CN110872240B - Method for extracting guanidine alkaloid of scorpion and scorpion alkaloid A and/or scorpion and scorpion alkaloid B in scorpion and medical application - Google Patents
Method for extracting guanidine alkaloid of scorpion and scorpion alkaloid A and/or scorpion and scorpion alkaloid B in scorpion and medical application Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C277/00—Preparation of guanidine or its derivatives, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups
- C07C277/08—Preparation of guanidine or its derivatives, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups of substituted guanidines
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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- C07C279/00—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups
- C07C279/04—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of guanidine groups bound to acyclic carbon atoms of a carbon skeleton
- C07C279/12—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of guanidine groups bound to acyclic carbon atoms of a carbon skeleton being further substituted by nitrogen atoms not being part of nitro or nitroso groups
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- C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
- C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
- C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D213/78—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, e.g. ester or nitrile radicals
- C07D213/81—Amides; Imides
Abstract
The invention discloses a method for extracting guanidine alkaloid scorpion alkaloid A and/or scorpion alkaloid B in scorpion and medical application thereof, wherein the chemical structural formula of the scorpion alkaloid A is as follows:
the chemical structural formula of the scorpion alkaloid B is as follows:
the scorpion alkaloid A and scorpion alkaloid B can effectively inhibit the activity of acetylcholinesterase and butyrylcholinesterase, are strongly combined with PAS sites of the acetylcholinesterase to have action characteristics, and have metal ion chelation capacity, so that the scorpion extract can be used as a medicine for treating neurodegenerative diseases such as Alzheimer disease and the like.
Description
Technical Field
The invention relates to the field of traditional Chinese medicine extraction and preparation, in particular to an extraction method and medical application of two guanidine alkaloids in scorpion.
Background
The scorpion, also known as Buthus martensii Karsch, Scorpio, sting, Tenebrio molitor, Scorpio, Buthus martensii Karsch, etc., is pungent, mild and toxic, and is a dried body of Buthus martensii Karsch, an animal belonging to family Buthidae. Scorpio is mainly produced in Shandong, Henan, etc. of China, and has been used as medicine in China, Korea, India and Africa for over 2000 years. Scorpio has been used as medicine in Song dynasty of China. The scorpion has the efficacies of analgesia, anticonvulsion, anti-epilepsy, anti-tumor and the like, and is clinically used for treating diseases such as ulcer, leukemia, asthma, chronic nephritis and the like. At present, no literature report on the research of the relevant action of the scorpion on Alzheimer's Disease (AD) is found.
The scorpion has rich resources in China and has wide scientific research on macromolecules and scorpion venom, but the research on chemical components and activity of some small molecules of the scorpion is relatively less at present, and relevant documents for extracting guanidine alkaloid components from the scorpion are not seen.
Alzheimer's disease is a neurodegenerative disease commonly found in the elderly, and its main clinical manifestations are memory impairment, apraxia, agnosia, visuospatial impairment, executive dysfunction, personality and behavioral abnormalities, severely affecting the patients' ability to live in daily life. The world health organization claims that 4600 million people currently suffer from Alzheimer's disease worldwide, and according to the latest report of Alzheimer's disease worldwide, nearly 100 million cases of Alzheimer's disease are expected to be newly increased every year by 2050. The current treatment of Alzheimer's disease is still one of the worldwide problems.
The exact pathogenesis of alzheimer's disease has not been fully elucidated to date, and the cholinergic system hypothesis, the amyloid hypothesis and the Tau protein hypothesis are now widely recognized. Since acetylcholinesterase (AChE) is widely distributed in nerve tissues in the human brain, it degrades the neurotransmitter acetylcholine in synaptic cleft, so by inhibiting AChE activity, normal level of acetylcholine in synaptic cleft can be maintained, cholinergic nerve signal transmission is normal, and cognitive impairment of AD patients is restored. At present, AChE inhibitors are mainly used in clinic as anti-AD drugs, and comprise AChE inhibitors of rivastigmine and galanthamine, and in China, huperzine A is also used.
These acetylcholinesterase inhibitors act mainly on the Catalytic Active Site (CAS) at the base of acetylcholinesterase and can relieve the symptoms of patients with mild to moderate AD. However, recent studies have found that the Peripheral Active Site (PAS) is also closely related to the hydrolysis of acetylcholine and can inhibit beta-amyloid aggregation. Therefore, functional molecules based on the inhibition of the double active center sites (CAS and PAS of AChE) have been the focus of research in recent years. Donepezil is currently the only two-site AChE inhibitory drug on the market for clinical treatment of anti-AD.
In recent years, the compensatory action of butyrylcholinesterase (BChE), an isozyme of acetylcholinesterase, has attracted attention. BChE was also found to be distributed in the brain and to degrade the neurotransmitter acetylcholine. There is substantial evidence that BChE compensates for AChE loss during AD progression and its function when AChE production is inadequate or its activity is inhibited. In fact, AChE levels in late AD patients are reduced by 90% compared to normal brain, while BChE levels are approximately twice normal and tend to increase continuously. This is the main reason for the development of resistance to selective AChE inhibitors in AD patients. BChE is therefore a fairly important drug target for the middle and late stages of AD. The only cholinesterase inhibiting drugs used for clinical AD treatment at present are rivastigmine which is a powerful inhibitor of BChE and AChE double esterase, and the only cholinesterase inhibiting drugs are related to the neglect of the effect of BChE for a long time; however, rivastigmine binds strongly to the CAS site of AChE only, and does not interact with its PAS site.
It has been found that AD patients contain excessive Cu in their senile plaques2+、Zn2+And Fe2+Plasma metal ions, which in turn promote beta amyloid aggregation and tau aggregation in the brain. Therefore, reduction of ion concentration in brain by metal ion chelation has also become one of the important strategies for the treatment of AD and is also used in the treatment of other neurodegenerative diseases (see Rapeng, et al. transition metal ions and neurodegenerative diseases. university chemistry 2006,21 (6): 32-35; and see Cristina; (see Cristina)Rodríguez-Rodríguez,et al.The art of building multifunctional metal-binding agents from basic molecular scaffolds for the potential application in neurodegenerative diseases.Coordination Chemistry Reviews,2012,256:2308–2332)。
Disclosure of Invention
The invention aims to provide a method for extracting two guanidine alkaloids in scorpion, namely scorpion alkaloid A and/or scorpion alkaloid B.
The second purpose of the invention is to provide the medical application of the scorpion alkaloid A and/or scorpion alkaloid B.
The third objective of the present invention is to provide a scorpion extract containing scorpion alkaloid A and scorpion alkaloid B.
The fourth purpose of the invention is to provide the medical application of the scorpion extract.
The fifth object of the present invention is to provide a pharmaceutical composition containing scorpion alkaloid A and/or scorpion alkaloid B.
The sixth purpose of the invention is to provide the medical application of the pharmaceutical composition.
The technical scheme of the invention is summarized as follows:
a scorpion alkaloid A has a structure of formula (I):
(II) the base B of Buthus martensii Karsch has the structure of formula (II):
the extraction method of scorpion alkaloid A and/or scorpion alkaloid B comprises the following steps:
(1) taking a scorpion dried body as a raw material, crushing, adding an ethanol water solution (or methanol water solution) with the volume fraction of 50% -90% which is 8-10 times of the mass of the raw material, extracting under reflux for 2-3 times, extracting for 2-3 hours each time, merging to obtain an extracting solution, recovering the solvent under reduced pressure, and concentrating to obtain a total extract, namely the scorpion extract;
(2) dispersing the total extract into 5-10 times of water by mass, extracting with ethyl acetate (or dichloromethane or chloroform), discarding the ethyl acetate (or dichloromethane or chloroform) extract, and concentrating the residual water layer under reduced pressure to obtain residual water layer extract;
(3) separating the residual water layer extract by silica gel column chromatography, and gradient eluting with dichloromethane-methanol at volume ratio of 20:1, 10:1 and 1:1 respectively to obtain fractions Fr.1, Fr.2, Fr.3, Fr.4 and Fr.5;
(4) separating fraction Fr.5 by silica gel column chromatography, eluting with dichloromethane-methanol at volume ratio of 10:1 at constant rate to obtain fractions Fr.5-1, Fr.5-2, Fr.5-3, Fr.5-4, Fr.5-5, and Fr.5-6;
(5) separating fraction Fr.5-5 by ODS column chromatography, and eluting with methanol-water at volume ratio of 1:9, 2:8, 4:6, 5:5 and 9:1 as eluent to obtain fractions Fr.5-5-1, Fr.5-5-2, Fr.5-5-3 and Fr.5-5-4;
(6) Separating fraction Fr.5-5-2 by ODS column chromatography, eluting with methanol-water at volume ratio of 1:4 as eluent at equal speed to obtain scorpion alkali A, i.e. N- (4-guanidine butyl) -4-hydroxybenzamide;
(7) separating fraction Fr.5-5-3 by ODS column chromatography, eluting with methanol-water at volume ratio of 1:4 as eluent at equal speed to obtain N- (4-guanidinobutyl) -2-pyridinecarboxamide.
Use of scorpion alkaloid A and/or scorpion alkaloid B in preparing medicine for treating Alzheimer disease, medicine for inhibiting acetylcholinesterase and butyrylcholinesterase, and metal ion chelating medicine is provided.
Comprises Scorpio extract containing Scorpio alkaloid A and Scorpio extract containing Scorpio alkaloid B.
The scorpion extract is applied to the preparation of medicaments for resisting Alzheimer disease, medicaments for inhibiting acetylcholinesterase and butyrylcholinesterase and metal ion chelating medicaments.
A pharmaceutical composition comprises scorpion alkaloid A and/or scorpion alkaloid B or pharmaceutically acceptable salts thereof, and pharmaceutically acceptable carriers and/or excipients.
The pharmaceutical composition is applied to the preparation of anti-Alzheimer's disease drugs, drugs for inhibiting acetylcholinesterase and butyrylcholinesterase and metal ion chelating drugs.
The invention has the advantages and beneficial effects that:
The scorpion alkaloid A and scorpion alkaloid B can effectively inhibit the activity of acetylcholinesterase and butyrylcholinesterase, are characterized by combining with PAS sites of the acetylcholinesterase to be advantageous, and have metal ion chelation capacity, and the scorpion extract can be used as a medicine for resisting Alzheimer disease.
Drawings
FIG. 1 is an absorption spectrum of the chelate of a base A of Buthus martensii Karsch with each metal ion.
FIG. 2 is an absorption spectrum of scorpion base B chelated with each metal ion.
Detailed Description
Example 1
The extraction method of scorpion alkaloid A and scorpion alkaloid B comprises the following steps:
(1) taking a dried body (2.0kg) of scorpion (Buthus martensii Karsch) as a raw material, crushing, adding a methanol water solution with volume fraction of 85% and 9 mass times of the raw material, carrying out reflux extraction for 3 times, extracting for 3 hours each time, mixing to obtain an extracting solution, recovering the solvent under reduced pressure, and concentrating to obtain a total extract (337g), namely the scorpion extract;
(2) dispersing the total extract into 8 mass times of water, extracting with equal volume of ethyl acetate, discarding ethyl acetate extract, and concentrating the residual water layer under reduced pressure to obtain residual water layer extract (110 g);
(3) separating the residual water layer extract by silica gel column chromatography, and gradient eluting with dichloromethane-methanol at volume ratio of 20:1, 10:1 and 1:1 respectively to obtain fractions Fr.1, Fr.2, Fr.3, Fr.4 and Fr.5(41 g);
(4) Separating fraction Fr.5 by silica gel column chromatography, eluting with dichloromethane-methanol at volume ratio of 10:1 at constant rate to obtain fractions Fr.5-1, Fr.5-2, Fr.5-3, Fr.5-4, Fr.5-5(6.6g), and Fr.5-6;
(5) separating fraction Fr.5-5 by ODS column chromatography, and eluting with methanol-water at volume ratio of 1:9, 2:8, 4:6, 5:5 and 9:1 as eluent to obtain fractions Fr.5-5-1, Fr.5-5-2(1.2g), Fr.5-5-3(0.8g) and Fr.5-5-4;
(6) separating fraction Fr.5-5-2 by ODS column chromatography, eluting with methanol-water at volume ratio of 1:4 as eluent at equal rate to obtain pale yellow solid Scorpio alkaloid A (53 mg);
(7) separating fraction Fr.5-5-3 by ODS column chromatography, eluting with methanol-water at volume ratio of 1:4 as eluent at equal rate to obtain yellow white solid Scorpio alkali B (18 mg).
The spectrum data of the scorpion alkali A is as follows:
HR-ESI-MS(positive mode)m/z:251.1514[M+H]+(calculated for C12H19N4O2,251.1508)。1HNMR(400MHz,CD3OD)δ:1.64(2H,m,H-3'),1.65(2H,m,H-2'),3.22(2H,t,J＝6.2Hz,H-4'),3.38(2H,t,J＝6.2Hz,H-1'),6.87(2H,d,J＝8.6Hz,H-3and H-5),7.71(2H,d,J＝8.6Hz,H-2and H-6)。13C NMR(100MHz,CD3OD) delta 170.4(C-7),161.2(C-4),158.2(C-5'),130.3(C-2and C-6),126.4(C-1),116.3(C-3and C-5),42.1(C-4'),40.2(C-1'),27.5(C-2'),27.0(C-3 '). Through modern spectroscopy techniques (HR-MS, 1D-NMR and 2D-NMR), combined with relevant data of the literature, the structure of the compound is identified as N- (4-guanadinebutyl) -4-hydroxybenzamide, and the scorpion alkaloid A is a newly discovered natural compound, and the chemical structure of the compound is shown as follows:
The spectrum data of the scorpion alkaloid B are as follows:
HR-ESI-MS(positive mode)m/z:236.1524[M+H]+(calculated for C11H18N5O,236.1511)。1HNMR(400MHz,CD3OD)δ:1.69(2H,m,H-3'),1.71(2H,m,H-2'),3.26(2H,t,J＝6.9Hz,H-4'),3.49(2H,t,J＝6.5Hz,H-1'),7.57(1H,dd-like,J＝4.4,7.7Hz,H-5),7.98(1H,t,J＝7.7Hz,H-4),8.11(1H,d,J＝7.7Hz,H-3),8.66(1H,d,J＝4.4Hz,H-6)。13C NMR(100MHz,CD3OD)δ:167.1(C-7),158.8(C-5'),151.1(C-2),150.0(C-6),139.0(C-4),127.9(C-5),123.2(C-3),42.3(C-4'),39.9(C-1'),27.9(C-2'),27.3 (C-3'). The structure of the compound is identified to be N- (4-guanidine butyl) -2-pyridine formamide through analysis by modern spectroscopy techniques (HR-MS, 1D-NMR and 2D-NMR), and the scorpion alkaloid B is a novel compound which is not reported in the literature and has the following chemical structure:
example 2
The extraction method of scorpion alkaloid A and scorpion alkaloid B comprises the following steps:
(1) crushing dried body of Scorpio, adding 50% ethanol water solution 8 times by mass of the raw materials, reflux-extracting for 2 times (3 hr each time), mixing to obtain extractive solution, recovering solvent under reduced pressure, and concentrating to obtain total extract as Scorpio extract;
(2) dispersing the total extract into water of which the mass is 5 times that of the total extract, extracting with dichloromethane of the same volume, discarding dichloromethane extract, and concentrating the residual water layer under reduced pressure to obtain residual water layer extract;
steps (3) to (8) were the same as those of steps (3) to (8) in example 1.
Example 3
The extraction method of scorpion alkaloid A and scorpion alkaloid B comprises the following steps:
(1) taking a scorpion dried body as a raw material, crushing, adding a methanol aqueous solution with the volume fraction of 90% which is 10 times of the mass of the raw material, extracting for 3 times by refluxing, extracting for 2 hours each time, mixing to obtain an extracting solution, recovering the solvent under reduced pressure, and concentrating to obtain a total extract, namely the scorpion extract;
(2) Dispersing the total extract into 10 mass times of water, extracting with chloroform of the same volume, discarding chloroform extract, and concentrating the residual water layer under reduced pressure to obtain residual water layer extract;
steps (3) to (8) were the same as those of steps (3) to (8) in example 1.
Example 4
Detection of cholinesterase inhibitory Activity of Buthus martensii Karsch alkali A and Buthus martensii Karsch alkali B
The acetylcholinesterase and butyrylcholinesterase inhibitory activities of scorpion A and scorpion B were measured by an improved Ellman method.
To a 96-well plate, 140. mu.L of 100mM phosphate buffer, 20. mu.L of 0.05U/mL AChE (or 20. mu.L of 0.05U/mL BChE), and 20. mu.L of the compound to be tested, either scorpion A or scorpion B, were added in this order, mixed well, and then incubated in an incubator at 25 ℃ for 15 minutes. Then, 10. mu.L of 10.0mM DNTB and 10. mu.L of 7.5mM ATCI (or 10. mu.L of 7.5mM BTCI) were added, and the resulting mixture was placed in a thermostat, and after incubation at 37 ℃ for 30 minutes, the absorbance at 412nm of the solution in a 96-well plate was measured. 20 μ L of 0.01mol/L DMSO was used as a negative control, 20 μ L of galantamine was used instead of the test compound as a positive control, and the blank was prepared by adding 20 μ L of 100mM phosphate buffer instead of the test compound. Each group of data was done in parallel 3 times and the average was taken.
The inhibition rate was calculated as follows:
inhibition ═ blank-experimental group)/blank × 100%
Activity results (IC)50Values) are shown in table 1.
The results show that the scorpion alkali A and the scorpion alkali B can effectively inhibit the activity of acetylcholinesterase and butyrylcholinesterase. Therefore, the scorpion alkaloid A and/or scorpion alkaloid B, the scorpion extract containing the scorpion alkaloid A and the scorpion extract containing the scorpion alkaloid B and the composition containing the scorpion alkaloid A and/or the scorpion alkaloid B can be prepared into the medicine for treating diseases related to insufficient acetylcholine. Diseases associated with acetylcholine deficiency include alzheimer's disease, myasthenia gravis, glaucoma, and the like.
TABLE 1 cholinesterase inhibition Activity results (IC) of Butyrosine A and B50Value)
Example 5
Propidium iodide displacement detection of scorpion alkali A and scorpion alkali B
5U of AChE and 150. mu.L of a 100. mu.M sample solution of scorpion alkaloid A or B were incubated in a 96-well plate at 25 ℃ for 6 hours. Then 50. mu.L of 1. mu.M propidium iodide solution was added thereto and cultured at room temperature for 10 min. The absorbance (OD) was measured with a microplate reader under the conditions that the excitation wavelength (. lamda.ex) was 535nm and the emission wavelength (. lamda.em) was 595 nm. Wherein the blank group is replaced by Tris-HCl solution, and the positive control group adopts donepezil standard substance. Each group of data was done in parallel 3 times and the average was taken.
The propidium iodide substitution was calculated according to the following formula:
replacement rate ═ 100% (1-OD sample/OD blank) ×
The results are shown in Table 2.
The result shows that the replacement capacity of the propidium iodide of the scorpion alkali A and the scorpion alkali B is similar to that of the positive control drug donepezil, and the result shows that the acetylcholinesterase inhibition of the scorpion alkali A and the scorpion alkali B has the function characteristic of strong inhibition with PAS sites.
TABLE 2 replacement of propidium iodide by scorpion A and B
Example 6
Chelation detection of metal ions by scorpion alkali A and scorpion alkali B
Step one, preparation of a sample: respectively preparing 20 mu M AlCl3，CuCl2，FeCl2，ZnCl2A methanol solution; a first scorpion alkaloid sample solution and a second scorpion alkaloid sample solution with the concentration of 20 mu M are respectively prepared.
Step two, determining the chelating capacity of the scorpion alkali A and the scorpion alkali B to the metal ions:
respectively mixing 2mL of the scorpion alkali A or scorpion alkali B sample solution with 2mL of the metal ion solution, placing the mixture in a cuvette and keeping the cuvette out of the sun for 30min, and then measuring the UV absorption spectrum with the wavelength range of 200-600nm by using an ultraviolet spectrophotometer. The results are shown in FIGS. 1 and 2.
The results show that Al is added into the sample solution of scorpion alkaloid A and scorpion alkaloid B3+、Cu2+、Fe2+And Zn2+The absorption intensity of the back UV spectrum is obviously reduced, which shows that the scorpion alkaloid A and scorpion alkaloid B have Al pair 3+、Cu2+、Fe2+And Zn2+The four metal ions have stronger chelating activity, thereby indicating that the scorpion alkaloid A and/or scorpion alkaloid B, the scorpion extract containing the scorpion alkaloid A and the scorpion alkaloid B and the composition containing the scorpion alkaloid A and/or the scorpion alkaloid B can be prepared to treat the Al3+、Cu2+、Fe2+And Zn2+Four drugs with diseases related to metal ion excess. Al (Al)3+、Cu2+、Fe2+And Zn2+The four diseases associated with excessive metal ions include Alzheimer's disease, Parkinson's disease, Huntington's disease, Prion diseases, etc.
The medical application of the composition containing the guanidine alkaloid scorpion alkaloid A and/or scorpion alkaloid B can be in the application forms suitable for oral administration or injection and the like, for example, the composition is added with pharmaceutically acceptable carriers and/or excipients to be prepared into tablets, capsules, powder, syrup, injection and the like according to the conventional technology.
The scorpion extract has pharmacological activity, so the composition containing the scorpion extract also has pharmacological activity.
The above description of the embodiments is only intended to facilitate the understanding of the method of the present invention and its central idea. It should be noted that it would be apparent to those skilled in the art that various changes and modifications can be made in the invention without departing from the principles of the invention, and such changes and modifications are intended to be covered by the appended claims.
Claims (6)
1. The method for extracting guanidine alkaloid scorpion alkaloid A and/or scorpion alkaloid B in scorpion is characterized by comprising the following steps:
(1) taking a scorpion dried body as a raw material, crushing, adding an ethanol water solution or a methanol water solution with the volume fraction of 50% -90% which is 8-10 times of the mass of the raw material, extracting for 2-3 times by reflux, extracting for 2-3 hours each time, merging to obtain an extracting solution, recovering the solvent under reduced pressure, and concentrating to obtain a total extract, namely the scorpion extract;
(2) dispersing the total extract into 5-10 times of water by mass, extracting with one of ethyl acetate, dichloromethane or chloroform, discarding the ethyl acetate, dichloromethane or chloroform extract, and concentrating the residual water layer under reduced pressure to obtain residual water layer extract;
(3) separating the residual water layer extract by silica gel column chromatography, and gradient eluting with dichloromethane-methanol at volume ratio of 20:1, 10:1 and 1:1 respectively to obtain fractions Fr.1, Fr.2, Fr.3, Fr.4 and Fr.5;
(4) separating fraction Fr.5 by silica gel column chromatography, eluting with dichloromethane-methanol at volume ratio of 10:1 at constant rate to obtain fractions Fr.5-1, Fr.5-2, Fr.5-3, Fr.5-4, Fr.5-5, and Fr.5-6;
(5) separating fraction Fr.5-5 by ODS column chromatography, and eluting with methanol-water at volume ratio of 1:9, 2:8, 4:6, 5:5 and 9:1 as eluent to obtain fractions Fr.5-5-1, Fr.5-5-2, Fr.5-5-3 and Fr.5-5-4;
(6) Separating fraction Fr.5-5-2 by ODS column chromatography, eluting with methanol-water at volume ratio of 1:4 as eluent at equal speed to obtain scorpion alkali A, i.e. N- (4-guanidine butyl) -4-hydroxybenzamide;
(7) separating fraction Fr.5-5-3 by ODS column chromatography, eluting with methanol-water at volume ratio of 1:4 as eluent at equal speed to obtain N- (4-guanidinobutyl) -2-pyridinecarboxamide.
2. The scorpion base B extracted by the method of claim 1, which has the structure of formula (II):
3. use of the scorpion alkaloid A and/or scorpion alkaloid B extracted by the method of claim 1 in the preparation of anti-Alzheimer's disease drugs, drugs for inhibiting acetylcholinesterase and butyrylcholinesterase, and metal ion chelating drugs.
4. Comprises the scorpion extract containing scorpion alkaloid A and scorpion alkaloid B extracted by the method of claim 1.
5. The scorpion extract of claim 4, which is used for preparing anti-Alzheimer disease drugs, drugs for inhibiting acetylcholinesterase and butyrylcholinesterase and metal ion chelating drugs.
6. An application of a pharmaceutical composition comprising the scorpion alkaloid A and/or scorpion alkaloid B extracted by the method in the claim 1 or pharmaceutically acceptable salts thereof and pharmaceutically acceptable carriers and/or excipients in preparing medicines for resisting Alzheimer disease, medicines for inhibiting acetylcholinesterase and butyrylcholinesterase and metal ion chelating medicines.
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