WO2011140676A1

WO2011140676A1 - Rhizoma gastrodiae plant extract used to prevent and treat alzheimer disease and vascular dementia and mixed type diseases thereof and preparative method thereof

Info

Publication number: WO2011140676A1
Application number: PCT/CN2010/000680
Authority: WO
Inventors: 王珂; 刘晔; 陈明霞; 苏晓会; 张翔; 刘素云; 赵大龙
Original assignee: 北京科莱博医药开发有限责任公司
Priority date: 2010-05-12
Filing date: 2010-05-12
Publication date: 2011-11-17

Abstract

The present invention discloses use of Rhizoma Gastrodiae plant extract containing parishin derivative in manufacture of medicaments for preventing and treating Alzheimer disease (AD), vascular dementia (VaD) and mixed type diseases thereof. The present invention also discloses the method for extracting and refining plant extract from Rhizoma Gastrodiae (Gastrodia elata Blume), wherein the effective constituent is parishin derivative, especially parishin A, B, C. The plant extract which is extracted by water or alcohol-water and refined and purified by chromatography contains parishin derivative whose content is more than 35%, especially more than 50%. The dosage forms of the present invention preferably include injection, tablets, capsules, soft capsules, oral liquid or drop pills etc.

Description

Gastrodia plant extract for controlling Alzheimer's disease and vascular dementia and mixed diseases thereof and preparation method thereof

The invention belongs to the technical field of natural medicine. Specifically, it relates to a plant extract of Gastronomy (Ga trod la el a ta Blume) containing parisin A (par i shin A ), pari shin B (par i shin B ), and pari shin C (par i shin C ) The use of the preparation for the prevention and treatment of diseases such as Alzhe imer Di sease (AD) and Vascular Dementia (VaD) and mixed diseases thereof. A plant extract obtained by extracting and refining from a Chinese herb Tianma and a method for extracting the same. Background technique

With the aging of China's population, the number of patients with Alzheimer's disease is increasing, which can be roughly divided into Alzheimer's disease (Alzheimer's disease, AD), vascular dementia (VD) and a mixture of the two. Modern medical research believes that Alzheimer's disease is a primary degenerative disease of the brain. It is characterized by progressive dementia and is the most common disease in brain degeneration. It is caused by a decrease in the function of various neurotransmitter systems centered on the cerebral cortex and the limbic system. The most significant change is cholinergic nerve. Systematic changes, a significant reduction in choline acetylase activity in the cerebral cortex and hippocampus. Vascular dementia is a multifactorial disease associated with factors such as hypertension, diabetes, and hyperlipidemia. Most scholars believe that the occurrence of vascular dementia is often associated with cortical lesions, especially left cortical ischemia and ischemic changes in the thalamus and hippocampus. In addition, bilateral, multiple cerebral infarction, ischemic infarction at important locations, and extensive brain damage play an important role in the development of this disease. Not only does the occurrence and development of stroke have a close causal relationship with VD, but long-term chronic brain ischemia can also cause VD.

Alzheimer's disease in general patients can not take care of themselves, always need family care, give patients People, families, and society bring a heavy burden. In addition to cerebral blood circulation improvers and brain energy metabolism activators, the research and development of senile dementia includes neurotransmitter precursors, neurotransmitter synthesis promoters, and neurotransmitter decomposition in addition to brain energy function improvers. Enzyme inhibitors and the like. In recent years, research on neuropeptides, nerve growth factors and Chinese herbal medicines has also been very active, and some progress has been made.

However, there is a problem that has to be faced with drugs for treating Alzheimer's disease. Because the medication cycle for preventing and treating memory and cognitive impairment is relatively long and may be lifelong for Alzheimer's patients, the safe use of drugs is crucial. It is necessary to extract and develop traditional Chinese medicines from the known safer Chinese herbal medicines.

Tianma alias tomorrow hemp, white dragon grass, red arrow root, is a perennial parasitic herb of Orchidaceae

^J^P^ Gas trod la elata i/zz/e tuber, plant, born under moist forest, now cultivated. In China, it is mainly produced in Sichuan, Yunnan, Guizhou, Hubei and Shaanxi. In traditional Chinese medicine, Gastrodia elata is mainly used for the treatment of stagnation of phlegm, phlegm and blood stasis, phlegm and pain relief and partial headache.

Parishin is a major chemical component in plant gastrodia. Document 5 reports that the structure of parishin A, B, C is as follows:

Parishin A parishin B parishin C Among them, parishin A is often referred to as parishin in the previous literature, and two other known Paclitaxin compounds (Parisin) B, C) Separate, call it parishin A.

references: 1. Li YM , Zhou Z.1. , Hong YF New phenolic derivatives from Galeola faberi. PI ant a Med.1 93, 59 ( 4 ) : 363-5.

2. Lin J. H., Liu Y. C., Hau J. P., et al. Parishins B and C from rhizomes of Gas t rod i a elata. Phytochemis try, 1996, 42 (2): 549-551.

3. Zhou Jun, Pu Xiangyu, Yang Yanbin. Nine phenolic ingredients of fresh gastrodia. Scientific Bulletin, 1981, 26 (18): 1118-1120.

4. Dahmen J., Leander K. Studies on Orchidaceae glucosides. Part 7. The structure of parishin, a glucos ide from Vanda parishii. Phytochemis try, 1976, 15(12): 1986-7.

5. Taguchi H., Yosioka I., Yamasaki K., et al. Studies on the constituents of Gas t rod i a elata Blume. Chem. Pharm. Bull., 1981, 29 (1): 55-62.

Although the literature reports a method of obtaining a monomeric compound of a Pelican derivative by repeated color separation by normal phase and reversed phase silica gel, it has never been reported that these monomeric compounds are involved in the action of dementia.

Due to the complex chemical composition of Gastrodia elata, and the polarity of Pelican compounds are quite different, it is more difficult to enrich high content (greater than 50%) of Paclitaxel derivatives by using single-color separation technology suitable for industrial production. There is no literature report on high content (greater than 50%) of the gastrodia plant extract of the Paclitaxel derivative, nor has it been reported to use the chromatographic separation technique to prepare the extract of the gastrodia elata plant rich in the product. Method, and it has never been found that a monomeric compound (especially Paclitaxel A, B, C) or a mixture of a gastrodin plant extract containing a Pelican derivative or a Pelican derivative has an increase in intelligence and prevention and treatment of Alzheimer's disease. (Alzheimer Disease, AD) and Vascular Dementia (VaD) and their mixed effects. Summary of the invention

The present invention provides a gastrodia plant extract or a Pelican monomer compound or a mixture containing a biclin derivative (especially Paclitaxel A, B, C) for the preparation of Alzheimer's disease (Alzheimer Di sease, AD) Use in medicines for diseases such as Vascular Dementia (VaD) and its mixed type. Among them, the extract has a total content of the Paclitaxel derivative of 35% or more, and preferably an extract having a total content of the Paclitaxel derivative of 50% or more. The Pelicin derivative may be any monomer of the Paclitaxel derivative, particularly Paclitaxel A, B, C, or a mixture of any of a plurality of monomeric compounds of the Paclitaxel derivative.

The results of pharmacodynamic examination showed that the effective doses of Gastrodia elata can improve the learning and memory ability of rats with vascular dementia in different degrees and alleviate the pathological damage of model rats. Its possible action links include: 1 Effectively inducing the synthesis of neuronal growth-related proteins during the repair of brain damage, inhibiting the excessive proliferation of astrocytes, and promoting the regeneration of neuronal processes after cerebral ischemic injury. 2 Regulation of synaptic activity of degraded glutamate neurons, reconstruction of Glu/GABA in the brain, learning and memory regulation system, steady state 3, improvement of function of brain cholinergic system 4 anti-lipid peroxidation, activation of anti-free radical oxidase SOD Activity, accelerate the clearance rate of free radicals in brain tissue, reduce the accumulation of free radical metabolite MDA, compared with the current first-line positive control drug donepezil, the efficacy is better than donepezil. It is indicated that the monomeric compound or mixture of the Paclitaxel derivative and the plant extract containing the Parylene derivative (especially Parylene A, B, C) have the effect of preventing and treating vascular senile dementia.

The "monomer" is defined as a single compound.

The present invention also provides a gastrodia plant extract containing a ricin derivative, wherein the total content of the pirin derivative in the extract is 35% or more, and preferably the total content of the pirin derivative is 50% or more, more preferably The total content of Pelicin derivatives is 50%-95%, preferably The Pelicin derivative is Paclitaxel A, B, C.

The present invention also provides a method for preparing a Gastrodia elata extract containing a Pelican derivative, wherein the total content of the Pelicin derivative in the extract is more than 35%, and the total content of the Pelicin derivative is preferably 50% or more. More preferably, the total content of the Paclitaxel derivative is from 50% to 95%, and preferably the Pelicin derivative is Paclitaxel A, B, C.

Through our research on the separation and purification process, we found that the extract of Gastrodia elata, which is rich in Patricia derivatives, can be obtained through a unique extraction and purification process.

The present invention also provides a pharmaceutical composition comprising a gastrodia plant extract and a preparation thereof, and the use of the gastrodia plant extract in the field of health care products and pharmaceuticals, in particular in the preparation of a treatment for Alzheimer's disease (Alzhe imer Di sease, AD) and Vascular Dementia (VaD) and their mixed use in the prevention and treatment of diseases and drugs for diseases such as Alzheimer's disease, wherein the total content of the Pelican derivative in the gastrodia plant extract is more than 35% Preferably, the total content of the Paclitaxel derivative is 50% or more, and more preferably the total content of the Paclitaxel derivative is 50% to 95%. Preferably, the Pelicin derivative is Paclitaxel A, B, C. The percentages are % unless otherwise specified.

Parisinin A parishin B parishin C

The process for preparing the gastrodia plant extract of the present invention is as follows:

i, extraction method: crush the plant gastrodia (tuber), and then use water or

An aqueous solution of 10-95 C1-C6 alkyl alcohol is extracted as a solvent at a temperature at which the O-solvent is refluxed (preferably at room temperature to the reflux temperature of the solvent), and the extract is often pressed or depressurized. The extract is condensed; wherein the C1-C6 alkyl alcohol is preferably decyl alcohol, ethanol, isopropanol or n-butanol or a mixed solvent thereof, and more preferably decyl alcohol or ethanol.

2. Refinement method:

The extract is purified by chromatographic method, purified and eluted with an elution solvent. The fractions rich in Paclitaxin A, B, and C compounds are collected, concentrated, filtered, dried, and detected and collected. Plant extract.

The chromatographic method for purification and purification is macroporous adsorption resin chromatography, normal phase silica gel and reverse phase silica gel chromatography, or a combination thereof, or the extracted extract is mixed in silica gel or diatomaceous earth, and dried. Afterwards, the elution solvent is refluxed; the elution solvent is water, C1-C6 alkyl alcohol, such as: methanol, ethanol, isopropanol or n-butanol, etc., lS hydrocarbon, such as: dichloromethane, Chloroform (trichloromethane), etc., ether solvents, such as: diethyl ether, mercapto tert-butyl ether, etc., ketone solvents, such as: acetone, 2-butanone, etc., ester solvents, such as: ethyl acetate, tannic acid B An ester or the like, and a mixed solvent composed of the above solvents, wherein a preferred elution solvent is one or a plurality of mixed solvents of chloroform (trichlorodecane), ethyl acetate, ethanol, methanol and water. In the purification method, when refining with a styrene-type macroporous adsorption resin, the elution solvent is preferably water and 10 to 75% of an aqueous solution of ethanol or methanol; when eluted with a reverse phase silica gel, the elution solvent is preferably It is water and 1 0-50% ethanol or methanol aqueous solution; when refined with normal phase silica gel, the elution solvent is preferably a mixed solvent of trichloromethane or ethyl acetate and 90-1 00% ethanol aqueous solution or methanol. Their proportion is preferably 10: 1-2: 1.

In the preparation method, the fractions rich in the Paclitaxel A, B, and C compounds are detected by thin layer chromatography (TLC), ultraviolet (UV), high pressure liquid chromatography (HPLC), etc., wherein A HPLC detection method is preferred.

It is preferred that the total content of the farnesin derivative in the gastrodia plant extract obtained by the present invention is detected by an HPLC method. Preferably, it is determined by a high performance liquid chromatography external standard method, and the total content of the perylene compound is calculated by using the Patricia A standard. The total content of the Pelican derivative in the gastrodia plant extract obtained by the above method is 25% or more, preferably the total content of the Pelicin derivative is 50% or more, and more preferably the total content of the Pelicin derivative is 50% to 95%. Preferably, the Pelicin derivative is Paclitaxel A, B, C.

The Gastrodia elata extract containing the Patricia derivative obtained according to the above method has a puzzle, augmented memory and treatment of Alzheimer Di sease (AD) and Vascular Dementia (VaD) and a mixture thereof. The role of the type.

The preparation method described in the present invention is one of the methods for preparing a gastrodia plant extract having a high content of the Paclixin derivative, and does not mean that only the high content of the Paclitaxel derivative obtained by the above method is a gastrodia plant extract. It can be used as a drug for the prevention and treatment of diseases such as Alzheimer Disease (AD) and Vascular Dementia (VaD) and their mixed type. It is conceivable that the gastrodia plant extract rich in the farnesin derivative obtained by other methods by those skilled in the art will have the same pharmacological action.

The plant extract of the traditional Chinese medicine Gastrodia elata is prepared by the above extraction and purification method, wherein the content of the active ingredient-parylene derivative in the plant extract can be as high as 50% or more. Among them, the macroporous adsorption resin method is more suitable for industrial production.

The plant extract obtained by the method of the present invention can be formulated into a pharmaceutical preparation together with a pharmaceutically acceptable carrier, for example, an injection, a tablet, a gelatin, an oral solution or a dropping pill. When the plant extract obtained by the method of the present invention is used to prepare various dosage forms of the desired drug, it can be prepared according to a conventional production method in the pharmaceutical field. Preferably, such pharmaceutical preparations of the invention may be administered orally or parenterally. Preferred formulations are administered orally or intravenously.

The sterile injectable form of the compositions of the invention may be aqueous or oily suspensions. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parentally acceptable diluent or solvent, for example A solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are often employed as a solvent or suspension medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or di-glycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, which are natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersing agents, such as carboxymethylcellulose or similar dispersing agents, which are commonly used in the formulation of pharmaceutically acceptable formulations, including emulsions and suspensions. . Other commonly used surfactants, such as Tweens, Spans, and other emulsifiers or bioavailability enhancers, which are commonly used in pharmaceutically acceptable solids, liquids, or other, may also be used for formulation purposes. The manufacture of the dosage form.

The pharmaceutical compositions of this invention may be orally administered in any orally acceptable dosage form, including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of oral tablets, commonly used carriers include lactose and corn starch. A lubricant such as magnesium stearate is usually also added. For oral administration in a gel form, useful diluents include lactose and dried corn starch. When an aqueous suspension is required for oral use, the active ingredient is combined with emulsifying and suspending agents. Some sweeteners, flavoring or coloring agents may also be added if desired.

If necessary, a maintenance dose of the compound, composition or combination of the invention can be administered once the condition of the patient has improved. Subsequently, the dose or frequency of administration or the dose and frequency can be reduced depending on the symptoms to a level at which the improved condition is retained. When the symptoms have been alleviated to the desired level, treatment should be discontinued. However, once any of the symptoms of the disease recur, the patient may need to receive intermittent treatment on a long-term basis.

The pharmaceutical preparation of the present invention may further comprise other active agents for treating Alzheimer's disease (AD) and vascular dementia (Vacular Dementia, VaD) and their mixed type or other treatments for Alzheimer's Disease (Alzheimer Disease, AD) is administered together with the vascular dementia (Vascular Dementia, VaD) and its mixed drugs.

It should also be understood that the specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the activity of the particular extract employed, age, weight, general health, sex, diet, time of administration, rate of excretion. , the combination of drugs, the judgment of the attending physician, and the severity of the particular disease being treated. detailed description

The following preparation examples and experimental examples are intended to further illustrate the invention but are not intended to limit the invention in any way. Example 1:

1. Extraction: Chinese medicine Tianma medicinal material l kg, pulverized and added to 50% ethanol for 3 times, 24 hours each time. The extracts were combined, ethanol was recovered and concentrated to a thick extract, heated by dissolving in distilled water, cooled to room temperature, allowed to stand for 24 hours, and centrifuged to obtain a clear filtrate.

2. Refining: The above filtrate is passed through a treated HPD-100 macroporous adsorption resin, eluted with water and 10%, 20%, 30%, 40%, 50%, 75% ethanol gradient, HPLC detection, collection rich The fractions of the Paclitaxel compound were combined, concentrated under reduced pressure, and spray dried to obtain 4.48 g of a plant extract. The content of the Pelicin compound was 51.8% as determined by high performance liquid chromatography.

Example 2:

1. Extraction: Chinese medicine Tianma medicinal material l kg, after pulverization, add water to soak and extract 3 times, each time for 12 hours. The extracts were combined, water was recovered and concentrated to a density of 1.05, allowed to stand for 24 hours, and centrifuged to obtain a clear filtrate.

2. Refining: The above filtrate passes through the treated HPD-100 macroporous adsorption resin. Water and 10%, 20%, 30%, 40%, 50°/. 75% ethanol gradient elution, HPLC detection, collection of fractions rich in Paclitaxin, combined, concentrated under reduced pressure, spray drying to obtain 4.48 g of plant extract. The content of the Pelicin compound was 56.34% as determined by high performance liquid chromatography.

Example 3:

1. Extraction: Chinese medicine Tianma medicinal material 1 kg, add water after pulverization, 50. C temperature soak extraction 3 times, 6 hours each time. The extracts were combined, water was recovered and concentrated to a thick extract, dissolved in distilled water, cooled to room temperature, allowed to stand for 24 hours, and centrifuged to obtain a clear filtrate.

2. Refining: The above filtrate is passed through a treated HP-20 macroporous adsorption resin, eluted with water and 10%, 20%, 30%, 40%, 50%, 75% ethanol gradient, HPLC detection, collection rich The fractions of the Paclitaxel compound were combined, concentrated under reduced pressure, and spray dried to obtain 4.42 g of a plant extract. The content of the pericin compound was 54.95% as determined by high performance liquid chromatography. Example 4:

1. Extraction: Chinese medicine Tianma medicinal material l kg, after pulverization, add 50% ethanol, 50. C temperature was soaked and extracted 3 times for 6 hours each time. The extracts were combined, ethanol was recovered and concentrated to thickened bone, heated to dissolve by adding distilled water, cooled to room temperature, allowed to stand for 24 hours, and centrifuged to obtain a clear filtrate.

2. Refining: The above filtrate is passed through a treated HP-20 macroporous adsorption resin, eluted with water and 10%, 20%, 30%, 40%, 50%, 75% ethanol gradient, HPLC detection, collection rich The fractions of the Paclitaxel compound were combined, concentrated under reduced pressure, and spray dried to obtain 4.42 g of a plant extract. The content of the Pelicin compound was 52.6% as determined by high performance liquid chromatography. Example 5:

1. Extraction: Chinese medicine Tianma medicinal material l kg, after pulverization, add 50% ethanol to soak overnight, then heat and reflux for 3 times, each time 3. 0 hours. The extracts were combined, ethanol was recovered and concentrated to a thick extract, dissolved in distilled water, cooled to room temperature, allowed to stand for 24 hours, and centrifuged to obtain a clear filtrate.

2. Refining: The above filtrate is passed through the treated HP-20 macroporous adsorption resin, washed with water, 10%, 20%, 30%, 40%, 50% and 75% concentration of decyl alcohol, HPLC detection, collection The plant extract is 4.68g. The plant extract is 4.68g. 4%。 The high-performance liquid chromatography, wherein the content of the parabens was 54.4%. Example 6:

1. Extraction: Chinese medicine Tianma medicinal material 1 kg, add water after pulverization, and dip three times at room temperature for 24 hours each time. The extracts were combined, water was recovered and concentrated to thickened bone, heated and dissolved by adding distilled water, cooled to room temperature, allowed to stand for 24 hours, and centrifuged to obtain a clarified filtrate. The concentrated solution was concentrated under reduced pressure.

2. Refining: The concentrated solution is passed through a reversed-phase silica gel column (C-18), eluted with water and 10% alcohol solution, and then eluted with a 50% alcohol solution. HPLC detection is performed to collect the polypyridyl-rich compound. The aliquot of the plant extract was 2.18 g. 7%。 The content of the Pelicin compound was 68.7%. Example 7

1. Extraction: Chinese medicine Tianma medicinal material l kg, after pulverization, add 75% ethanol, leaching 3 times at room temperature, each time 24. 0 hours. The extracts were combined, ethanol was recovered and concentrated to thickened bone, heated to dissolve by adding distilled water, cooled to room temperature, allowed to stand for 24 hours, and centrifuged to obtain a clear filtrate. The concentrated solution was concentrated under reduced pressure. 2. Refining: The concentrate is passed through a reversed-phase silica gel column (Cl 8 ), eluted with water and a 10% alcohol solution, and then eluted with a 50% alcohol solution. HPLC detection is performed to collect a stream rich in pyrene-like compounds. The mixture was extracted, and the plant extract was 2.18 g. 2%。 The high-performance liquid chromatography, wherein the content of the parabens was 66.2%. Example 8

1. Extraction: Chinese medicine Tianma medicinal material l kg, after pulverization, add 75% ethanol soaked overnight, 75. C heat extraction 3 times, each time 6. 0 hours. The extracts were combined, ethanol was recovered and concentrated to a thick extract.

2. Refining: Dip the silica gel sample, elute through a normal phase silica gel (100-200 mesh) column, elute with chloroform-nonanol (9:1), and then elute with chloroform-nonanol (5:1). The HPLC extract was collected, and the mixture was concentrated, concentrated under reduced pressure, and dried to obtain a plant extract of 4. 51 g. 3%。 The content of the compound is 50.3%. Example 9:

1. Extraction: Chinese medicine Tianma medicinal material l kg, after pulverization, add 75% ethanol to soak overnight, then heat and reflux for 3 times, each time 3. 0 hours. The extracts were combined, ethanol was recovered and concentrated to a thick extract.

2. Refining: Dip the silica gel sample, elute through a normal phase silica gel (100-200 mesh) column, ethyl acetate-95% ethanol (10:1), and then ethyl acetate-95% ethanol (3: 1) After the extraction with ethyl acetate-95% ethanol (2:1), HPLC, the fractions containing the paclitaxin-like compound were collected, combined, concentrated under reduced pressure, and dried to obtain a plant extract 4. 88 g. 0%。 The content of the parabens was 56.0%. Example 10: 1. Extraction: Chinese medicine Tianma medicinal material l kg, pulverized and added with 95% ethanol at room temperature for 3 times, each time 24. 0 hours. The extracts were combined, ethanol was recovered and concentrated to a thick extract, heated to dissolve by adding distilled water, cooled to room temperature, allowed to stand for 24 hours, and centrifuged to obtain a clear filtrate. The concentrated solution was concentrated under reduced pressure.

2. Refining: The concentrate is eluted through a reversed phase silica gel (C-18) column, water and 10% ethanol solution, and then eluted with 50% alcohol solution. HPLC detection is performed to collect the polypyridyl compound. The mixture was extracted, dried, and concentrated under reduced pressure. 9%。 The content of the parabens was 60.9%. Example 11

1. Extraction: Chinese medicine Tianma medicinal material l kg, after crushing, add 95% ethanol, 75. C warm immersion extraction 3 times, each time 8. 0 hours. The extracts were combined, ethanol was recovered and concentrated to a thick extract.

2. Refining: Dip the silica gel sample, elute with normal phase silica gel (100-200 mesh) column, chloroform-nonanol (9:1), and elute with chloroform-nonanol (5:1). HPLC 。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。 1%。 The content of the parabens compound was 55.1%. Example 12:

1. Extraction: Chinese medicine Tianma medicinal material l kg, after pulverization, add 95% ethanol to soak overnight, then heat and reflux for 3 times, each time for 4. 0 hours. The extracts were combined, ethanol was recovered and concentrated to a thick extract.

2. Refining: Dip the silica gel sample, elute through a normal phase silica gel (100-200 mesh) column, ethyl acetate-95% ethanol (10:1), and then ethyl acetate-95% ethanol (5: 1) eluted with ethyl acetate-95% ethanol (3:1), detected by HPLC, collected rich in Paclitaxel The fractions of the compound were combined, concentrated under reduced pressure, and dried to yield 1.86 g. The content of the Pelicin compound was 55.8% as determined by high performance liquid chromatography. Experimental example

Effect of Gastrodia elata plant extract on puzzle and prevention and treatment of senile dementia

The abbreviation "clb" used hereinafter is a gastrodia elata extract which is assigned to Lixin A, B, C and/or its derivative content of more than 50%.

Experimental Example 1: Learning and memory impairment model induced by scopolamine in mice [Experimental materials and instruments]

1. Face animals: Male Kunming mice, weighing 20-22g, were provided by the Animal Laboratory of the Chinese Drug Control Institute. The animals were housed in the animal room for two days before the experiment.

2. Test drug: parishin A and the plant extract clb obtained by the above Preparation Example 2 (product of Example 2, content: 56.34%) were dissolved in distilled water.

3. Route of administration: gavage, control group, equal volume of distilled water

4. Dose: parishin: 0.25, 0.5, 1.0 mg/kg body weight; clb: 2.5, 5 10 mg/kg body weight

5. Positive control drug: donepezil: 2.5mg/kg body weight

6. Instrument: Mouse water maze program automatic control instrument: SMG-2

7. Reagents: scopolamine, purchased from Sigma Company Results:

The detection of parishin A and clb in the range of 0.25-1.0 mg/kg and 2.5-10 mg/kg, respectively, can significantly prolong the incubation period of the animal jumping off the platform for the first time, and the number of downtimes (the number of errors) within 5 minutes is reduced. , indicating that clb can effectively improve scopolamine-induced learning and memory impairment in mice, the intensity of action and yang The drug drug donepezil is close. The results are shown in Table 1.

Table 1 c 1 b is a small model of scopolamine-induced dementia

The impact of the number of errors

Group dose (mg/kg) latency (seconds) number of errors (times) normal control 259 ± 18 0.3 ± 0.2

Model group 142±28# 1.4 ± 0.3##

Par ishin A 0.25 233 ±22* 0.5 ±0.2"

Parishin A 0.5 255 ± 16* 0.2 ±0.1*

Parishin A 1.0 262 ± 18** 0.3 ± 0.2*

Clb 2.5 212 ± 15* 0.3± 0.1"

Clb 5 237 ± 19* 0.2 ± 0.1*

Clb 10 275 ± 17** 0.4 ± 0.2*

Donepezil 3 241 ± 25* 0.3 ±0.2**

The latency and number of errors in the table are expressed as: Mean ± SEM (mean ± standard deviation).

The model group is compared with the normal control group # Ρ<0· 05, ##Ρ<0.01,

The drug-administered group was compared with the model group *Ρ<0.05, **Ρ<0.01. Experimental Example 2: Mouse darkness test

Animals: SPF Kunming male mice, weighing 19-20 g, were provided by the Animals Laboratory of the Chinese Drug Control Institute. They were housed in the animal room for two days before the experiment.

Reagents and preparation

Clb (product of Example 2, content 56.34%) was diluted with distilled water to 2.5 mg/10 ml (2.5 mg/kg), 10 mg/lOml (10 mg/kg), and administered by intragastric administration.

Donepezepam: 2.5 mg/kg, administered in 2.5 mg/5 ml with distilled water. Instrument: SBA-2 Mouse Concealer, provided by the Institute of Materia Medica, Chinese Academy of Medical Sciences. Methods: SPF Kunming mice, weighing 19-20g, male, randomly divided into 5 groups, normal control group, model group, donepezil group (2.5mg/kg), clb group (2.5mg/kg and 10mg/kg) ). The normal control group was given water, the other administration was administered, and the administration was 8 and The mouse darkening experiment was carried out for 9 days. On the 8th day, 20 minutes before the study, intraperitoneal injection of scopolamine hydrobromide (5mg/kg).

The experimental device is a conditional reflection box, which is divided into two parts: light and dark. There is a small hole in the two chambers to allow the mice to pass freely. The bottom of the box has a copper grid, and the copper grid at the bottom of the dark chamber is connected with 36V. During the experiment, the mice were placed in the bright room and turned back to the dark room. Because the mice have the habit of darkness and drilling holes, they will be drilled into the dark room several times during the real face training. When the mouse enters the dark room, the four-legged contact copper grid When you get an electric shock, this behavior is an error, and its normal reaction is to immediately try to exit the darkroom. On the first day of training, the number of times the mice entered the darkroom in 3 minutes and the time to enter the dark box for the first time were recorded. After 24 hours, an experiment was conducted to observe the incubation time of the mouse entering the black box for the first time within 5 minutes, and the number of times the mouse entered the black box was the number of errors.

The experimental results were counted and the latency of the first time each group of mice entered the darkroom and the number of times of entering the darkroom were counted. It can be seen from the table below that in the mouse avoidance test, the model group has a very significant difference compared with the normal group, and the experimental method makes the model successful. Compared with the model group, the dopenezide group, the c lb low dose (2.5 mg/kg) group, and the high dose (10 mg/kg) or medium dose (5 mg/kg) group all significantly increased the first entry. The incubation period of the darkroom and the number of times of entering the darkroom are the number of errors, indicating that donepezil and c lb have a role in preventing memory impairment caused by scopolamine hydrobromide in the mice. See the table below for the results. Table 2 Effect of c lb on memory impairment caused by scopolamine hydrobromide in mouse darkness test

Group dose number of animals latency period (s) number of errors (times)

Compared with the normal group ##P<0.01, compared with the model group * P<0.05, **Ρ<0.01 clb in the mouse darkness test against the memory impairment caused by scopolamine hydrobromide

Group dose animal latency ( s ) number of errors (times) Normal control group 12 155.69 ±110.45 1.08 soil 1.00 model group 13 71.82 ± 64.54# 4.85 ± 1.63## donepezil group 2.5 mg/kg 13 161.88 ± 111.21* * 1.22±1· 18** clb 2.5 mg/kg 12 183.21 ± 103.96** 1.75 ± 1.42** clb 5 mg/kg 11 143.84 ± 99.31** 1.61 ± 1.30** Compared with normal group ##Ρ<0.01, Comparison with the model group * Ρ < 0.05 , ** Ρ < 0.01 Experimental Example 3: Model of memory formation disorder induced by cyclohexene imine (CHX) in mice: SPF Kunming mice, body weight 19-20 g, male, Randomly divided into 5 groups, normal control group, model group, donepezil group (2.5 mg/kg), clb group (product of example 1, content 51.8%) (2.5 mg/kg, 5.0 mg/kg and 10mg/kg). The normal control group was given water, the rest were administered, and the platform was tested on the 9th and 10th day. Intraperitoneal injection of cyclohexeneimine (5mg/kg) 20 minutes before the 9th day of study

RESULTS: The detection of clb at 2.5mg/kg, 5mg/kg and 10mg/kg by probosping method can effectively prolong the incubation period of the first time in mice, and reduce the number of mice in the lower stage, suggesting that clb is induced by cyclohexeneimine. Memory formation disorders have an improved effect, which is equivalent to donepezil. Table 4. Effect of clb on CHX-induced memory consolidation disorder in mice (jumping experiment) (X soil SEM)

Group dose (mg/kg) latency (S) number of errors (times) Control 250 ± 12 0.4 ± 0.1

CHX (model) 187 ± 25 2.0± 0.

Clb 2.5 167±24 0.5 ± 0.2 clb 5 209 ±20 0.3± 0· 1** clb 10 233 soil 30 0.4 ± 0.1* donepezil 5 205 soil 19 0.6 ± 0.2 model group compared with normal control group ### P<0.001

The drug-administered group was compared with the model group *P<0.05, **P<0.01. Effect of Gastrodia elata Plant Extract on Spatial Learning and Memory Ability in Rats with Vascular Dementia

The observation and evaluation of learning and memory ability is an indispensable indicator in clinical and experimental research of dementia. In order to objectively evaluate the learning and memory ability of drugs in model animals, this experiment is specially used to test animal learning and memory ability. Water maze. It detects that in many trainings, the rat learns to find a hidden platform of fixed position and forms a stable spatial position cognition. This spatial cognition is a reference cognition with different references. The memory formed is a spatial reference memory. From the perspective of the processing and extraction of information, this spatial reference memory enters the consciousness system, and its storage mechanism mainly involves the limbic system (such as hippocampus) and the brain area related to the cerebral cortex, which should belong to declarative memory, and clinical forgetfulness and dementia. The patient, it is the declarative memory that is firstly damaged and more prominent. Therefore, from the detection of the memory properties, it is more appropriate to use the Morris water maze to screen the effective drugs for the prevention and treatment of such diseases.

1 experimental material

The experimental drug clb is the plant extract clb obtained in Example 2 (Example 2 The product, content 56.34%), was dissolved in distilled water. The experimental animal clean grade SD rats, male, weighing 190-210 g, were supplied by Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., and the certificate number was scxk (Beijing) 2002-0003. Under the experimental conditions, the diet was taken after three days of adaptation to the environment.

Instrument Morris Water Maze DMS-2 System is provided by the Institute of Materia Medica, Chinese Academy of Sciences

2 experimental methods

2.1 Preparation of rat model of vascular dementia

Male Sprague-Dawley rats, weighing 190-210 g, 10% chloral hydrate were intraperitoneally injected, placed supine, and the anterior neck was decomposed and disinfected. The bilateral common carotid arteries were isolated, the double-wire was ligated, and the wound was sutured. In the sham operation group, only the bilateral common carotid arteries were freed, and each group of rats was raised under the same conditions.

2.2 Grouping of experimental animals

Animals were randomly divided into sham operation group (n=10), model group (n=15), clb low dose group (n=15), clb high dose group (n=15), clb medium dose group (n-15). , P-type drug donepezil group (n = 15), positive drug nimodipine group (n = 15).

2.3 administration

The rats were administered 24 hours after operation. The rats in the low-dose clb group were administered with rats in the high dose group of 0.8 mg/kg and clb according to the 7.5 mg/kg, clb medium dose group at 2.5 mg/kg; the positive drug donepezil The group was administered with 0.58 mg/kg and the nimodipine group was administered orally at 9 mg/kg. The sham operation group and the model group were given an equal volume of normal saline. Morris water maze training was performed from day 42 for 40 days of continuous administration.

The Morris water maze is mainly composed of a circular pool and an automatic camera and computer analysis system. Through the camera system above the maze, the whole process of exploring the rat in the maze is carried out. Shooting, such as the rat's trajectory, movement time, distance of movement, movement, etc., avoids the influence of human factors. The platform is placed in the southwest quadrant (mth quadrant) from the center of the pool

At 22cm, the water was injected into the pool before the test, the water depth was 30cm (that is, the water surface was lcm higher than the surface of the platform, so that the rats could not see the platform), the water temperature was controlled between 20~21, room temperature 25 X:. Various experimental conditions were required to remain unchanged during the experiment. The whole experimental process is divided into two parts: hidden-platform acquisition training and probetrial testing.

Experimental Example 1: Effect on the evasion latency of locating navigational face in vascular dementia rats

The results are shown in Table 1. The rats in each group had shorter and shorter time to search for the platform. There was no significant difference in the escape latency between the animals on the 1st and 2nd day. From the 3rd day of swimming training, the escape latency of the model group was significantly longer than that of the sham operation group. (P<0.05), indicating that there was significant spatial learning and memory impairment in the model rats; the escape latency of the rats in each group of c 1 b was reduced to a different extent than that in the model group, especially in the low-dose group for 3 or 4 days of swimming. It was significantly shorter than the model group (P<0.05); the positive drug nimodipine was also significantly shorter than the model group on the 4th day of swimming (Ρ<0.05).

Table 1 Effect of drugs on the escape latency of Morr i s water maze in rats with vascular dementia ((X Shi s)

Group search platform latency time / s

1st 曰 2nd 曰 3rd 曰 4th sham operation group 79. 99 ± 19. 88 51. 55 soil 27. 03 25. 19 ± 16. 36*

14. 49 ± 7. 63*

Model 84. 52 ± 12. 69 69. 90 ± 18. 19 48. 70 ± 22. 37

41. 15 ± 25. 05

Clb large 83. 98 ± 14. 87 67. 26 ± 22. 55 45. 67 ± 19. 09

29. 55 ± 17. 65

In clb 74. 25 ± 26. 24 55. 76 ± 24. 72 37. 72 ± 19. 28

25. 25 ± 12. 33*

Clb small 84. 63 ± 12. 57 63. 65 ± 18. 77 30. 15 ± 14. 50*

17. 27 ± 9. 40*

Donepezil 80. 47 ± 20. 26 51. 32 ± 23. 43 35. 84 ± 19. 48

44. 27 士 27. 28

Nimodipine 86· 75 ± 9. 53 68. 81 ± 24. 54 37. 78 ± 15. 27

23. 75 士 11. 37*

Note: Compared with the model group: *P<0. 05

Experimental Example 2: Effect of positioning navigation test swimming path on vascular dementia rats

The results are shown in Table 2. After swimming training, the path of the animals searching for the platform was getting shorter and shorter. There was no significant difference in the path of the first and second days of swimming training in each group of rats, but the swimming of the model group began from the third day of swimming training. The path was significantly longer than that of the sham-operated group (P<0.05). The swimming path of the clb group was reduced to a different extent than that of the model group, especially in the low-dose group, which was significantly shorter than the model group during swimming for 3 or 4 days. (P<0.05). Table II Effect of drugs on swimming path of Morris water maze swimming training in vascular dementia rats (X soil S)

Search platform path /cm

Chapter 1 Section 2 Section 3 Section 4 Prosthetic Hand 994.29 ± 223.45 849.99 488.26 356.88 ± 272.41* 251.05 ±133.12* Model 1007.243 ±164.55 953.75 ± 375.31 790.95 ±310.28 585.66 ± 354.81

1210-A Large 1148· 25 ± 273.52 929.58 ±273.64 723.10 ± 320.34 436.54 ±241.24

994.37 ± 387.08 809.51 ± 375.47 540.83 ± 346.27 392.83 ± 222.17 in 1210-A

1210-A small 1010.831 ± 316.59 935.74 ± 266.21 472.50 ± 209.11 * 275.25 ± 141.09 * more « Qi 1169.31 ± 357.16 834.87 ± 502.62 728.26 ± 525.09 751.53 ± 593.48 Floor level 1112.201 ± 202.04 953.47 ± 372.92 491.43 ± 203.56 317.40 ± 162.07 Note: With the model Group comparison: *P<0.05

Claims

Rights request

1. Use of an extract extracted from Gastrodia elata in the prevention and treatment of diseases such as Alzheimer Disease (AD) and Vascular Dementia (VaD) and mixed diseases thereof.

2. The extract according to claim 1 mainly comprising a pirin derivative, which is a Paclitaxin A, B, C.

Among them, the structure of par i shin A, B, C is as follows:

Parisinin A parishin B parishin C

3. The total content of the Paclitaxel derivative according to claim 2 is 35% or more, preferably the total content of the Paclitaxel derivative is 50% or more, and more preferably the total content of the Pelicin derivative is 50°/ο-95°. _/ ο β may be the derivative thereof as a brinzolamide Xin Yansheng monomeric compound may be a mixture of any of brinzolamide Xin Yansheng was more compounds;

The method for producing a gastrodia plant extract according to any one of claims 1 to 3, wherein the extract is obtained by the following steps:

(1) pulverizing the plant gastrodia tuber, and then extracting with water or an aqueous solution of 10-95% C1-C6 alkyl alcohol as a solvent at room temperature to reflux temperature, and extracting the extract under normal pressure or reduced pressure to obtain an extract;

(2) purifying and purifying the extract by chromatographic method, eluting with an elution solvent, collecting a fraction rich in the paclitaxel derivative, concentrating, filtering, and drying to obtain an extract containing a pirin derivative, wherein The total content of the Paclitaxel derivative is 35% or more, and the total content of the Parylene derivative is preferably 50% or more, and more preferably the total content of the Pelicin derivative is 50%-95%;

Wherein the pyrene derivative is par i sh in A, B, C, and its structural formula is as follows:

Parisinin A parishin B parishin C

The preparation method according to claim 4, wherein the raw material plant Tianma is Chinese herbal medicine Gastrodia elata.

The process according to claim 4, wherein the C1-C6 alkyl alcohol is methanol, ethanol, isopropanol or n-butanol or a mixed solvent thereof.

The preparation method according to claim 4, wherein the chromatographic method for purification and purification is macroporous adsorption resin chromatography, normal phase silica gel, reverse phase silica gel chromatography, or a combination thereof, or the extract is mixed with In silica gel or diatomaceous earth, air dry and elute with organic solvent or water.

The preparation method according to claim 4, characterized in that the solvent used for elution is water, CI-C6 alkyl alcohol, halogenated hydrocarbon, ether solvent, ketone solvent, ester solvent and composed of the above solvent. Mixed solvent.

9. The elution solvent according to claim 8 comprising decyl alcohol, ethanol, isopropanol, n-butanol, dichlorodecane, chloroform (chloroform), diethyl ether, methyl tert-butyl ether, C A ketone, 2-butanone, ethyl acetate, ethyl formate, and a mixed solvent composed of the above solvents.

The preparation method according to claim 8, wherein the solvent used for elution is preferably one or a mixture of chloroform (trichloromethane), ethyl acetate, ethanol, decyl alcohol and water; in particular, In the purification method, when refining with a styrene-type macroporous adsorption resin, the elution solvent is preferably water and 10-75% ethanol or methanol aqueous solution; when purified by reverse-phase silica gel chromatography, the elution solvent is preferably water. And 10-50% ethanol or methanol aqueous solution; when refined with normal phase silica gel, the elution solvent is preferably a mixed solvent of trichloromethane or ethyl acetate and 90-100% aqueous ethanol solution or methanol, and their ratio is preferably For 10: 1-2: 1.

11. A pharmaceutical composition for preventing and treating diseases such as Alzhe imer D i sease (AD) and Vascular Dementia (VaD) and a mixed type thereof, which is rich in Paclitaxel a gastrodia plant extract of a derivative, and a pharmaceutically acceptable carrier, the composition preferably being formulated into a pharmaceutical preparation such as a tablet, an injection, a capsule, a soft capsule, an oral solution or a dropping pill, preferably a gastrodia plant extract The total content of the middle ricin derivative is 35% or more, and the total content of the ricin derivative is preferably 50% or more, and more preferably the total content of the pirin derivative is 50°/. - 95%;

The pyrene derivative thereof is par i shin A, B, C, and its structure