CN113975322B

CN113975322B - Uncaria rhynchophylla total alkaloids and extraction and purification method and application thereof

Info

Publication number: CN113975322B
Application number: CN202010734471.0A
Authority: CN
Inventors: 张庆英; 蒲小平; 赵欣; 屠鹏飞; 杨红帅; 郭强; 刘秋怡
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2020-07-27
Filing date: 2020-07-27
Publication date: 2022-08-30
Anticipated expiration: 2040-07-27
Also published as: CN113975322A

Abstract

The invention discloses total alkaloids of uncaria, an extraction and purification method and application thereof. The extraction and purification method of the uncaria rhynchophylla total alkaloids comprises the following steps: ethanol is used as an extraction solvent to obtain an ethanol extract, and after acid and alkali treatment, the ethanol extract is purified by macroporous adsorption resin column chromatography and MCI GEL column chromatography in sequence. The invention further establishes an HPLC-UV chemical composition analysis method based on 45 main alkaloids. The uncaria rhynchophylla total alkaloids prepared by the method have high yield, definite components and controllable quality. The uncaria total alkaloids prepared by the invention can obviously improve the behavioral manifestations of 1-methyl-4-phenyl-1, 2,3, 6-tetrahydropyridine induced C57BL/6J mice such as autonomic activity, fatigue resistance, action coordination, behavior disorder and the like, protect the integrity of dopaminergic neurons, improve the content of dopamine and metabolites thereof, have exact pharmacological action for preventing and treating neurodegenerative diseases and obviously improve the pharmacodynamic action.

Description

Uncaria rhynchophylla total alkaloids and extraction and purification method and application thereof

Technical Field

The invention relates to active ingredients of uncaria, in particular to uncaria total alkaloids and extraction and purification methods and an analysis method of chemical ingredients of the uncaria total alkaloids.

Background

Parkinson's Disease (PD) is the second most common neurodegenerative disease in humans, and its incidence is second only to Alzheimer's Disease (AD). Parkinson's disease is a chronic degenerative disease of the central nervous system caused by extravertebral dysfunction, and the main pathological change is degeneration of neurons in the Substantia nigra compacta (SNc) Dopamine (DA) energy. When the neuronal degeneration reaches a certain degree, symptoms begin to appear, mainly manifested as resting tremor, bradykinesia and muscular rigidity, in addition to gait and posture abnormalities, cognitive impairment and other symptoms. The etiology of parkinson's disease is currently poorly understood, and although a variety of etiological theories have been proposed, such as the dopamine deficiency theory, the excitotoxicity theory, the oxidative free radical theory, the mitochondrial dysfunction theory, and the like, only the dopamine deficiency theory is recognized by most scholars. The theory holds that parkinson's disease is due to a reduction in striatal DA, which is primarily due to impaired degeneration of the substantia nigra.

At present, the clinical treatment of the Parkinson's disease mainly comprises drug treatment and operation treatment, wherein the drug treatment is the main treatment. The drug therapy is mainly based on western medicine therapy, and is most commonly used with levodopa (L-DOPA), which mainly relieves symptoms, but cannot prevent the development process of diseases. The long-term use of L-DOPA can cause the gradual weakening of the efficacy and serious toxic and side effects, such as 'on-off' phenomenon, akinesia, mental disorder and the like.

With the advent of aging society, the development of new drugs for the treatment of Parkinson's disease with high efficiency and safety has become especially urgent and important. In recent years, the research and development mode of western medicine ' single component-single target ' has gradually revealed the defects, and the concept of multi-component, multi-target and overall treatment of traditional Chinese medicine is more in line with the essence of complex diseases such as Parkinson's disease and the like and multiple pathological links, and belongs to one of the traditional characteristics and advantages fields of traditional Chinese medicine. Therefore, the development of the high-efficiency and low-toxicity drug for resisting Parkinson's disease from the common traditional Chinese medicines for clinically treating Parkinson's disease has wide application prospect and important significance.

The Uncaria (Uncaria) plants are perennial climbing shrubs of Rubiaceae (Rubiaceae), about 34 species exist in the world, and are mainly distributed in southeast Asia, southeast America, Africa and other tropical regions. The plant of the genus uncaria in China has 11 types, 1 type, and is mainly distributed in provinces and regions such as Guangdong, Guangxi, Yunnan, Sichuan, Hubei, Hunan, Guizhou, Fujian, Jiangxi, Shanxi, Gansu, Tibet, Taiwan and the like. The ramulus Uncariae cum uncis is dried stem branch with hook of Uncaria rhynchophylla, Uncaria macrophylla, Uncaria hirsuta, Uncaria sinensis or Uncaria sessilifolia. Ramulus Uncariae cum uncis is cool in nature and sweet in taste, enters liver and pericardium channels, has effects of calming endogenous wind, arresting convulsion, clearing heat and calming liver, and can be used for treating liver wind stirring, convulsive epilepsy, convulsion, febrile convulsion, common cold with convulsion, infantile cry, preeclampsia, headache, giddiness, etc. Modern researches show that uncaria has related activities of a cardiovascular system, a cerebrovascular system and a central nervous system, and comprises the effects of resisting hypertension, Alzheimer disease, Parkinson disease, anxiety and the like. Although the research on uncaria is more at present, the research objects are only limited to uncaria water decoction, alcohol extract (such as methanol extraction and ethanol extraction), total alkaloid (such as crude total alkaloid part obtained by acid-base treatment) and individual main monomer alkaloid (such as uncarine, isocourine, and kentuckazine methyl ether) and the like, and a preparation and purification method of uncaria total alkaloid with the activity of resisting the parkinson disease under the tracking of the activity and chemical components, a system characterization method of the chemical components and research reports of the uncaria total alkaloid with the activity of resisting the parkinson disease and the like are not found.

The existing literature and the previous experimental results of the inventor show that the uncaria alkaloid shows the activity of resisting the Parkinson disease. Although alkaloid components in uncaria are rich in structure, the content of the alkaloid components is low, and the extraction method of the uncaria total alkaloids for carrying out the anti-Parkinson disease activity research in the literature does not carry out effective purification and enrichment and chemical component characterization on the alkaloid, so that the components of the uncaria total alkaloids with activity are unclear, the substance basis is unclear, and the quality is difficult to control and needs to be improved.

Disclosure of Invention

One of the purposes of the invention is to provide a method for extracting and purifying uncaria rhynchophylla total alkaloids with anti-Parkinson disease activity;

the invention also aims to provide an HPLC-UV chemical component analysis method aiming at the uncaria rhynchophylla total alkaloids;

the third purpose of the invention is to apply the prepared and purified total alkaloids of uncaria in preventing and treating neurodegenerative diseases.

The above object of the present invention is achieved by the following technical solutions:

an extraction and purification method of uncaria rhynchophylla total alkaloids comprises the following steps:

(1) reflux-extracting ramulus Uncariae cum uncis with organic solvent such as alcohols; treating the extract with acid and alkali, subjecting to HP20 macroporous adsorbent resin column chromatography, sequentially eluting with water, 95% ethanol and acetone, and collecting 95% ethanol eluate to obtain crude product of total alkaloids fraction of ramulus Uncariae cum uncis; (2) dissolving the crude product of the total alkaloids part of the uncaria with 50% methanol, carrying out MCI GEL column chromatography, sequentially eluting with 0.02% ammonia water, methanol-0.02% ammonia water with the volume ratio of 3:7 and methanol-ammonia water with the volume ratio of 1000:0.2, and collecting the elution part of the methanol-ammonia water with the volume ratio of 1000:0.2 to obtain the purified total alkaloids of the uncaria.

When the crude product of the total alkaloid part of the uncaria rhynchophylla is extracted from the uncaria rhynchophylla, different extraction methods are considered, including the extraction effects of the rhynchophylla alkaloid, such as solvent type, material ratio, reflux time, extraction times and the like, and the result shows that the factor which has the greatest influence on the extraction efficiency of the rhynchophylla alkaloid is the extraction solvent, wherein the extraction is best by using 70% ethanol; when reflux extraction is carried out, the mass ratio of uncaria and ethanol can be 1 (5-12), and preferably 1 (5-8); the reflux extraction times are 1-3 times, preferably 2 times; the extraction time is 15-90min, preferably 30-60 min.

The method for performing acid-base treatment on the extracting solution in the step (1) comprises the following steps: concentrating the extractive solution under reduced pressure, adjusting pH to acidity, vacuum filtering or centrifuging to obtain acid water solution and precipitate; adjusting pH of the acid water solution to alkaline with alkaline solution, and concentrating under reduced pressure to obtain concentrated solution; preferably, the extracting solution is decompressed and concentrated, the pH value is adjusted to 2, and the acid water solution and the sediment are obtained through decompression suction filtration or centrifugation; adjusting pH of the acid water solution to 8-9 with ammonia water, and concentrating under reduced pressure to obtain concentrated solution.

Dissolving the crude product of the total alkaloid part of the uncaria with 50 percent methanol and then carrying out MCI GEL column chromatography.

When the extracted uncaria rhynchophylla total alkaloid part crude product is purified, the purification effect of different chromatographic fillers comprising ODS, Sephadex LH-20, cation exchange resin, MCI GEL and the like on the uncaria rhynchophylla total alkaloid part crude product is considered, the result shows that neither ODS, Sephadex LH-20 nor cation exchange resin can effectively remove impurities in the uncaria rhynchophylla total alkaloid part crude product, but the MCI GEL is eluted by 0.02% ammonia water and methanol-0.02% ammonia water (v: v,3:7) to well remove the large chromatographic peak at the front edge of a solvent of the uncaria rhynchophylla total alkaloid part crude product, so that the methanol-ammonia water (v: v,1000:0.2) eluting part is efficiently enriched with uncaria rhynchophylla alkaloid.

According to the method, through systematic methodology investigation and optimization, the extraction and purification method of the uncaria rhynchophylla total alkaloids is determined, most of non-alkaloid impurities in the uncaria rhynchophylla total alkaloids prepared by the method are removed, and the yield of the uncaria rhynchophylla total alkaloids is about 5.5-8.5 g/kg of uncaria rhynchophylla medicinal materials.

The invention further provides an HPLC-UV chemical component analysis method for the prepared uncaria rhynchophylla total alkaloids based on 45 uncaria rhynchophylla alkaloid reference substances obtained by separation from uncaria rhynchophylla, and the HPLC-UV chemical component analysis method for the uncaria rhynchophylla total alkaloids is established by determining the conditions and the detection wavelengths of a mobile phase through systematic investigation of chromatographic conditions including the mobile phase, the detection wavelengths and the like. Under the analysis condition, 45 uncaria alkaloid reference substances are well separated, the chromatographic peak is uniformly distributed, and the baseline is stable.

The invention provides an HPLC-UV chemical component analysis method for uncaria rhynchophylla total alkaloids, which comprises the following steps of carrying out detection analysis on the uncaria rhynchophylla total alkaloids by HPLC-UV, wherein the detection conditions of HPLC are as follows: the column was Waters Xbridge C18 (250X 4.6mm,5 μm); mobile phase acetonitrile (containing 0.02% ammonia water) (B) -0.02% ammonia water (A), gradient elution program is 0-30min, 5% -20% B, 30-50min, 20% -50% B, 50-70min, 50% -100% B; the flow rate is 1.0 mL/min; the column temperature is 30 ℃; the sample injection volume is 5 mu L; the detection conditions of the ultraviolet detector comprise: the detection wavelength was 220 nm.

The established HPLC-UV analysis method is adopted to analyze the chemical components of the uncaria rhynchophylla total alkaloids, the result shows that the prepared uncaria rhynchophylla total alkaloids contain more than 70 chromatographic peaks, and the sum of the relative peak areas of the chromatographic peaks is 70.3% through area normalization calculation. Compared with 45 alkaloid reference substances, the structures of 28 alkaloids are identified from the uncaria rhynchophylla total alkaloids (URA), and the HPLC-UV chemical component analysis method of the uncaria rhynchophylla total alkaloids can be well used for representing various chemical components in the uncaria rhynchophylla total alkaloids, can be used for determining the chemical component profiles in the uncaria rhynchophylla total alkaloids, and ensures that the quality of the uncaria rhynchophylla total alkaloids is controllable.

The uncaria rhynchophylla total alkaloids extracted and purified by the method can obviously improve the autonomous activity, fatigue resistance, action harmony and behavior disorder of a Parkinson mouse caused by MPTP after administration, and have dose dependence; the MCI-N part has no improvement effect on the behavioral indexes of the Parkinson mice caused by the MPTP.

The brain area DA neuron Tyrosine Hydroxylase (TH) immunohistochemical staining experiment result shows that the TH positive cell number in substantia nigra and striatum can be obviously increased after the prepared and purified uncaria rhynchophylla total alkaloids are administrated, and the uncaria rhynchophylla total alkaloids have a protection effect on DA neurons; whereas the MCI-N moiety has not been shown to have protective effects on DA-competent neurons.

The experimental result of the content of dopamine and metabolites thereof in striatum measured by HPLC-ECD method shows that the uncaria rhynchophylla total alkaloids prepared and purified by the invention can improve the content of DA, DOPAC and HVA in striatum of mice of MPTP model group after administration; while the MCI-N part has no influence on the content of DA and metabolites thereof.

The experimental result shows that the uncaria rhynchophylla total alkaloids extracted and purified by the invention have exact pharmacological activity for preventing or treating Parkinson's disease, and can be applied to treating neurodegenerative diseases including Parkinson's disease.

Therefore, in another aspect, the present invention provides a pharmaceutical composition for preventing or treating neurodegenerative diseases, which is prepared by combining an effective amount of total alkaloids from uncaria with a pharmaceutically acceptable carrier, that is, combining the effective amount of total alkaloids from uncaria with a pharmaceutically acceptable carrier or diluent, and then preparing the combined product into any suitable pharmaceutical composition according to conventional preparation methods in the art. Generally, the compositions are suitable for oral administration, as well as other methods of administration. The composition can be in the form of liquid preparation such as tablet, capsule, powder, granule, pill or oral liquid, or topical preparation such as ointment, cataplasma, etc. In order to increase the dissolution and absorption of the drug, a solid dispersion may also be prepared. In addition, the composition can also be prepared into a slow-release control agent, a nano preparation and an intelligent drug delivery system. The pharmaceutical composition of the present invention may contain 0.1 to 99% by weight, preferably 10 to 60% by weight of total alkaloids of uncaria rhynchophylla according to various administration routes and administration methods.

The oral solid preparation of the total alkaloids of uncaria comprises active ingredients of the total alkaloids of uncaria and a carrier serving as a dispersing agent, wherein the carrier material is selected from one or a mixture of more of crosslinked polyvinylpyrrolidone, superfine silica gel powder, polyethylene glycol, dextrin and derivatives thereof, lactose, pregelatinized starch, microcrystalline cellulose and the like. The ratio of carrier to drug is generally in the range of 0.5-20:1(w/w), preferably 1-5:1(w/w), and most preferably 2:1 (w/w).

The diluent added can be one or more components which increase the weight and volume of the tablet; common diluents include lactose, starch, pregelatinized starch, microcrystalline cellulose, sorbitol, mannitol, and inorganic calcium salts; the most common of them are lactose, starch, microcrystalline cellulose.

The externally added disintegrating agent can be one or a mixture of more of crosslinked polyvinylpyrrolidone (with the total weight ratio of 2-6%), crosslinked sodium carboxymethylcellulose (with the total weight ratio of 2-6%), alginic acid (with the total weight ratio of 2-5%), and microcrystalline cellulose (with the total weight ratio of 5-15%). Wherein the content of crospolyvinylpyrrolidone (2-7 wt% of total weight) and croscarmellose sodium (2-6 wt% of total weight) are preferred, and the content of crospovidone (2-6 wt% of total weight) is most preferred.

The lubricant comprises one or more of stearic acid, sodium stearate, magnesium stearate, calcium stearate, polyethylene glycol, talcum powder and hydrogenated vegetable oil; magnesium stearate is most preferred. The amount of the lubricant is in the range of 0.10 to 1% (by total weight), and is generally 0.25 to 0.75%, and preferably 0.5 to 0.7%.

The binder employed may be one or several ingredients which facilitate granulation. It can be starch slurry (10-30% of total weight of binder), hydroxypropyl methylcellulose (2-5% of total weight of binder), and polyvinylpyrrolidone (2-20% of total weight of binder), preferably ethanol water solution of polyvinylpyrrolidone, and more preferably 50% ethanol water solution of polyvinylpyrrolidone.

The glidant can be one or more of micropowder silica gel, talcum powder and magnesium trisilicate.

The preparation process of the uncaria rhynchophylla total alkaloid solid dispersion can be a melting method, a solvent-melting method or a grinding method. The water soluble carrier is PEG, polyvinylpyrrolidone, poloxamer, and saccharide. The water-insoluble carrier is generally ethyl cellulose, chitosan, acrylic resin, lipids, or the like. Enteric materials are typically hydroxypropyl methylcellulose phthalate and acrylics.

The surfactant used may be one or more components that improve wetting and increase drug dissolution. Sodium lauryl sulfate (commonly used in the range of 0.2-6% by weight, based on total weight) is commonly used. Generally, the recommended dose of the uncaria rhynchophylla total alkaloids for treating neurodegenerative diseases is 1-200mg/kg, preferably 5-100mg/kg, and most preferably 10-60 mg/kg. The above dosage may be administered in a single dose or in multiple doses.

On the basis of a large number of experiments, the preparation and purification method of the uncaria rhynchophylla total alkaloids with anti-Parkinson activity is finally determined, the HPLC-UV chemical component characterization method based on 45 main alkaloids is established, and the uncaria rhynchophylla total alkaloids prepared by the method are high in yield, clear in components and controllable in quality. The uncaria rhynchophylla total alkaloids prepared and purified by the invention can obviously improve the behavioral manifestations of 1-methyl-4-phenyl-1, 2,3, 6-tetrahydropyridine (MPTP) -induced C57BL/6J mice such as autonomous activity, fatigue resistance, action coordination, behavior disorder and the like, protect the integrity of dopaminergic neurons, improve the content of dopamine and metabolites thereof, have exact pharmacological action for preventing and treating neurodegenerative diseases, and obviously improve the pharmacodynamic action.

Drawings

FIG. 1 is a flow chart of the extraction and purification of total alkaloids from uncaria rhynchophylla.

FIG. 2 is an HPLC-UV (220nm) chromatogram of each eluted fraction of a crude uncaria rhynchophylla total alkaloid part after ODS column chromatography.

FIG. 3 shows HPLC-UV (220nm) chromatograms of elution parts of crude uncaria rhynchophylla total alkaloid part after Sephadex LH-20 column chromatography.

FIG. 4 is HPLC-UV (220nm) chromatogram of each elution part of the crude product of total alkaloids of uncaria through ion exchange column chromatography.

FIG. 5 is an HPLC-UV (220nm) chromatogram of the crude uncaria rhynchophylla total alkaloid part after MCI GEL column chromatography.

Figure 645 chemical structures of the uncaria alkaloid reference.

FIG. 7 is HPLC-UV (220nm) chromatograms of crude product (A) and total alkaloid fraction (B) of ramulus Uncariae cum uncis.

FIG. 845 HPLC-UV (220nm) chromatograms of a mixed reference substance (A) and total alkaloids (B) of Uncaria rhynchophylla.

FIG. 9 Effect of Uncaria rhynchophylla Total alkaloid on MPTP-induced autonomic Activity count and Activity distance in Parkinson mice (mean. + -. SEM, n-6; compare blank group) ^## p<0.01， ^### p<0.001; comparison with model group ^* p<0.05， ^** p<0.01)。

Fig. 10 ramulus Uncariae cum uncisEffect of Total Alkaloids on the Motor latency and the number of droppings in rotarod Activity in Parkinson mice due to MPTP (mean. + -. SEM, n 12; compare blank group) ^### p<0.001; comparison with model group ^* p<0.05， ^*** p<0.001)。

FIG. 11 Effect of Total Alkaloids of Uncaria rhynchophylla on time to turn around, climb completely long time and mean stride in MPTP Parkinsonian grids (mean + -SEM, n 10; compare with blank group) ^### p<0.001; comparison with model group ^* p<0.05， ^*** p<0.001)。

FIG. 12 staining of TH-positive cells in substantia nigra A Control B Model C Selegiline D-F URA (10,20,80mg/kg) G MCI-N (80 mg/kg).

FIG. 13 staining of TH-positive cells in the striatum A Control B Model C Selegiline D-F URA (10,20,80mg/kg) G MCI-N (80 mg/kg).

FIG. 14 Effect of Uncaria rhynchophylla Total alkaloid on TH-positive cells in substantia nigra and striatum of Parkinson mice due to MPTP (mean. + -. SEM, n-6; compare blank group) ^## p<0.01， ^### p<0.001; comparison with model group ^* p<0.05， ^** p<0.01， ^*** p<0.001)。

FIG. 15 Effect of Total Alkaloids of Uncaria rhynchophylla on the levels of DA and metabolites in the striatum of Parkinson mice due to MPTP (mean. + -. SEM, n ═ 6; compare blank group) ^### p<0.001; comparison with model group ^* p<0.05， ^*** p<0.001)。

Detailed Description

The invention is further described below in conjunction with specific experimental examples, and the advantages and features of the invention will become more apparent as the description proceeds. However, these examples are only illustrative and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and substitutions are intended to be within the scope of the invention.

Experimental example 1 extraction and purification of Uncaria rhynchophylla Total alkaloid

1. Test materials and reagents

Ramulus Uncariae cum uncis (stem and branch of ramulus Uncariae cum uncis of U.H. N.H. C.H. H. H.) was purchased from Shennong Chinese medicinal decoction piece factory in Liuzhou city of Guangxi, and its origin is Guangxi and is identified by Shennong Chinese medicinal decoction piece factory in Liuzhou city of Guangxi.

Ethanol, methanol, ammonia water, hydrochloric acid and the like used for conventional extraction and purification are analytically pure, and are products of Beijing-Tong broad-spectrum fine chemical company; HPLC analysis was performed using acetonitrile as HPLC chromatogram, a product of Fisher Scientific, Inc. (Fisher Chemicals, NJ, USA); HPLC analysis was performed using Milli-Q Synthesis A10(Bedford, MA, USA) as water. Dianion HP-20 macroporous adsorbent resin is TSK Corporation (TSK Corporation, JP); MCI GEL CHP20/P120 resin is a product of Mitsubishi Chemical Corporation (JP); ODS-A-HG is YMC (YMC CO. LTD., JP) Co; amberlyst 15(H) type ion exchange resins are available from Alfa Aesar (Alfa Aesar, USA).

2. Experimental methods and results

2.1 extraction of Uncaria Total alkaloid

The extraction effect of the rhynchophylla alkaloids by different extraction methods including solvent types, material ratio, reflux time, extraction times and the like is investigated, and the result shows that the factor which has the greatest influence on the extraction efficiency of the rhynchophylla alkaloids is the extraction solvent, wherein the extraction with 70% ethanol is the best, and the influence of other factors on the extraction efficiency is not great.

The method comprises the following steps: collecting 1375g of ramulus Uncariae cum uncis, pulverizing, reflux-extracting with anhydrous ethanol (material ratio 1:8) for 30min, and reflux-extracting twice; decocting the residue with distilled water (material ratio 1:8) for 15min, mixing extractive solutions, and concentrating under reduced pressure until no alcohol smell is detected to obtain fluid extract. Dispersing the fluid extract with appropriate amount of water, adjusting pH to 2 with 2% hydrochloric acid, vacuum filtering or centrifuging to obtain acid water solution and precipitate. Adjusting pH of the acidic water solution to 8-9 with ammonia water, and concentrating under reduced pressure to obtain concentrated solution. Subjecting the concentrated solution to HP20 macroporous adsorbent resin column chromatography, eluting with water, 95% ethanol and acetone sequentially, collecting 95% ethanol part, and concentrating under reduced pressure to dryness to obtain ramulus Uncariae cum uncis total alkaloid part crude product (37.7g) (FIG. 1).

The method 2 comprises the following steps: taking 1000g of ramulus Uncariae cum uncis, pulverizing, extracting with 70% ethanol (material ratio 1:8) under reflux for 30min, and extracting under reflux twice; concentrated under reduced pressure until no alcohol smell (about 600 mL). Adjusting pH to 2 with 2% hydrochloric acid, vacuum filtering or centrifuging to obtain acid water solution and precipitate. Adjusting pH of the acid water solution to 8-9 with ammonia water, and concentrating under reduced pressure to obtain concentrated solution. Subjecting the concentrated solution to HP20 macroporous adsorbent resin column chromatography, eluting with water, 95% ethanol, and acetone sequentially, collecting 95% ethanol fraction, and concentrating under reduced pressure to dry to obtain crude product (35.0g) of total alkaloids fraction of ramulus Uncariae cum uncis (FIG. 1).

2.2 Uncaria rhynchophylla Total alkaloid purification method investigation

The purification effect of the crude product of the total alkaloid part of the uncaria rhynchophylla is examined on different chromatographic fillers comprising ODS, Sephadex LH-20, cation exchange resin, MCI GEL and the like.

2.2.1 purification of Marsdenia tenacissima alkaloid by ODS

Taking 200mg of uncaria rhynchophylla total alkaloid part crude product, performing ODS column chromatography, sequentially performing gradient elution by using 0.02% ammonia water and methanol with different volume fractions, namely 0.02% ammonia water (v: v,1:19,1:4,1:1,4:1), collecting elution fractions, and performing HPLC-UV analysis on each concentrated fraction respectively, wherein the result shows that ODS cannot effectively remove impurities in the uncaria rhynchophylla total alkaloid part crude product (figure 2).

2.2.2 Sephadex LH-20 purification of Marsdenia tenacissima alkaloids

Taking 200mg of crude uncaria rhynchophylla alkaloid crude product, performing Sephadex LH-20 column chromatography, sequentially performing gradient elution by using 20% methanol, 50% methanol and methanol with different volume fractions, collecting elution fractions, and performing HPLC-UV analysis on the concentrated fractions respectively, wherein the result shows that impurities in the crude uncaria rhynchophylla total alkaloid part cannot be effectively removed by Sephadex LH-20 (figure 3).

2.2.3 purification of Marsdenia tenacissima alkaloid by ion exchange resin

A200 mg uncaria rhynchophylla crude alkaloid sample is taken, is eluted by water, 0.1% ammonia water and methanol-0.1% ammonia water (v: v,1:1) sequentially through cation exchange resin column chromatography, elution fractions are collected, and HPLC-UV analysis is respectively carried out on the concentrated fractions, and the result shows that the cation exchange resin cannot effectively remove impurities in a crude uncaria rhynchophylla total alkaloid part (figure 4).

2.2.4 purification of Marsdenia tenacissima alkaloid by MCI GEL

Taking 200mg crude uncaria rhynchophylla alkaloid product, carrying out gradient elution by using 0.02% ammonia water, methanol-0.02% ammonia water (v: v,3:7) and methanol-ammonia water (v: v,1000:0.2) in sequence through MCI GEL column chromatography, collecting elution fractions, and carrying out HPLC-UV analysis on the concentrated fractions respectively, wherein the result shows that the MCI GEL 0.02% ammonia water and the methanol-0.02% ammonia water (v: v,3:7) can well remove a large chromatographic peak at the front edge of a solvent in the crude uncaria rhynchophylla alkaloid part, so that uncaria rhynchophylla alkaloid is efficiently enriched in the elution part of the methanol-ammonia water (v: v,1000:0.2) (figure 5).

2.3 determination of purification method of Uncaria rhynchophylla total alkaloids

Dissolving the crude product (10g) of the total alkaloids fraction of ramulus Uncariae cum uncis obtained by 2.1 extraction with a small amount of 50% methanol, subjecting to MCI GEL column chromatography, and sequentially eluting with 0.02% ammonia water, methanol-0.02% ammonia water (v: v,3:7), and methanol-ammonia water (v: v,1000: 0.2). Collecting 0.02% ammonia water fraction and methanol-0.02% ammonia water (v: v,3:7) fraction, concentrating to dryness, and mixing to obtain fraction named MCI-N (about 7.2 g); concentrating methanol-ammonia water (v: v,1000:0.2) to dry to obtain ramulus Uncariae cum uncis total alkaloid (URA, about 2.1-2.5 g) (figure 1). The purification method of the uncaria alkaloid is determined based on the HPLC-UV analysis result and combined with an animal pharmacodynamic test.

Through systematic methodology investigation and optimization, the preparation and purification method of the uncaria rhynchophylla total alkaloids is determined, most of non-alkaloid impurities of the uncaria rhynchophylla total alkaloids prepared and purified by the method are removed, and the yield of the uncaria rhynchophylla total alkaloids is about 5.5-8.5 g/kg of uncaria rhynchophylla medicinal materials.

Experimental example 2 characterization experiment of chemical Components of Uncaria rhynchophylla Total alkaloid

1. Laboratory instruments, materials and reagents

1.1 instruments

High Performance Liquid Chromatography (HPLC) Aglient 1260HPLC (Aglient Co., U.S.A.).

1.2 materials and reagents

2.1 part of uncaria rhynchophylla total alkaloids in experimental example 1 and 2.3 parts of purified uncaria rhynchophylla total alkaloids in experimental example 1. 45 alkaloid controls (FIG. 6) were isolated from Uncaria rhynchophylla for this subject group and their structures were identified by spectroscopic methods.

HPLC analysis was performed using acetonitrile as HPLC chromatogram, a product of Fisher Scientific, Inc. (Fisher Chemicals, NJ, USA); HPLC analysis was performed using water prepared from Milli-Q SynthesisA10(Bedford, MA, USA); other ethanol, methanol and the like used for conventional extraction and dissolution are analytically pure reagents, and are produced by Beijing Tong GuangZhong Fine chemical company.

2. Experimental methods

2.1 preparation of control solutions

Taking 45 alkaloids separated from ramulus Uncariae cum uncis as reference substances, and mixing 45 alkaloids separated from ramulus Uncariae cum uncis to obtain 0.01mg/mL mixed standard to obtain reference substance solution.

2.2 preparation of test solutions

Taking the crude product of the total alkaloid part of the uncaria rhynchophylla and about 5mg of the total alkaloid part of the uncaria rhynchophylla, dissolving the crude product and the total alkaloid part of the uncaria rhynchophylla in 1mL of methanol by ultrasound, and filtering to obtain a test solution.

2.3 sample analysis

The column was Waters Xbridge C18 (250X 4.6mm,5 μm); mobile phase acetonitrile (containing 0.02% ammonia water) (B) -0.02% ammonia water (A), gradient elution program is 0-30min, 5% -20% B, 30-50min, 20% -50% B, 50-70min, 50% -100% B; the flow rate is 1.0 mL/min; the detection wavelength of the ultraviolet detector is 220 nm; the column temperature is 30 ℃; the injection volume was 5. mu.L.

3. Results of the experiment

Based on 45 uncaria alkaloid reference substances separated from uncaria, a HPLC-UV characterization method of uncaria total alkaloids is systematically researched. Through system investigation of chromatographic conditions including mobile phase, detection wavelength and the like, the mobile phase conditions and the detection wavelength are determined, and the HPLC-UV chemical component characterization method of the uncaria rhynchophylla total alkaloids is established. Under the analysis condition, 45 uncaria alkaloid reference substances are well separated, the chromatographic peak is uniformly distributed, and the baseline is stable.

The uncaria rhynchophylla total alkaloids prepared in the experimental example 1 are analyzed by an established HPLC-UV method, and the result shows that the uncaria rhynchophylla total alkaloids contain more than 70 chromatographic peaks, and the sum of relative peak areas of the chromatographic peaks is 70.3% through area normalization calculation. The structures of 28 alkaloids were identified from uncaria total alkaloids (URA) by comparison with 45 alkaloid controls (fig. 7-8).

The HPLC-UV chemical component analysis method of the uncaria rhynchophylla total alkaloids, which is disclosed by the invention, can be well used for representing chemical components in the uncaria rhynchophylla total alkaloids, determining the outline of the chemical components in the uncaria rhynchophylla total alkaloids and ensuring the quality of the uncaria rhynchophylla total alkaloids to be controllable.

Experimental example 3 behavioral experiments on Uncaria rhynchophylla Total alkaloid

1. Laboratory instruments, materials and reagents

1.1 instruments

A vertical grid instrument, a YLS-4C model mouse rotating rod fatigue tester (produced by laboratory instruments of the department of medicine of Beijing university); JZZ 98-CM model small animal autonomous activity detector (manufactured by Chinese academy of medical sciences).

1.2 animals

C57BL/6J mice, male, 8 weeks old, 20-22g in body weight, were raised at constant temperature at room temperature, diet and drinking water were not limited, and 12 hours of alternate illumination were provided by the laboratory animal center of Beijing university department of medicine. The environment was adapted for one week before the test.

1.3 materials and reagents

1-methyl-4-phenyl-1, 2,3, 6-tetrahydropyridine (MPTP) is a product of Sigma company; selegiline (selegiline) is a product of orlian corporation, finland.

2. Experimental methods

2.1 animal experiment grouping and processing method

C57BL/6J mice were raised at room temperature and constant temperature, diet and water were not limited, and the mice were alternately illuminated for 12h and adapted to the environment for one week before the test. The mice were randomly divided into 7 groups of 12 mice each, namely a blank control group, a model control group, a positive drug selegiline group (15mg/kg), a URA low dose group (10mg/kg), a URA medium dose group (20mg/kg), a URA high dose group (80mg/kg) and an MCI-N group (80 mg/kg). Administration: the gavage was continued for 14d, and the blank control group and the model control group were given equal volumes of distilled water. Molding: the behavioral index test was performed 1h after the last 1 administration, 5 consecutive days after the beginning of the gavage administration of MPTP30mg/kg (control group was given an equal volume of physiological saline) in the abdominal cavity 1h before the 10 th administration. 1d after the behavioral indexes are tested, the mice are killed by blood bleeding after the head is broken, the brains of the mice are taken, the substantia nigra and the striatum are separated and stored at the temperature of minus 80 ℃ for immunohistochemical analysis and content determination of dopamine and metabolites thereof.

2.2 method for determining Experimental indices

General behavioral observations: general behavior of mice after being subjected to MPTP modeling in the abdominal cavity is observed, abnormal reactions occur, and differences among groups are compared and analyzed.

And (3) autonomous activity experiment: the spontaneous activity of the mice was measured and counted using a JZZ 98-CM model mouse locomotor, the locomotor activity of the mice was automatically recorded by a recorder by placing the mice in locomotor activity boxes (1 mouse per activity box, 4 mice were measured at the same time each time), the number of times and distance of activity of each mouse within 5min were measured, and statistical analysis was performed.

Rotating rod experiment: the YLS-4C type rotating rod type fatigue instrument is used for testing the rotating rod behavior of the mouse and researching the influence of the medicament on the action coordination and the anti-fatigue property. Training is continuously carried out for 3d before testing, 2 times every day, the rotating speed is 12r/min, and the training time is 180 s. In the formal test, a mouse is placed on a rotating rod, the rotating speed is set to be 35r/min, the time from the rotation of the rotating rod to the leaving of the rotating rod of the test mouse is used as the mouse movement latency, in addition, the number of times that the mouse falls off the rotating rod is recorded, and the test time is 180 s. Each mouse was tested 3 times with 30min intervals, and the results were averaged for statistical analysis 3 times.

Vertical grid experiment: the vertical grid instrument is a vertical open box with the specification of 55cm multiplied by 8cm multiplied by 5cm, and a metal square grid with the size of 0.6cm multiplied by 0.6cm is arranged on the rear side of the box. During testing, the head of the mouse is kept upward and placed on the metal net of the metal grid instrument, the distance between the head of the mouse and the metal net is about 3cm, the mouse can turn by itself to climb down towards the bottom after being held by the hand, and the time that the mouse turns around and climbs down and the time that the mouse climbs the whole length are recorded. Training for 2d in advance before formal testing, training each mouse for 3 times every day, during the formal testing, if the mouse cannot climb down within 60s, placing the mouse again for measurement, if the mouse slides down or falls down midway, recording the maximum climbing time as 60s, measuring each mouse for 3 times, wherein the interval of each test is 10min, and averaging the results of 3 times for statistical analysis.

3. Results of the experiment

3.1 general behavioural experiments

Starting 5min after the intraperitoneal administration of MPTP, the general behavior of MPTP model mice was abnormal compared to the placebo group, and most of the mice showed the following changes: tail lifting, hair erecting, increased salivary secretion, accelerated respiration, hypotonia, sensitivity to external environment stimulation, dental tremor and the like, and the duration of the treatment is generally 2-3 h. And the uncaria rhynchophylla total alkaloids group and the selegiline group have light symptom and short duration.

3.2 autonomic Activity experiments

Compared with the blank group, the MPTP model group has obviously reduced activity frequency and activity distance of the mice (p is less than 0.01, and p is less than 0.001). After 14 days of continuous gavage administration, compared with the MPTP model group, the number of times of activity and the activity distance of the mice of the positive drug selegiline group are obviously increased (p is less than 0.01). The number of mouse activities in the URA low dose (10mg/kg) group was increased compared with the MPTP model group, but it was not statistically significant, and the number of mouse activities in the URA medium dose (20mg/kg) group and the URA high dose (80mg/kg) group was significantly increased compared with the MPTP model group (p < 0.05); the mice in the URA low dose (10mg/kg) group and URA medium dose (20mg/kg) group had increased distance traveled compared to the MPTP model group, but not statistically significant, and the mice in the URA high dose (80mg/kg) group had significantly increased distance traveled compared to the MPTP model group (p < 0.05). The MCI-N (80mg/kg) group had a slight increase in the number of times and distance of movement of mice compared to the MPTP model control group, but had no statistical significance (FIG. 9).

3.3 rod rotation experiment

Compared with the blank group, the MPTP model group has obviously shortened movement latency (p <0.001) and obviously increased dropping times (p < 0.001). After 14 days of continuous gavage, compared with the MPTP model group, the mice of the positive drug selegiline group have obviously increased motor latency (p <0.001) and obviously reduced dropping times (p < 0.001). The mice in the URA low dose (10mg/kg) group had increased motor latency compared with the MPTP model group, but the mice in the URA medium dose (20mg/kg) group and the URA high dose (80mg/kg) group had significantly increased motor latency compared with the MPTP model group (p <0.05, p < 0.001); the mice dropped less frequently in the URA low dose (10mg/kg) group than in the MPTP model group, but not statistically significantly in the URA medium dose (20mg/kg) group and the URA high dose (80mg/kg) group (p <0.05, p < 0.001). The MCI-N (80mg/kg) group showed no significant difference in motor latency and number of droppings compared to the MPTP model control group (FIG. 10).

3.4 vertical grid experiment

The MPTP model group compared to the blank group, mice had significantly increased turn around time and crawl completely long time (p <0.001), with significantly decreased average steps (p < 0.001). After 14 days of continuous gavage, the turning-around time and climbing completion time of mice in the positive drug selegiline group were significantly reduced (p <0.001) and the average stride was significantly increased (p <0.001) compared to the MPTP model group. The u-turn time and creeping time of mice in the URA low dose (10mg/kg) group are reduced compared with those in the MPTP model group, but the u-turn time and creeping time of mice in the URA medium dose (20mg/kg) group and the URA high dose (80mg/kg) group are obviously reduced compared with those in the MPTP model group (p is less than 0.001); there was no significant difference in mean stride between URA low dose (10mg/kg) group and MPTP model group, the mean stride between URA (20mg/kg) and MPTP model group was increased, but there was no statistical significance, and the mean stride between URA high dose (80mg/kg) and MPTP model group was significantly increased (p < 0.05). The mice in the MCI-N (80mg/kg) group showed no significant difference in turning-around time, climbing completely long time and average stride compared to the MPTP model control group (FIG. 11).

4. Conclusion of the experiment

After the uncaria rhynchophylla total alkaloids are administrated, the autonomous activity, the fatigue resistance, the action coordination and the behavior disorder of a Parkinson mouse caused by MPTP can be obviously improved, and the uncaria rhynchophylla total alkaloids have dose dependence; the MCI-N part has no improvement effect on the behavioral indexes of the Parkinson mice caused by the MPTP.

EXAMPLE 4 brain area DA neuronal Tyrosine Hydroxylase (TH) immunohistochemical staining

1. Laboratory instruments, materials and reagents

1.1 instruments

A cryomicrotome (model CM1850UV, Leica, Germany), a high resolution bright field fluorescence digital slide scanning system (Nano Zoomer model S60, Korea).

1.2 materials and reagents

1-methyl-4-phenyl-1, 2,3, 6-tetrahydropyridine (MPTP) is a product of Sigma company; selegiline (selegiline) is a product of orlian corporation, finland. The tyrosine hydroxylase generation antibody (SC-25269) is a product of Santa Cruz company; the mouse two-step method detection kit (PV-9002) and the Diaminobenzidine (DAB) color developing solution are purchased from Beijing China fir Jinqiao biotechnology limited; bovine Serum Albumin (BSA), hematoxylin stain, PBS buffer and 3% hydrogen peroxide were purchased from Seville Google Biotech, Inc.; the xylene is a product of chemical reagents of national drug group.

2. Experimental methods

The mice 1d after the behavioral indexes in the experimental example 3 were tested were anesthetized with 10% chloral hydrate, then perfused with normal saline, then with 4% paraformaldehyde buffer, and the brain tissue was taken out and placed in 4% paraformaldehyde buffer for storage at room temperature. The substantia nigra and striatum were paraffin sectioned (10 μm thick) and immunohistochemically stained for Tyrosine Hydroxylase (TH). Positive reaction of cells was observed by using TH polyclonal antibody and mouse two-step kit, DAB color development. The specific operation steps are as follows:

(1) paraffin section dewaxing and water washing: placing the slices in xylene I15 min-xylene II 15 min-xylene III 15 min-absolute ethyl alcohol I5 min-absolute ethyl alcohol II 5 min-85% ethyl alcohol 5 min-75% ethyl alcohol 5 min-distilled water washing.

(2) Antigen retrieval: placing the tissue slices in a repairing box filled with citric acid antigen repairing buffer solution (pH 6.0) in a microwave oven for antigen repairing, stopping heating for 8min until boiling, maintaining the temperature for 8min, and turning to low and medium heat for 7min to prevent excessive evaporation of the buffer solution. After natural cooling, the slides were washed 3 times in PBS (pH 7.4) on a destaining shaker for 5min each.

(3) Blocking endogenous peroxidase: the sections were placed in 3% hydrogen peroxide solution, incubated for 25min at room temperature in the dark, and the slides were washed 3 times 5min each time in PBS (pH 7.4) with shaking on a destaining shaker.

(4) And (3) sealing serum: 3% BSA was added dropwise to the tissue in the formation ring to cover the tissue uniformly, and the tissue was blocked at room temperature for 30 min. (Primary antibody was goat-derived blocked with rabbit serum, other sources with BSA)

(5) Adding a primary antibody: gently removing the confining liquid, dripping PBS (phosphate buffer solution) on the slices to prepare primary antibodies according to a certain proportion, and flatly placing the slices in a wet box for incubation at 4 ℃ overnight. (Small amount of water added in wet box to prevent evaporation of antibody)

(6) Adding a secondary antibody: slides were washed 3 times in PBS (pH 7.4) with shaking on a destaining shaker for 5min each time. After the section is slightly dried, the second antibody cap tissue corresponding to the first antibody is dripped into the ring, and the section is incubated for 50min at room temperature.

(7) DAB color development: slides were washed 3 times in PBS (pH 7.4) with shaking on a destaining shaker for 5min each time. After the section is slightly dried, a DAB color developing solution which is prepared freshly is dripped into the ring, the color developing time is controlled under a microscope, the positive color is brown yellow, and the section is washed by tap water to stop color development.

(8) Counterstaining cell nuclei: counter-staining with hematoxylin for about 3min, washing with tap water, differentiating with hematoxylin differentiation solution for several seconds, washing with tap water, returning the hematoxylin to blue, and washing with running water.

(9) Dewatering and sealing: placing the slices in 75% alcohol for 5 min-85% alcohol for 5 min-anhydrous alcohol I for 5 min-anhydrous alcohol II for 5 min-xylene I for 5min, dehydrating, removing the slices from xylene, air drying, and sealing with neutral gum.

(10) And (5) image acquisition and analysis.

3. Results of the experiment

In the experiment of immunohistochemistry of mouse substantia nigra and striatum TH, a large amount of immunostaining positive cells can be seen in the substantia nigra of a blank group of mice, cytoplasm is rich, cell axons are clear, and a large amount of dense immunostaining positive nerve fibers can be seen in the striatum. Compared with the blank group, the number of immunostaining-positive cells in the substantia nigra of the model group mice is obviously reduced (p <0.001), the axon is broken, and the average optical density of the immunostaining-positive nerve fibers in the striatum of the model group mice is obviously reduced (p < 0.01). After 14 days of continuous gavage administration, compared with the MPTP model group, the number of immunostaining positive cells in the substantia nigra of the mice of the positive drug selegiline group is remarkably increased (p is less than 0.001), and the average optical density of the immunostaining positive nerve fibers in the striatum of the mice of the positive drug selegiline group is remarkably increased (p is less than 0.01). Compared with the MPTP model group, the URA low-dose (10mg/kg) group has more immunostaining positive cells in the substantia nigra of mice, but has no statistical significance, and compared with the MPTP model group, the URA medium-dose (20mg/kg) group and the URA high-dose (80mg/kg) group have significantly more immunostaining positive cells in the substantia nigra of mice (p is less than 0.001); the mean optical density of immunostaining-positive nerve fibers in the striatum of mice was increased in the URA low dose (10mg/kg) group and the URA medium dose (20mg/kg) group compared to the MPTP model group, but not statistically significant, and the mean optical density of immunostaining-positive nerve fibers in the striatum of mice was significantly increased in the URA high dose (80mg/kg) group compared to the MPTP model group (p < 0.05). The MCI-N (80mg/kg) group showed no significant difference in the number of immunostaining-positive cells in the substantia nigra of mice and the average optical density of immunostaining-positive nerve fibers in the striatum of mice, compared to the MPTP model group (FIGS. 12-14).

4. Conclusion of the experiment

After the uncaria rhynchophylla total alkaloids are administrated, the number of TH positive cells in substantia nigra and striatum can be obviously increased, and the uncaria rhynchophylla total alkaloids have a protection effect on DA energy neurons; whereas the MCI-N moiety does not appear to have a protective effect on DA-competent neurons.

Experimental example 5 content experiment for measuring dopamine and metabolite thereof in striatum by HPLC-ECD method

1. Laboratory instruments, materials and reagents

1.1 instruments

High performance liquid chromatography (HPLC 1260, Inc., USA), JY 96-II ultrasonic cell crusher (Ningbo Xinzhi Biotech, Inc.), SIGMA 3-18K centrifuge (German Sigma), high performance liquid chromatography (LC-6A, Japan Shimadzu corporation), BASLC-4B type electrochemical detector, Dalianjiang high performance chromatography workstation JS-3070, high performance liquid chromatography column: DIKMA Diamond (R) C18 column (150X 4.6mm,5 μm).

1.2 materials and reagents

Dopamine (DA), 3, 4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and Isoproterenol (IP) are products of Sigma; citric acid, disodium ethylene diamine tetraacetate and n-dibutylamine are produced by Beijing chemical plants; anhydrous sodium acetate is produced by Tianjin chemical reagent factory.

2. Experimental methods

2.1 sample pretreatment

1d of mice after the behavioral indexes in the experimental example 3 are tested are taken, decapitation and bloodletting are performed to kill the mice, and brains of the mice are taken, striatum is separated, and the mice are stored at the temperature of minus 80 ℃ and used for measuring the content of dopamine and metabolites thereof.

The tissue sample treatment process is carried out under ice bath condition, and the principle of low temperature and rapidness is followed. Adding solution A into tissue sample of 1mg at a ratio of 10 μ L A (0.6mol/L perchloric acid solution with IP added to give final concentration of 0.375 μ g/mL, and storing at 4 deg.C) according to W: V (1:10), cutting striatal tissue, ultrasonically preparing homogenate at 20000g, centrifuging at 4 deg.C for 20 min, sucking a certain amount of supernatant, adding half volume of solution B (containing 20mM potassium citrate, 300mM dipotassium hydrogen phosphate, and 2mM disodium edetate, storing at 4 deg.C), mixing, standing, 20000g, centrifuging at 4 deg.C for 20 min, sucking a certain amount of supernatant, storing at 4 deg.C, and testing.

2.2 chromatographic conditions

The mobile phase is sodium acetate-citric acid buffer solution (containing 85mM of citric acid, 100mM of anhydrous sodium acetate and 0.2mM of ethylene diamine tetraacetic acid), 850mL is prepared firstly, then 150mL of methanol is added, if the volume is less than 1L, triple distilled water is added to 1L, the pH value is adjusted to 3.68 by using sodium hydroxide, after suction filtration, a proper amount of SOS (90 mg is added firstly and then determined according to the separation condition) and n-dibutylamine (15 mu L is added firstly) are added to completely separate peaks, the flow rate is 1.0mL/min, the detection sensitivity of an electrochemical detector is 10nA, a glassy carbon working electrode, an Ag/AgCl reference electrode, the detection potential is 0.76V, the column temperature is 25 ℃, and the sample injection amount is 80 mu L. The content of dopamine and metabolites is expressed in μ g/g wet tissue weight, and statistical analysis is performed after data are determined.

3. Results of the experiment

The DA, DOPAC and HVA levels in the striatum of the model group mice were significantly reduced compared to the blank group (p < 0.001). After 14 days of continuous gavage, the contents of DA, DOPAC and HVA in striatum of mice with positive drug selegiline are obviously increased compared with that of MPTP model group (p is less than 0.05, and p is less than 0.001). The low URA dose (10mg/kg) group, the medium URA dose (20mg/kg) group, and the high URA dose (80mg/kg) group had increased levels of DA, DOPAC, and HVA in the striatum of mice compared to the MPTP model group, but had no statistical significance. The MCI-N (80mg/kg) group showed no significant difference in DA, DOPAC and HVA content in the striatum of mice compared to the MPTP model group (FIG. 15).

4. Conclusion of the experiment

After the uncaria total alkaloids are administrated, the contents of DA, DOPAC and HVA in striatum of mice in an MPTP model group can be increased, but the effect is not obvious; while the MCI-N part has no influence on the content of DA and metabolites thereof.

Claims

1. A method for extracting and purifying total alkaloids of uncaria rhynchophylla is characterized by comprising the following steps:

(1) reflux-extracting ramulus Uncariae cum uncis with 70% ethanol as extraction solvent; treating the extract with acid and alkali, subjecting to HP20 macroporous adsorbent resin column chromatography, sequentially eluting with water, 95% ethanol and acetone, and collecting 95% ethanol eluate to obtain crude product of total alkaloids fraction of ramulus Uncariae cum uncis; (2) subjecting the crude product of the total alkaloids part of the uncaria rhynchophylla to MCI GEL column chromatography, sequentially eluting with 0.02% ammonia water, methanol-0.02% ammonia water with the volume ratio of 3:7 and methanol-ammonia water with the volume ratio of 1000:0.2, and collecting the elution part of the methanol-ammonia water with the volume ratio of 1000:0.2 to obtain the purified total alkaloids of the uncaria rhynchophylla.

2. The extraction and purification method according to claim 1, wherein in the reflux extraction in the step (1), the mass ratio of the uncaria to the ethanol is 1: 5-12; the reflux extraction times are 1-3 times; the extraction time is 15-90 min.

3. The extraction and purification method according to claim 2, wherein in the step (1), the mass ratio of the uncaria to ethanol is 1: 5-8; the reflux extraction times are 2 times; the extraction time is 30-60 min.

4. The extraction and purification method according to claim 1, wherein the method for acid-base treatment of the extract in the step (1) comprises: concentrating the extractive solution under reduced pressure, adjusting pH to acidity, vacuum filtering or centrifuging to obtain acid water solution and precipitate; then the pH of the acid water solution is adjusted to be alkaline by using an alkaline solution, and the concentrated solution is obtained by decompression and concentration.

5. The extraction and purification process according to claim 4, wherein the extract is concentrated under reduced pressure, then the pH is adjusted to 2, and the filtrate is filtered under reduced pressure or centrifuged to obtain an acid water solution and a precipitate; adjusting pH of the acid water solution to 8-9 with ammonia water, and concentrating under reduced pressure to obtain concentrated solution.

6. The extraction and purification method according to claim 1, wherein in the step (2), the crude uncaria rhynchophylla total alkaloid part is dissolved by 50% methanol and then subjected to MCI GEL column chromatography.

7. Uncaria rhynchophylla total alkaloids obtained by the extraction and purification method according to any one of claims 1-6.

8. The HPLC-UV chemical component analysis method of uncaria rhynchophylla total alkaloids according to claim 7, comprising: detecting and analyzing the uncaria rhynchophylla total alkaloids by a high performance liquid chromatography-ultraviolet detector, wherein the detection conditions of HPLC comprise: the chromatographic column is Waters Xbridge C18; the mobile phase contains B-A; the phase B is 0.02% ammonia acetonitrile, and the phase A is 0.02% ammonia;

the gradient elution procedure was: 0-30min, 5-20% B, 30-50min, 20-50% B, 50-70min, 50-100% B; the flow rate is 1.0 mL/min;

the detection conditions of the ultraviolet detector comprise: the detection wavelength is 220 nm;

other detection conditions include: the column temperature is 30 ℃; the injection volume was 5. mu.L.

9. Use of the uncaria rhynchophylla total alkaloids in claim 7 in preparation of medicines for preventing or treating neurodegenerative diseases.

10. The use according to claim 9, wherein said neurodegenerative disease comprises parkinson's disease.

11. A pharmaceutical composition for preventing or treating neurodegenerative diseases, which comprises the uncaria rhynchophylla total alkaloids in claim 7 in an amount which is effective in preventing or treating and a pharmaceutically acceptable carrier or auxiliary material.