNF-α, IL-1β, IL-6 ELISA kits were purchased from Shanghai Senxiong Technology Industrial Co., Ltd. ELISA Thermol Labsystems, Finland

Others are the same as embodiment 9.

2 Experimental methods

2.1 AD model construction, animal grouping and administration are the same as in Example 9.

2.2 The animal materials are the same as in Experimental Example 8.

2.3 Detection indicators and methods

2.3.1 The immunohistochemical detection method of microglial cells in rat brain tissue is the same as the third part of experiment 1

Microglial cells were labeled with microglial cell-specific marker antibody OX-42, and the expression of microglial cells in brain tissue was observed under a microscope. Positive reaction products are brownish-yellow particles, and the stronger the reaction, the darker the color. Negative reactions are colorless. Observe under a light microscope, and count microglial cells under a high-power field of view (x400 times) using an image analysis system.

2.3.2 Detection of TNF-α, IL-1β, IL-6 activity in rat brain tissue

Enzyme-linked immunosorbent assay (ELISA), the same as in Example 12.

2.4 Statistical processing is the same as in Example 9.

3 results

3.1 Activation of microglial cells in the brain tissue of rats in each group

The brain of normal rats is generally in a resting state, and the anti-OX-42 immunohistochemical staining is negative, and it is difficult to find or the cell shape is not clear on the slice. After traumatic stimulation in the sham operation group, the activation transformed into an early response state, and OX-42 lightly stained positive cells could be seen, the cell shape was visible, but irregular, and the number was not different from that of the normal group (P>0.05); the model group OX-42 deeply stained , the shape of OX-42 positive cells was clear, the cell bodies became larger, and the number increased significantly, which was significantly different from the normal group (P<0.01); the reaction of each treatment group gradually weakened, OX-42 was deeply stained, and the number of positive cells gradually decreased. The shape becomes blurred. In terms of quantity, there was a significant difference between the medium group and the model group (P<0.01), there was no difference between the Western medicine group and the high group and the model group (P>0.05), and there was also a difference between the high group and the middle group (P<0.05). 0.01).

n)Table 11-1 The activation of microglial cells in the brain tissue of rats in each group (

n)

Note: ^△△ P<0.01 compared with normal group and ^▲▲ P<0.01 compared with model group

Compared with the western medicine group ^★★ P＜0.01 Compared with the moderate group ^☆☆ P＜0.01

3.2 Levels of cytokines in brain tissue of rats in each group

In this experiment, the levels of IL-1β, IL-6 and TNF-α in brain tissue of rats in each group were measured. It can be seen from Table 11-2 that the content of each index in the blank group increased slightly, but statistically there was no difference in the content of IL-1β, IL-6 and TNF-α between the normal group and the sham operation group (P>0.05); the model group IL-1β , IL-6 and TNF-α levels were significantly higher than those in the normal group (P<0.01); there was no difference between the western medicine group and the model group (p>0.05). The low and high dose groups of the invented drug can reduce the IL-1β content of the model group (P<0.05), and the middle dose has a better effect (P<0.01). Compared with the model group, there was a significant difference (P<0.01) in the content of IL-6 between the middle and low dose groups and the model group, but there was no difference between the high dose group and the model group (p>0.05). The results of TNF-α showed that there was only a difference between the middle dose group and the model group (P<0.05), and there was no difference between the other treatment groups and the model group (P>0.05).

pg/mg)Table 11-2 Results of IL-1β, IL-6 and TNF-α levels in brain tissue of rats in each group (

pg/mg)

Note: Compared with the normal group, ^△△ P<0.01 Compared with the model group, ^▲ P<0.05 ^▲▲ P<0.01

Compared with western medicine group ^★ P＜0.05 ^★★ P＜0.01

4 discussions

The results of this experiment show that the brain of normal rats is generally in a resting state, and there is no activation of microglial cells. After the animal model was established, the microglial cells had a clear shape, the cell body became larger, and the number increased significantly. It can be seen that there was a significant activation, which was consistent with the appearance of senile plaques and neurofibrillary tangles. The medium-dose group of the invented drug can effectively inhibit the excessive activation of microglia and protect the brain tissue from damage, but the low and high doses have no effect. It may be that the low dose is difficult to achieve the most effective drug concentration, while the high dose may be due to Excessive concentration is a kind of stimulation to the body and causes the activation of microglial cells.

The experimental results show that the invented drug can better regulate the cytokines of the immune system in the model group, can effectively reduce the levels of IL-1β, IL-6 and TNF-α in the brain tissue of animals, and reduce the damage to the brain tissue. And there is a certain dose-effect relationship, and the effect of the middle dose is obviously better than that of the high and low dose groups. The mechanism may be related to the inhibition of overactivated microglia.

Example 15 Experiment of research on the protective effect of PC12-induced AD cell model neurons on Aβ _25-35 by drug-containing serum of the present invention

1 Experimental materials

1.1 Cell lines

PC12 (mouse pheochromocytoma cell line) was provided by the Institute of Cells, Shanghai Branch, Chinese Academy of Sciences.

1.2 Main reagents

1640 medium, product of GibcoBRL; fetal bovine serum (FBS), horse serum (HS), product of Hangzhou Sijiqing Institute of Bioengineering Materials; tetramethylazolazolium blue (MTT), product of Fluka; Aβ25-35, trypsin, Sigma products. Caspase-3 mouse IgG and ready-to-use SABC staining kit, Wuhan Boster Bioengineering Co., Ltd. The rest of the reagents were of domestic analytical grade.

1.3 Main instruments and equipment

CO2 incubator, American Sheldon Company; ultra-clean bench, Suzhou Purification Equipment Co., Ltd.; inverted phase-contrast microscope, Japan OLYMPUS (CK-TKc-3); enzyme-linked immunoassay detector, Finland Thermol Labsystems; flow cytometry

FACSCLibur, American BD Asia Co., Ltd.; vertical electric pressure steam sterilizer, LDZX-40 type.

1.4 Experimental animals

Clean-grade Wistar rats, weighing 250±20 g, were provided by the Experimental Animal Center of Tongji Medical College, Huazhong University of Science and Technology.

1.5 Experimental Drugs

The medicine of Example 3 was prepared and stored at 4°C for future use.

2 methods

2.1 Culture of PC12 cells

After the cells were bought back, quickly put them into a 37°C, 5% CO ₂ incubator for static adaptive culture for 1 day, and then half of the medium was changed. After the cells in the original culture bottle reached the passage standard, the cells were inoculated in a 25cm ² culture bottle , the cell density is 2×10 ⁵ /ml, cultured with DMEM medium supplemented with 10% fetal calf serum. According to the growth of the cells, the culture medium was replaced every 1-2 days, and the cells were passaged once every 2-3 days. Observe and count under an inverted microscope, dilute the cells to a cell suspension of 1×10 ⁶ cells/ml with complete 1640 medium, inoculate in culture flasks and culture plates, culture at 37°C, 5% CO ₂ , 2-3 When the cells adhere to the wall, they can be used for experiments.

2.2 Establishment of AD cell model

When used in the experiment, the culture wells in the 96-well culture plate where the PC12 cells are confluent in a monolayer (each well containing about 1×10 ⁶ cells) were selected, and replaced with serum-free medium (except the normal control group). Add different final concentrations of Aβ _25-35 , and randomly divide them into 6 groups with 8 wells in each group: ①Complete medium group (I); ②Blank PBS control group (H); ③0.1μM group (III); ④1μM group (IV); ⑤5μM group (V); ⑥10μM group (VI). ⑦The 20μM group (VII) was co-incubated for 12h, 24h, and 48h, followed by trypan blue staining and morphological observation.

The prepared Aβ _25-35 solution was incubated at 37°C for 4 days to promote its aging, and the concentration of Aβ _25-35 in the culture medium was 5 μmol/L as the experimental concentration. Cells entering the logarithmic growth phase after subculture were selected, Aβ _25-35 fragments at a final concentration of 5 μmol/L were added, and the AD cell model was established after continuing to culture for 1 day.

2.3 Preparation of drug-containing mouse serum and blank mouse serum:

Thirty Wistar rats were divided into administration group (A) and control group (B), 15 in each group.

Group A is based on the human-animal body surface area equivalent dose ratio table conversion algorithm ^[1-2] and the serum pharmacology experiment method, and the serum donor is based on clinical pharmacology or the relevant effective dose of the whole model animal (8 times the clinical dose in humans). -10 times the amount) administration, the drug water decoction was invented by gavage to rats, administered twice a day (8:00-9:00 in the morning, 3:00-4:00 in the afternoon), the equivalent crude drug amount is 25g /kg·d. For 7 consecutive days, 2 hours after the last administration (drinking water was prohibited 12 hours before administration), after intraperitoneal injection of pentobarbital sodium (40mg/kg) for anesthesia, blood was collected from the heart of the rat by aseptic operation, and the serum was separated by centrifugation at 1000rpm , and then the sera of 10 rats were mixed and inactivated at 56°C for 30 minutes. Sterilize by filtration with a 0.22 μm microporous membrane, and store at -20°C for later use. This method prepares the medicated serum. When used in experiments, RPMI1640 medium was used to dilute the drug-containing serum into three different concentrations of 5%, 10%, and 20%, respectively.

Group B was fed with 0.9% normal saline for 7 consecutive days, and the administration time, method and method of obtaining serum were the same as group A. This method prepares the blank serum. Dilute the blank serum into different concentrations of serum in the same way.

2.4 Detection indicators and methods

2.4.1 Cells were directly placed under a microscope to observe their morphology and count

Preparation of cell suspension: Terminate the culture, aspirate the medium, and wash the culture once gently with PBS. Add 1ml of 0.25% trypsin to the culture flask, digest at 37°C for 3-5min, observe under the microscope that the cells become round and close to detachment, add 4.5ml of medium, and repeatedly blow gently with a pipette to detach the cells to make a cell suspension.

Cell counting: place a dedicated cover glass for counting in the center of the blood cell counting plate, draw 0.1ml of cell suspension with a glass siphon, and let the siphon flow out of the suspension from the groove of the counting plate on the upper or lower side of the cover glass to the cover glass The underside of the tablet is filled with liquid. Count the total number of cells in the squares under a microscope. For the cells that press the line, only the ones on the upper line and the left line are counted. Count according to the following formula: cell density=(total number of cells/4)×10 ⁴ (cells/ml)

2.5.2 MTT detection method

4 hours before the end of 12 hours of drug-containing serum, add MTT with a final concentration of 0.5 mg/ml to each well, continue to cultivate for 4 hours for color reaction, then suck off the original medium, add 200 μl of 100% dimethyl sulfoxide to each well, Mix well until the formazan crystal particles are dissolved, let stand for 4 hours, place on a microplate reader, and detect the absorbance of each well at the absorbance value of 560nm. The drug-containing serum was used to calculate the proliferation rate of PC12 cells according to the following formula:

Proliferation rate (%)=OD _{drug-containing serum} -OD _{drug-free serum control} /OD _{drug-free serum control} ×100%.

In the same way, observe the effect of drug-containing serum on the proliferation of PC12 cells for 24 hours and 48 hours.

2.5.2 The effect of drug-containing serum on the proliferation of PC12 cells

After the PC12 cells were cultured into a single layer in the culture flask, the original medium was discarded, and the PBS solution was gently washed once. Add 1ml of 0.25% trypsin, digest at 37°C for 2-3min, and when the monolayer of cells loosens and floats in clumps, add blood-free medium to stop the digestion. Pipet several times with a pipette to disperse the cells individually, count them under an inverted phase-contrast microscope, dilute the cells to 1× ¹⁰⁶ cells/ml with serum-free medium, and inoculate them in a 96-well culture plate, 200 μl per well.

The PC12 cells inoculated in the 96-well culture plate were randomly divided into 7 groups, each group with 8 holes, administered according to the following scheme: 1. the first group added complete medium group (I); 2. the second group added 5% blank serum ( II); ③ the third group plus 10% blank serum (III); ④ the fourth group 20% blank serum (IV); ⑤ the fifth group plus 5% drug-containing serum (V); ⑥ the sixth group plus 10% containing Drug-containing serum (VI); ⑦ Add 20% drug-containing serum (VII) to the seventh group. After co-incubating for 12, 24, and 48 hours, the cell morphology was observed by an inverted phase-contrast microscope, the cell count was performed, and the cell proliferation rate was detected by MTT.

2.5.3 The protective effect of the drug of the present invention on the neurotoxicity of the PC12 cell model

Select the culture wells with PC12 cells covered with a single layer, and divide them into 8 groups randomly, with 8 wells in each group: ① control group (I); ② Aβ model group (II); ③ Aβ+5% blank serum group (III); ④ Aβ+ 10% blank serum group (IV); ⑤Aβ+20% blank serum group (V); ⑥Aβ+5% drug-containing serum group (VI); ⑦Aβ+10% drug-containing serum group (VII); ⑧Aβ+20% drug-containing serum group Serogroup (VIII). Aspirate the original medium, after gently washing with PBS once, add 1ml of serum-free medium to each well, after 30min of action, add Aβ to each group at a final concentration of 20μM for 48h, then add different concentrations of drug-containing serum and blank serum. After co-incubating for 24 hours, morphological observation, MTT, flow cytometry detection and immunohistochemical staining were carried out.

According to the following formula, calculate the survival rate of each group of blank serum against PC12 cell injury caused by Aβ _25-35 :

Survival rate=OD _{blank serum group} -ODAβ/OD _{complete medium} -OD _Aβ ×100%

According to the following formula, the survival rate of Wendan Decoction-containing serum on PC12 cell damage caused by Aβ _25-35 was calculated: Survival rate=OD _{Aβ+ medicated serum} -OD _{Aβ+blank serum group} /OD _{blank serum group} -OD _{Aβ+blank serum Group} x 100%.

2.5.4 The effect of the drug of the present invention on the apoptosis of NG108-15 cell model induced by Aβ _25-35

2.5.4.1 Flow cytometry detection method is the same as that in Example 8.

2.5.4.2 Immunohistochemical staining of Caspase-3 protein is the same as that in Example 8.

2.6 Statistical processing is the same as in Example 9.

3 results

3.1 The effect of drug-containing serum on the proliferation of PC12 cells

3.1.1 Morphological observation

Observed under an inverted phase-contrast microscope, the PC12 cells in the complete medium group could rapidly adhere to the wall 24 hours after inoculation, and most of them were spindle-shaped, and a few of them were triangular. Translucent, the cells are villous and have short protrusions around the cells. There are no obvious spots and deposits on the cell surface, and there are no or only a few debris in some pores. The growth status of the cells in the blank serum group was similar to that in the complete medium group. After adding different concentrations of drug-containing serum, PC12 cells had no obvious changes in cell morphology, no damage, and grew well. When cultured with drug-containing serum for 48 hours, the cells grew vigorously, growing in a fusiform and triangular shape, covering a monolayer, and cultured for 72 hours. The growth rate gradually slows down. See attached drawing 12.

3.1.2 MTT automatic trace method detection results

The results are shown in Table 12-1. The drug-containing serum was added to the medium for 12h, 24h, and 48h, all of which could promote the growth of PC12 cells in a dose-dependent manner. There was no significant difference between the complete medium group and the blank serum group (P>0.05); the drug-containing serum group was significantly higher than the complete medium group and the blank serum group, and there was a significant difference (P<0.01). Among the three different concentrations of drug-containing serum groups, the cell inhibition rate of 5% drug-containing serum was the lowest, and the cell proliferation rate of 20% drug-containing serum was the highest.

n＝8)Table 12-1 The comparison of different concentrations of drug-containing serum to the proliferation rate of PC12 cells (

n=8)

Note: Compared with the complete medium, ^△ P<0.05, ^△△ P<0.01 Compared with the blank serum control group, ^★ P<0.05, ^★★ P<0.01

3.2 The effect of the drug of the present invention on the neurotoxicity protection of the PC12 cell model induced by Aβ _25-35

3.2.1 Observation of cell morphology

When the cells were cultured without Aβ _25-35 fragments, the cells in the normal rat serum group and the drug-containing serum groups all grew well, the cytoplasm was uniform and bright, with obvious halos, the nuclei were clear, and the cell bodies had good refraction and plumpness. And full of three-dimensional sense, the cells are villous and have short protrusions around them, and they increase into a network. There are no obvious spots and deposits on the cell surface, but the cells in the drug-containing serum group are relatively better than the normal group. Under the toxic effect of Aβ, the cells cultured in the Aβ group accumulated into clusters, most of the neuron cell bodies shrank, and the cell processes were shortened and less, thickened, broken or disappeared. In the normal group, the number of PC12 cells treated with the serum control medium was significantly reduced, and the cell edges were dull and speckled, and blackened and ruptured cells could be seen. Compared with the blank serum group, the growth status of the cells in the drug-containing serum group was better, and there were relatively few darkened and broken cells. See attached drawing 13.

3.2.2 Effect of drug-containing serum on cell death rate (trypan blue staining method)

Under the microscope, the dead cells were observed to be blue, and the PC12 cell death rate was calculated by the hemocytometer after 20 μM Aβ _25-35 was treated for 48 hours, and the serum containing different concentrations was treated for 24 hours. See Table 12-2.

％)The influence (n=8,

%)

Note: Compared with the complete medium group, ^△ P<0.05 ^△△ P<0.01 Compared with the model group, ^▲ P<0.05 ^▲▲ P<0.01

Compared with blank serum control group ^★ P＜0.05 ^★★ P＜0.01

3.2.3 Effect on survival rate of PC12

After PC12 cells were treated with 20 μM Aβ _25-35 for 48 hours, MTT was measured, and the OD value was converted into cell viability (%). It was found that 20μM Aβ _25-35 decreased the cell viability, and the drug-containing serum had a significant protective effect on the cytotoxicity caused by Aβ _25-35 .

n＝8)Table 12-3 Different concentrations of drug-containing serum on the impact of cell viability (%) (

n=8)

Note: Compared with the complete medium group, ^△ P<0.05 ^△△ P<0.01

Compared with the model group ^▲ P＜0.05 ^▲▲ P＜0.01

Compared with the corresponding blank serum control group ^★ P＜0.05 ^★★ P＜0.01

3.3 The effect of the medicine of the present invention on the apoptosis of PC12 cell AD model induced by Aβ _25-35

3.1.1 Flow cytometry results

It can be seen from Table 12-4 that after PC12 cells were treated with Aβ _25-35 , a hypodiploid peak appeared on the left side of the G1 peak, and the apoptosis rate of the drug-containing serum group was significantly lower than that of the model group (P<0.01) . And there are different degrees of reduction among different concentration groups (P<0.01). Compared with the corresponding blank serum groups, each group of drug-containing serum has a more obvious effect, and there is a significant difference (P<0.01). See attached drawing 14.

％)Table 12-4 Effects of different concentrations of drug-containing serum on the apoptosis rate (n=8,

%)

Note: Compared with the complete medium group, ^△ P<0.05 ^△△ P<0.01

Compared with the model group ^▲ P＜0.05 ^▲▲ P＜0.01 Compared with the blank serum control group ^★ P＜0.05 ^★★ P＜0.01

3.1.2 Caspase-3 immunohistochemical detection results

Caspase-3-positive neurons were stained with brownish-yellow cytoplasm and membrane. In this experiment, it was observed that caspase-3 was positively expressed in PC12 cells treated with Aβ _25-35 . There is also a small amount of positive expression in the normal saline control group, but the color is lighter. The expression of caspase can be partially reduced after being treated with drug-containing serum. The counts of positive neurons in 5 random fields of vision under a high-power microscope are shown in Table 12-5, and the t-test shows statistical significance. See attached drawing 15.

)PC12 cell Caspase-3 expression comparison (n=8,

)

Note: Compared with the complete medium group, ^△ P<0.05 ^△△ P<0.01

Compared with the model group ^▲ P＜0.05 ^▲▲ P＜0.01 Compared with the blank serum control group ^★ P＜0.05 ^★★ P＜0.01

4 discussions

In this experiment, the pre-aging Aβ _25-35 was used to directly induce PC12 cell apoptosis to establish an AD cell model, and the morphology, flow cytometry and other indicators supported the view that apoptosis is involved in the pathogenesis of AD. Then use the traditional Chinese medicine serum pharmacology method to explore the therapeutic mechanism of the invented drug on AD. The results showed that the drug-containing serum could proliferate PC12 cells and protect the AD model induced by Aβ _25-35 . We observed the proliferation rate of PC12 cells cultured with different concentrations (5%, 10%, 20%) of drug-containing serum, and the results showed that, compared with the situation of the above-mentioned AD cell model, different concentrations of drug-containing serum could to a certain extent Resist the damage effect of Aβ _25-35 on cells; but each group is different, in the range of 5%-20% concentration, its protective effect increases with the increase of concentration, showing a certain concentration dependence.

At the same time, from the morphology and MTT method, we observed that after the neurons were treated with Aβ, the cell survival rate decreased, and the flow cytometry showed a hypodiploid peak; and the drug-containing serum could inhibit the PC12 cell apoptosis induced by Aβ , increased Aβ fragment toxicity-induced PC12 cell survival. The results confirmed that the drug-containing serum can reduce the neurotoxic response of cells to Aβ, indicating that the invented drug exerts a therapeutic effect on AD by antagonizing the pathological development of AD.

Flow cytometry detection of apoptosis showed that the apoptosis rate in the model group was significantly increased, suggesting that the pathological basis of AD may be derived from neuronal apoptosis caused by Aβ. However, the Wendan Decoction containing cerebrospinal fluid and drug-containing serum group had a higher cell survival rate and a lower cell apoptosis rate than the model group (p<0.05). From the perspective of serum pharmacology, it must be found that the drug protects cells induced by Aβ _25-35 . The role of nerve cell apoptosis provides a theoretical basis for the clinical application of the invention of drugs to prevent and treat senile dementia.

In addition, we observed the protein expression of caspase-3 induced by Aβ _25-35 in PC12 cells, and the results showed that Caspase-3 was positively expressed in the AD cell model group. It was also found that the drug-containing serum can reduce Aβ-induced apoptosis and the positive expression of caspase-3, suggesting that the drug-containing serum may inhibit apoptosis by activating the expression of caspase-3. This provides a new basis and a new research approach for the invention of drugs for the treatment of AD.

Example 16 The effect of the drug of the present invention on Aβ _25-35 NG108-induced AD cell model neuron protection experiment

1 Experimental materials

1.1 Cell lines

The NG108-1 neuroma cell line was provided by the Chinese Type Culture Collection Center of Wuhan University.

1.3 Main reagents and instruments

Rabbit anti-rat Bcl-2 and Bax polyclonal antibody, Wuhan Boster Company. All the other are the same as embodiment 15.

1.4 Experimental animals

Twelve healthy and clean white-eared rabbits, weighing 2.5-3kg, male or female, were purchased from the Experimental Animal Center of Tongji Medical College, Huazhong University of Science and Technology.

1.5 Experimental Drugs

The medicine of Example 3 was prepared and stored at 4°C for future use.

1.6 The preparation of the main solution is the same as in Example 15.

2 methods

2.1 Culture of NG108-15 cells

After the cells were bought back, quickly put them into a 37°C, 5% CO ₂ incubator for static adaptive culture for 1 day, and then half of the medium was changed. After the cells in the original culture bottle reached the passage standard, the cells were inoculated in a 25cm ² culture bottle , the cell density is 2×10 ⁵ /ml, cultured with DMEM medium supplemented with 10% fetal bovine serum. According to the growth of the cells, the culture medium was replaced every 1-2 days, and the cells were passaged once every 2-3 days. Count the cells under an inverted microscope, adjust the cell density, inoculate the cells in culture flasks or culture plates, observe the cell adhesion under the microscope, and the 8-12 cells can be used in the experiment when they enter the logarithmic growth phase.

2.2 Establishment of AD cell model is the same as in Example 15.

2.3 Preparation of drug-containing rabbit serum and drug-containing rabbit cerebrospinal fluid

2.3.1 Preparation of drug-containing rabbit serum

2.5-3kg big-eared white rabbits were randomly divided into two groups after adaptive feeding for 3 days: blank group and traditional Chinese medicine group. The Chinese medicine gavage group was given Chinese medicine concentrated decoction 4.5g/kg·d orally, twice a day for 3 consecutive days. Fast for 12 hours before the last administration, but water is not allowed. 1 hour after the last gavage, anesthetize with 10% chloral hydrate (1ml/kg), collect 50ml blood from the sterile heart, collect blood in a centrifuge tube overnight at 4°C, and wait for its serum Fully analyze, centrifuge at 3000rpms×15min, collect serum, inactivate complement at 56°C for 30min, filter through a 0.22μm microporous membrane, and store at -20°C for later use. The blank group was gavaged with the same volume of normal saline, and after the same time, the serum was extracted by the same method, and the resulting blank serum was obtained.

2.3.2 Collection of drug-containing rabbit cerebrospinal fluid

After infusion of traditional Chinese medicine for intracardiac blood collection, quickly connect the No. 7 needle with a disposable 1ml syringe, and insert the needle vertically and slowly from the foramen magnum in the rabbit under aseptic conditions. 800-1000 μl of cerebrospinal fluid was collected from each rabbit, quickly injected into a sterilized 1.5ml EP tube, and frozen at -70°C for later use. The cerebrospinal fluid of the blank group was collected by the same method, and the obtained cerebrospinal fluid was blank.

2.4 Experimental detection method

2.4.1 Cells were directly placed under a microscope to observe their morphology and count

Preparation of cell suspension: Terminate the culture, aspirate the medium, and wash the culture once gently with PBS. Add 1ml of 0.25% trypsin to the culture flask, digest at 37°C for 3-5min, observe under the microscope that the cells become round and close to detachment, add 4.5ml of medium, and repeatedly blow gently with a pipette to detach the cells to make a cell suspension.

Cell counting: place a dedicated cover glass for counting in the center of the blood cell counting plate, draw 0.1ml of cell suspension with a glass siphon, and let the siphon flow out of the suspension from the groove of the counting plate on the upper or lower side of the cover glass to the cover glass The underside of the tablet is filled with liquid. Count the total number of cells in the squares under a microscope. For the cells that press the line, only the ones on the upper line and the left line are counted. Count according to the following formula: cell density=(total number of cells/4)×10 ⁴ (cells/ml)

2.4.2 MTT detection method

4 hours before the end of 12 hours of drug-containing serum, add MTT with a final concentration of 0.5 mg/ml to each well, continue to cultivate for 4 hours for color reaction, then suck off the original medium, add 200 μl of 100% dimethyl sulfoxide to each well, Mix well until the formazan crystal particles are dissolved, let stand for 4 hours, place on a microplate reader, and detect the absorbance of each well at an absorbance value of 560NM. The drug-containing serum was used to calculate the proliferation rate of PC12 cells according to the following formula:

Proliferation rate (%)=OD _{drug-containing serum} -OD _{drug-free serum control} /OD _{drug-free serum control} ×100%.

In the same way, observe the effect of drug-containing serum on the proliferation of PC12 cells for 24 hours and 48 hours.

2.5 The protective effect of the medicament of the present invention on the N6108-15 cell model

Six experimental groups were established: blank group, model group, blank cerebrospinal fluid group, drug-containing cerebrospinal fluid group, blank serum group, and drug-containing serum group. In the cerebrospinal fluid group, 100 μl of blank cerebrospinal fluid and drug-containing cerebrospinal fluid were added, and then the complete medium was added to 5 ml. In the serum group, the original culture medium was replaced with the complete culture medium containing 10% blank serum and 10% drug-containing serum respectively. Except the blank group, AD cell models were established in other groups in advance, and 5 μmol/L Aβ fragments were added after drug intervention to maintain the AD model. After 24 hours of corresponding treatment in each group, the cell growth state was observed under an inverted phase-contrast microscope and photographed to detect relevant indicators.

2.6 The effect of the medicine of the present invention on the apoptosis of NG108-15 cell model induced by Aβ _25-35

2.6.1 Flow cytometry detection method is the same as that in Example 15.

2.6.2 The immunohistochemical staining of Bcl-2 and Bax protein is the same as that in Example 15.

2.7 Statistical processing is the same as in Example 9.

3 results

3.3 The effect of the invented drug on the NG108-15 cell model induced by _Aβ25-35

3.3.1 Effects on cell morphology

The growth background of the cells in the blank group without Aβ _25-35 is clear, the cells grow well, the cell body is full and the refraction is good, the cytoplasm is uniform and translucent, the nucleus is clear, there are short protrusions around some cells, and there are no obvious spots and deposits on the cell surface. Under the neurotoxic effect of Aβ protein fragments, the growth background of the cells in the other groups was poor, and some dead cells and cell fragments could be seen floating. Dim, with spots, many cells detached and floated, and the number of nerve cells with protruding protrusions was significantly reduced. Among them, the cells in the drug-containing cerebrospinal fluid and drug-containing serum groups had better growth status, relatively fewer dead, blackened, and fragmented cells, and more cells with protrusions and elongation. The ratio of the number of cells with protrusions to the total number of cells in each group was counted, and the results are shown in Table 13-1. See attached drawing 16.

Table 13-1 Invention drug on the impact of NG108-15 cell protrusion rate (%)

Note: Compared with the blank group: ^★ p<0.05; compared with the model group: ^▲ p<0.05.

The inter-group t-test results showed that compared with the blank group, the proportion of cells with protrusions in the other groups decreased, indicating that the neurotoxic effect of Aβ _25-35 inhibited the growth of protrusions in NG108-15 cells. Compared with the model group, the drug-containing serum group and the drug-containing cerebrospinal fluid group all increased the cell protrusion rate, and the difference was significant (p<0.05). Alleviates the inhibition of cell processes by Aβ.

3.3.2 Effect on cell viability

The cell viability was detected by the MTT method, and the absorbance (OD value) of each well was used to represent the cell activity of the well. The higher the OD value, the higher the cell viability, see Table 2-17. The results showed that the OD values of the drug-containing serum group and the drug-containing cerebrospinal fluid group were significantly higher than those of the model group (p<0.05), indicating that both the drug-containing serum and the drug-containing cerebrospinal fluid of the invention can protect NG108-15 cells from damage induced by neurotoxicity of Aβ fragments . Compared with the drug-containing serum group, the OD value of the drug-containing cerebrospinal fluid group tended to increase, but there was no statistical significance (p>0.05).

Table 13-2 Invention drug on the impact of NG108-15 cell viability (OD value)

Note: Compared with the blank group: ^★ p<0.05; compared with the model group: ^▲ p<0.05.

3.2.1 Effect on apoptosis

In the histogram of flow cytometry, a hypodiploid apoptotic peak (Ap peak) appeared before the DNA peak (G1 peak) of normal diploid cells in all groups except the blank group. The results showed that the neurotoxicity of Aβ _25-35 fragments could induce abnormal apoptosis of NG108-15 cells. The apoptosis rate of each group is shown in Table 13-3. Compared with the model group, the apoptosis rate of the drug-containing cerebrospinal fluid and drug-containing serum groups were both reduced (p<0.01), and the neuron apoptosis rate of the drug-containing cerebrospinal fluid group was lower than that of the drug-containing serum group (p<0.05). The results support the results of MTT detection of cell viability. Figure 17.

Table 13-3 The influence of the medicine of the present invention on the apoptosis rate (%) of NG108-15 cells

Note: Compared with blank group: ^★ p＜0.05 ^★★ p＜0.01;

Compared with the model group: ^▲ p<0.05 Compared with the drug-containing serum group: ^△ p<0.05

3.2.2 Effects on expression of apoptosis-related genes Bcl-2 and Bax

The cytoplasm of the cells is brownish-yellow, and the brownish-yellow granules are bcl-2 positive cells. Immunohistochemical staining of Bax was in the cytoplasm, and the positive staining of Bax was inhomogeneous brown-yellow fine granules or brown clumps in the cytoplasm. The number of Bcl-2 and Bax-positive cells and the ratio of Bcl-2/Bax in each group are shown in Table 13-4. Figure 18.

The influence of table 13-4 medicine of the present invention on NG108-15 cell Bcl-2, Bax protein expression

Note: Compared with the blank group: ^★ p<0.05 Compared with the model group: ^▲ p<0.05 ^▲▲ p<0.01

Compared with the blank group, the expression of Bcl-2 in each group with the effect of Aβ _25-35 decreased, while the expression of Bax increased (p<0.05), indicating that the neurotoxic effect of Aβ on apoptosis was related to the reduction of the apoptosis inhibitory gene Bcl- 2 expression, while increasing the expression of the pro-apoptotic gene Bax. Compared with the model group, there was no significant difference in the positive expression of neurons Bcl-2 in each serum group and cerebrospinal fluid group (p>0.05), while the expression of Bax in the drug-containing cerebrospinal fluid group and the drug-containing serum group decreased (p<0.05), Bcl -2/Bax ratio increased. There was no significant difference between the two groups (p>0.05).

4 discussions

From the morphology and MTT method, we observed that after the neurons were treated with Aβ, the cell survival rate decreased, and the hypodiploid peak was seen in flow cytometry; and the drug-containing cerebrospinal fluid could inhibit the apoptosis of NG108-15 cells induced by Aβ in the AD model , increased Aβ fragment toxicity-induced NG108-15 cell survival. The results confirmed that the drug-containing serum can reduce the neurotoxic response of cells to Aβ, indicating that the invented drug exerts a therapeutic effect on AD by antagonizing the pathological development of AD.

Compared with the blank group, the expression of Bcl-2 in each group with the effect of Aβ _25-35 decreased, while the expression of Bax increased, suggesting that one of the possible pathways of Aβ _25-35- induced neuronal cell apoptosis is the activation of the Bcl-2 gene family. regulation, which is consistent with the above point of view. The experimental results show that the invented drug can significantly increase the bcl-2/bax ratio of AD model cells and reduce the apoptosis rate. In addition, compared with the model group, there was no significant difference in the number of bcl-2 positive expression in each serum group and cerebrospinal fluid group, and the expression of bax in the drug-containing cerebrospinal fluid group and the drug-containing serum group was significantly reduced, suggesting that the invention drug protects the blood cells caused by Aβ. The effect of NG108-15 cell apoptosis may be related to its down-regulation of bax gene expression.

Example 17 Effect experiment of the drug of the present invention on the signal transduction of AD cell model induced by Aβ _25-35 and NG108

1 Experimental materials

1.1 Animals are the same as in Example 16.

1.2 The cell strain is the same as in Example 16.

1.3 Experimental drugs are the same as in Example 16.

1.4 The main reagents are the same as in Example 16.

1.5 The main instruments are the same as in Example 16.

2 Experimental methods

2.1 Reagent preparation is the same as in Example 16.

2.2 The cultivation of NG108 cells is the same as in Example 16.

2.3 Grouping

Blank group: adding blank cerebrospinal fluid with a final concentration of 10% for 24 hours.

Model group: add pre-aging 4d with a final concentration of 5 μmol/LAβ _25-35 fragments and culture for 24 hours, then add blank cerebrospinal fluid with a final concentration of 10% and continue to culture for 24 hours.

High-dose group of traditional Chinese medicine: 5 μmol/LAβ _25-35 fragments aged 4 days were added to culture for 24 hours, and then cerebrospinal fluid containing 20% drug was added to continue culture for 24 hours.

Medium dose of traditional Chinese medicine group: add pre-aged 4d concentration of 5 μmol/LAβ _25-35 fragments and culture for 24 hours, then add cerebrospinal fluid with a final concentration of 10% drug and continue to culture for 24 hours.

Low-dose traditional Chinese medicine group: add pre-aged 4d concentration of 5 μmol/LAβ _25-35 fragments and culture for 24 hours, then add cerebrospinal fluid with a final concentration of 5% drug and continue to culture for 24 hours.

SP600125 group: pre-treated with final concentration of 25 μmol/LSP600125 for 30 minutes, added pre-aged 4 days of 5 μmol/LAβ _25-35 fragments and cultured for 24 hours, and then added cerebrospinal fluid with a final concentration of 10% drug for 24 hours.

2.4 Duplication of AD model

The prepared Aβ _25-35 solution was incubated at 37°C for 4 days to promote its aging, and the concentration of Aβ _25-35 in the culture medium was 5 μmol/L as the experimental concentration. Cells entering the logarithmic growth phase after subculture were selected, Aβ _25-35 fragments at a final concentration of 5 μmol/L were added, and the AD cell model was established after continuing to culture for 1 day.

2.5 The extraction of the drug-containing cerebrospinal fluid of the present invention is the same as in Example 16.

2.6 Detection indicators

2.6.1 Immunohistochemical detection of the expression of P-JNK and P53

In quiescent cells, JNK is located in the cytoplasm and nucleus, and once activated by phosphorylation, it will move to the nucleus, promote gene expression and new protein synthesis through phosphorylation of transcription factors, and promote or cause apoptosis. Death. The p53 localized in the nucleus is one of the target gene substrates of JNK-induced apoptosis. Through the color reaction of the antigen-antibody complex, it can be seen that the nucleus of the positively expressed cells is dark brown.

2.6.2 Detection of cell cycle by flow cytometry

Collect the NG108-15 cells cultured in each group, centrifuge at 1000rpm for 5min to collect the cells, wash twice with PBS, fix with 70% ice ethanol at 0°C for 24h, centrifuge at 1000rpm for 5min before using the machine to remove ethanol, wash twice with PBS , then add 0.5ml of propidium iodide staining solution, place in the dark for 30min, filter with 300-mesh nylon mesh, and measure the cell cycle distribution on the machine, and detect 10,000 cells per sample.

2.7 The statistical method is the same as in Example 9.

3 results

3.1 The influence of the drug-containing cerebrospinal fluid of the present invention on the expression of p-JNK and p53

As shown in Table 14-1, the expressions of p-JNK and p53 in the model group were significantly higher than those in other groups (P<0.01). Close but still significantly different, there was no statistically significant difference between the SP600125 group and the blank group.

％)Table 14-1 Analysis of the positive rate of immunohistochemical staining in each group (

%)

Note: Compared with the model group ^▲ P<0.01 Compared with the SP600125 group ^★ P<0.01 ^☆ P<0.05

4 discussions

The drug-containing cerebrospinal fluid of the invention has the effect of protecting NG108-15 nerve cells from damage induced by Aβ _25-35 . The JNK signal transduction pathway is involved in the apoptosis of NG108-15 nerve cells induced by _Aβ25-35 , and activates its downstream substrate p53, so that the cells start the self-repair program, and the cells that cannot be repaired undergo apoptosis. The cell cycle is blocked, and the invented drug-containing cerebrospinal fluid can improve the internal environment of the cells, down-regulate the expression levels of p-JNK and p53, thereby reducing the damage degree of Aβ _25-35 to NG108-15 nerve cells, and this protection The effect was dose-dependent. However, from the comparison between the high-dose group and the inhibitor group, there are significant differences between them, indicating that the NG108-15 nerve cell injury process induced by Aβ _25-35 has not been completely reversed.

Embodiment 18 The influence experiment of the disassembled prescription of the medicine of the present invention on the learning memory and cholinergic system of AD model rats

1 material

1.1 Experimental animals

100 15-month-old Wistar rats, half male and half male. Weight 400±50g, provided by the Experimental Animal Center of Tongji Medical College, Huazhong University of Science and Technology.

1.2 Drugs, reagents and instruments

The invented medicine is composed of Radix Polygoni Multiflori, Shichangpu, Bamboo Ginseng, Ligusticum Chuanxiong, Zhiyuanzhi, etc. Bamboo Ginseng was purchased from Enshi City, Hubei Province, and the rest were purchased from the Expert Clinic of Hubei University of Traditional Chinese Medicine, with excellent quality. Haberin, Henan Zhongsheng Pharmaceutical Co., Ltd. Yuzhong Pharmaceutical Factory, batch number: 060321. Amanita acid (Ibotenic acid, IBO) and Aβ _25-35 were ordered from Sigma Company. The rat Morris water maze automatic experiment recorder was provided by the Chinese Academy of Medical Sciences. Ach, AchE and ChAT radioimmunoassay kits were purchased from the Academy of Military Medical Sciences. Stereotaxic instrument was provided by American TPI Company.

2 methods

2.1 AD model construction is the same as that in Example 9.

2.2 Animal grouping and administration

100 15-month-old Wistar rats. They were randomly divided into 10 groups, namely normal group, sham operation group, model group, Haberin group, Xinnao Yizhi original prescription group (Shou Shou Wu, Shi Chang Pu, Bamboo Ginseng, Chuan Xiong, Zhi Yuan Zhi), Kidney tonifying group ( Radix Polygoni Multiflori, Bamboo Ginseng), Phlegm-Resolving Group (Shichangpu, Polygala), Blood-Promoting Group (Bamboo Ginseng, Ligusticum Chuanxiong), Kidney-Resolving Phlegm Group (Shouwu, Bamboo Ginseng, Shichangpu, Polygala) and Kidney-Activating Blood Group (Shouwu, Bamboo Ginseng, Ligusticum Chuanxiong), 10 rats in each group. The medicines of each group were decocted with water to make a concentrated solution (equivalent to 1 g/ml of crude drug). Administration was administered 2 weeks after the intracerebral injection. The 6 groups of traditional Chinese medicine were administered to the rats with water decoction of this group of drugs in the stomach respectively; the Haberine suspension (0.036mg/ml) was used for intragastric administration of the Haberine group; the normal group, the sham operation group, and the model group were given equal volume normal saline gavage. The dosage is determined according to the ratio of the body surface area coefficients of humans and rats, and the rats in each group are administered orally at 1ml/100g·d. 28 consecutive days.

2.3 Detection indicators and methods

2.3.1 Morris water maze test is the same as Experimental Example 6.

2.3.2 Determination of Ach, AchE and ChAT activities in rat hippocampus

Using radioimmunoassay. The hippocampal tissue was separated, weighed, and homogenized to make a 10% homogenate. Take the supernatant for testing.

2.4 Statistical processing is the same as in Example 9.

3 results

3.1 The medicine of the present invention disassembles the prescription to the influence of rat learning and memory level

Two days before the experiment, the animals in each group basically swam around the pool wall, seldom swam to the vicinity of the platform, and their movement trajectories were randomly distributed in each quadrant. However, as the experiment progressed, the time for the rats in each group to find the platform location by relying on spatial cues gradually shortened, and their movement trajectories also gradually located in the platform quadrant, or searched in the left and right quadrants adjacent to the platform quadrant, and their latency decreased rapidly. But the rats in the model group basically maintained the situation of the previous two days, with a slight improvement, except for a very small number of rats who could find the platform. To analyze information acquisition ability, the total latency of the 4-day training and the post-latency of the last 2 days of training were measured. Statistical results showed that there was no significant difference between the normal group and the sham operation group, while the incubation period of the model group was higher than that of other groups, with a significant difference (P<0.05). The blood circulation group was higher than that of the Huperin group, and there was a significant difference (p<0.05). There was no significant difference (p>0.05) between the kidney-tonifying group, phlegm-resolving group, and kidney-activating-blood group and Haberine group (p>0.05), while there were significant differences between the kidney-resolving phlegm group and the original formula group (P<0.05), but There was no difference between the two groups (p>0.05).

In the positioning navigation test, the swimming distance of the experimental rats in the quadrant of the test platform accounted for the percentage of the total distance, and the swimming distance percentages of the 20% and 40% areas of the pool wall were observed. It can be seen from the experiment that the normal group can find the location of the platform relying on spatial clues, and its movement trajectories are located in the quadrant of the platform at most, while the animals in the model group rarely swim near the platform, and their movement trajectories are randomly distributed in each quadrant. Each medication group mainly searched in the left and right quadrants adjacent to the platform quadrant, and most of them could find the platform, but there were certain differences among the groups. Statistical results show that each group shows basically the same trend as the incubation period in the above table. The difference is that in the 20% area, there is a significant difference between the Kidney-tonifying group and the Huberin group (P<0.05), but it is worse than the original formula group and the Bushen-resolving phlegm group (P<0.05). There was no difference between the blood circulation group and the model group (p>0.05).

s)Table 15-1 The results of the learning and memory ability of the rat positioning navigation test (

s)

In the spatial search experiment, the swimming distance of the platform quadrant accounted for the percentage of the total distance, and the results of the percentage of swimming distance in the 20% and 40% areas of the pool wall showed a consistent trend with each group in the positioning navigation test, and there was a significant difference between the model and other groups (P< 0.05). There was no significant difference between the kidney-tonifying group, the phlegm-resolving group, the kidney-tonifying and blood-activating group and the Huberin group (p>0.05). The number of times rats in each group crossed the original platform position was significantly lower than that in the positioning navigation test. Only a small number of rats in the model group were able to pass through, and the number of times the drug groups traveled in the platform quadrant and crossed the platform was significantly more than that of the model group, showing better memory ability than the model group.

Table 15-2 The results of the learning and memory ability of the rat space search test

3.2 Influence of the disassembled prescription of the invented drug on the cholinergic system in the hippocampus

In this experiment, the activities of Ach, ChAT and AchE in the hippocampus of the rats in each group were measured. The statistical results showed that the Ach and ChAT in the hippocampus of the model group were significantly reduced compared with each group (P<0.05), while the activity of AchE was significantly increased (P<0.05). <0.05). There was no significant difference between the normal group and the sham operation group (p>0.05). There was no significant difference between each group of traditional Chinese medicine and Haberin group (p>0.05). However, there was no significant difference between the kidney-tonifying and phlegm-resolving group, the original formula group and the sham operation group (p>0.05), which was close to the level of the sham operation group (p>0.05), but not yet reached the level of the normal group (P<0.05). But there was no difference between the two groups (p>0.05).

Table 15-3 Results of Ach, ChAT and AchE activities in rat hippocampus

4 discussions

The results of this experiment show that each disassembled prescription of the medicine of the present invention can improve the behavioral learning level of AD model rats to a certain extent, and each group of traditional Chinese medicines also shows a better effect in improving the damage of the cholinergic system. However, the original prescription of the invented medicine and the invented medicine had the best effect, and there was no significant difference between them. Based on the experimental results, we believe that the most effective components of this prescription are the kidney-resolving phlegm-resolving medicines Shouwu, Bamboo Ginseng, Shichangpu, and Polygala, which can be used as the best prescription for further research.

Example 19

Clinical curative effect research of medicine of the present invention on senile dementia patients

Senile dementia is a chronic progressive encephalopathy characterized by cognitive dysfunction in old age.

1 Study Standard

1.1 Diagnostic criteria

Diagnostic criteria of Western medicine: adopt the diagnostic criteria of Alzheimer's Disease (AD) and Vaseular dementia (VD) in the American Psychiatric Association's "Diagnostic and Statistical Manual of Mental Disorders" CDSM-IV 4th edition ^[ , ① Cognitive dysfunction manifests in two aspects, That is, memory impairment and cognitive impairment have at least one of the following (aphasia, apraxia, agnosia, impairment of abstract thinking or judgment). ② The above two types of cognitive dysfunction obviously interfere with occupational and social activities, or are significantly reduced compared with the individual in the past. ③Not only occurs in the course of delirium. ④ The above damage cannot be explained by other mental and emotional diseases (such as depression, schizophrenia, etc.).

Diagnostic criteria of traditional Chinese medicine: adopt the "Standards for Diagnosis, Syndrome Differentiation and Judgment of Curative Effect of Alzheimer's Disease" formulated by the Geriatrics Society of the All-China Association of Traditional Chinese Medicine in 1990-05.

1.2 Degree Judgment Criteria

Using the clinical dementia rating scale, CDR=0 means no dementia, CDR=0.5 means suspected dementia, CDR=1 means mild dementia, CDR=2 means moderate dementia, and CDR=3 means severe dementia.

1.3 Identification standard

①The Hachinski Ischemia Scale (HIS) was used to distinguish between vascular dementia (VD) and Alzheimer's disease (AD). Those with a score of 7 were classified as vascular dementia; those with a score of 5 and 6 were classified as mixed dementia; those with a score of 4 were classified as Alzheimer's disease Alzheimer's disease. ②Use the Cornell Depression Scale (CSDD) to exclude depression, ≥8 points for depression.

1.4 Inclusion criteria

Clinical data All cases are in line with DSM-IV-R diagnostic criteria and NINCDS-ADRDA is mild to moderate AD, MMSE score is between 15 and 25, HIS (Hachinsk) ischemia scale ≤ 4; CSDD depression scale score < 8 points; In TCM syndrome differentiation, those with marrow sea deficiency, phlegm turbidity blocking orifices, and qi stagnation and blood stasis are all treated as observation and treatment objects.

1.5 Exclusion criteria

①Patients with severe dementia (CDR=3) or severe neurological impairment, such as various aphasia, agnosia, etc.; ②Patients with serious primary diseases such as heart, brain, liver, kidney, and hematopoietic system, and mental illness; ③Included Cases with drug allergic reactions or other serious adverse reaction events, or complications should not continue to accept the test cases; ④ other reasons lead to the case that the entire course of treatment is not completed, which affects the efficacy or safety judgment. However, those who have exceeded 1/2 course of treatment will count the curative effect.

2 clinical data

All cases come from the inpatient departments of Hubei Affiliated Hospital of Traditional Chinese Medicine, Hubei Shashi Psychiatric Hospital, Enshi Autonomous Prefecture Central Hospital, and Wuhan First Hospital. Select altogether 56 cases of senile dementia patients meeting the above criteria, and divide the patients into two groups by a completely random method, 30 cases of the drug granule treatment group of the present invention, including 18 males and 12 females; mean age 72 ± 8.3 years, average The course of disease was 1.26±0.13 years, the education level was 9.36±4.73 years, 10 cases had mild dementia, and 20 cases had moderate dementia. Naofukang control group included 26 cases, including 15 males and 11 females; the average age was 71±11.5 years, the average disease duration was 1.31±0.28 years, and the education level was 9.8±4.85 years; 9 cases were mild and 17 cases were moderate. After statistical analysis, there was no significant difference between the two groups in age, gender, average course of disease, education level, and disease severity (P>0.05), which were comparable.

3 Research Methods

3.1 Treatment methods

The treatment group took the medicine of the present invention (the product of Preparation Example 1. Produced by the preparation room of Hubei Provincial Hospital of Traditional Chinese Medicine, supervised by the Geriatrics Research Office of Hubei College of Traditional Chinese Medicine, batch number: 2001006), 2 times a day, 6g each time; the control group took Naofukang Tablets (produced by the Northeast Pharmaceutical General Factory of Dongyi Group, batch number: 200006066, each containing 0.4g of piracetam), 0.4-0.8g each time, 3 times a day, 3 months as a course of treatment. For those with diabetes and hypertension, hypoglycemic and antihypertensive drugs can be added, and other cerebral vasodilators and nootropics should be stopped during treatment. All patients cooperate with psychological counseling and intellectual training; if a warm family care is established, the family members of the patients are required to communicate with them frequently, comfort and care for the patients; and according to the patient's education level, hobbies, etc., encourage listening to music, watching TV, doing housework, and reading , Reading newspapers, etc.

3.2 Determination of observation indicators

3.2.1 Safety observation

General inspection items: blood, urine, and stool routine tests; heart, liver, and kidney function tests.

3.2.2 Curative effect observation

3.2.2.1 Detection of intelligence and living ability

Before and after treatment, the 20 items of the Mini-Mental State Examination (MMSE) and the Self-care of Daily Life (ADL) (ADL ⁾ were checked and scored respectively.

3.2.2.2 Check the main biochemical indicators of blood

Determination of blood lipids by enzymatic method (serum cholesterol - by CHOD-PAP method, triglyceride - by GPO-PAP method, high-density lipoprotein - by magnesium phosphotungstate precipitation method, the kit is provided by Beijing Zhongsheng Bioengineering High-Tech Co., Ltd. Provided), the cone and plate method was used to measure the viscosity of whole blood and the viscosity of plasma hematocrit. The content of lipid peroxide (LPO) was determined by TBA method, and the serum SOD was determined by purine oxidase method. The kit was purchased from Union Hospital Affiliated to Huazhong University of Science and Technology, and operated according to the instructions.

4 Efficacy criteria and treatment results

4.1 Curative effect standard

4.1.1 Clinical curative effect standard

According to the "Evaluation Criteria for the Efficacy of Alzheimer's Disease" revised in May 1990 by the Society of Geriatrics and the Society of Internal Medicine of the Chinese Society of Traditional Chinese Medicine. Significantly effective: the main symptoms are basically recovered, the consciousness is clear, the orientation is sound, the questions are answered correctly, the response is sensitive, the daily life is self-care, and the general social activities can be carried out; But the response is still slow, and there are still some obstacles in intelligence and personality; invalid: the main symptoms do not change or the disease progresses, the life cannot take care of itself, the answers to the questions are not correct enough, and the mind is stupid.

4.1.2 Scoring of the Mini-Mental State Examination

An increase of ≥5 in the score compared with before treatment is considered markedly effective, an increase of 2 to 4 is considered effective, and an increase of <2 or decrease in the score is considered ineffective.

4.1.3 Score of 20 items of ADL in daily life self-care ability table

After treatment, the total score decreased by more than 6 points as markedly effective, decreased by ≥3 points as effective, and decreased by <3 points or increased as ineffective.

5 statistical methods

表示，组间比较及前后对照，用t检验，计数资料采用X²检验。Measurement data with mean ± standard deviation

Indicates that the comparison between groups and before-and-after control was performed by t test, and the count data was tested by X ² test.

6 Treatment Results

6.1 The curative effect comparison between the two groups is shown in Table 16-1

Table 16-1 Comparison of curative effects between the two groups [n(%)]

Note: Comparison between two groups ^※ P＜0.05

6.2 Comparison of MMSE and ADL points between the two groups before and after treatment is shown in Table 16-2

)Table 16-2 Comparison of MMSE and ADL scores between the two groups before and after treatment (points

)

Note: Compared with this group before treatment ^※※ P＜0.01

Compared with the control group after treatment, ^△ P<0.05 ^△△ P<0.01

6.3 Comparison of main biochemical indicators in blood before and after treatment of the two groups of patients, see Table 16-3

Table 16-3 Comparison of main biochemical indicators in blood between the two groups before and after treatment

Note: Compared with this group before treatment ^※ P＜0.05 ^※※ P＜0.01

Compared with the control group after treatment, ^△ P<0.05 ^△△ P<0.01

6.4 The changes of hemorheology indexes before and after treatment in the two groups are compared in Table 16-4

Table 16-4 Comparison of changes in hemorheology indexes between the two groups before and after treatment

Note: Compared with this group before treatment ^※ P＜0.05 ^※※ P＜0.01

Compared with the control group after treatment, ^△ P<0.05 ^△△ P<0.01

6.5 The changes of main clinical symptoms after treatment in the two groups are shown in Table 16-5

Table 16-5 Comparison of changes in main clinical symptoms between the two groups after treatment

Note: Compared with this group before treatment ^※ P＜0.05 ^※※ P＜0.01

Compared with the control group after treatment, ^△△ P<0.01

7 Conclusion

Rehabilitation treatment was carried out on 30 cases of senile dementia mainly by nourishing the kidney and replenishing essence, promoting blood circulation and resolving phlegm. In the control group, 26 cases received rehabilitation treatment with Naofukang combined with intellectual training. Clinical observation found that the TCM treatment group had better effects on patients' dementia symptoms and daily living ability, and the overall curative effect was better than that of the control group. The main blood biochemical indicators, blood rheology and other inspection items were performed on each patient before and after treatment. The results showed that after treatment, serum cholesterol and triglycerides decreased significantly, high-density lipoprotein, red blood cell SOD activity significantly increased, and plasma LP decreased significantly. . According to examination results and modern pharmacological research, it can be deduced that the mechanism of action of the Chinese medicine composition of the present invention for the treatment of senile dementia is as follows: (1) lowering blood fat, preventing and treating arteriosclerosis, softening blood vessels is beneficial to blood circulation; (2) scavenging free radicals, inhibiting The formation and accumulation of lipofuscin in the brain improves and delays the aging of the brain; (3) expands cerebral blood vessels, increases cerebral blood flow, resists hypoxia, dissolves thrombosis, and inhibits thrombosis; (4) enhances energy metabolism of the brain, improves The cell vitality of brain neurons can enhance the memory of the brain and improve the intelligence of the brain.

Embodiment 20 The preferred research of the present invention's medicine

1 Experimental materials

1.1 Drugs

All the medicines were purchased from Hubei Provincial Traditional Chinese Medicine Company and sent to Professor Chen Keli of Hubei College of Chinese Materia Medica for inspection. Calculate the mouse equivalent dose according to the coefficient of human and animal body surface area (0.0026).

Positive control drug: Naofukang Tablets. Crush Naofukang tablets, prepare a suspension containing 24 mg of drug per ml with normal saline, and refrigerate at 0-4°C for later use.

1.2 Equipment

Morris water maze, Institute of Materia Medica, Chinese Academy of Sciences.

2 Experimental methods

2.1 Drugs, animals and groups

200 3-month-old mice, weighing 18-22 g, were randomly divided into 20 groups, 10 in each group.

Ginger Pinellia, Zhuru, and Citrus Citrus are used as the basic prescriptions, which are tentatively designated as A0 prescriptions. In addition, kidney tonifying (rehmannia glutinosa, Gastrodia elata), phlegm resolving (polygala, shichangpu, white mustard seed), blood circulation (chuanxiong), qi tonifying (ginseng ) Therapeutic principles and medicines were formulated separately. They are respectively coded as A0, A1, A2, A3, and A4. According to the combination principle, a total of 16 different compatibility methods were preliminarily determined, and a model control group, a positive drug control group, a normal control group and the original Wendan decoction group were set up. After the experiment, the best team was tentatively determined to be party B.

2.2 Detection of learning and memory level

Each group was administered for 10 days, and on the 11th day, the animals in each group were trained in the water maze once a day for 4 consecutive days. Test on the 5th day, 40min after intragastric administration of each group of mice, intraperitoneal injection of scopolamine, dosage is 3mg/kg (except normal group), test with Morris water maze after 30min, record its time to find the platform in 1min (escape incubation period). On the 6th day, the platform was removed, and the rat was put into any water entry point, and the number of times the rat crossed the original platform position within 1 min was recorded. The two experiments also recorded the swimming distance of the rats in the platform quadrant as a percentage of the total distance, and the swimming distance percentages of the 20% and 40% areas of the pool wall.

2.3 Statistical methods

表示，SPSS 17.0统计软件分析，采用单因素ANOVA分析。Each group of data is used

Said, SPSS 17.0 statistical software analysis, using one-way ANOVA analysis.

3. Experimental results

It can be seen from the table below that, compared with the model group, the incubation period of the normal group and the Naofukang group has a very significant difference (p<0.01). Compared with the model group, Chinese medicine 7, 14, 15, 17, 18, and 20 have very significant differences (p<0.01), and Chinese medicine 4, 5, 6, 8, 9, and 16 groups have significant differences (p<0.05) . Select the traditional Chinese medicine group with very significant difference for comparison between the two groups. There is no statistical significance among 7, 14, 15, 17, and 18 (p>0.05), and there is a significant difference between group 20 and other groups (p<0.05) .

4 Conclusion

After screening experiments, the most effective prescriptions were obtained: 10 parts of Jiang Pinellia, 12 parts of Zhuru, 10 parts of Zhishi, 12 parts of Rehmannia glutinosa, 10 parts of Polygala, 10 parts of Shichangpu, 10 parts of Chuanxiong, and 6 parts of ginseng.

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.

Images