阅读说明：本技术 全蝎中胍类生物碱钳蝎碱甲和/或钳蝎碱乙的提取方法及医药用途 (Method for extracting guanidine alkaloid of scorpion and scorpion alkaloid A and/or scorpion and scorpion alkaloid B in scorpion and medical application ) 是由 刘玉明 范晶婧 艾双艳 冯亚东 于 2019-12-05 设计创作，主要内容包括：本发明公开了全蝎中胍类生物碱钳蝎碱甲和/或钳蝎碱乙的提取方法及医药用途,钳蝎碱甲的化学结构式为：<Image he="194" wi="700" file="DDA0002304066470000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>钳蝎碱乙的化学结构式为：<Image he="207" wi="700" file="DDA0002304066470000012.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>本发明的钳蝎碱甲和钳蝎碱乙能有效地抑制乙酰胆碱酯酶和丁酰胆碱酯酶的活性,并以与乙酰胆碱酯酶的PAS位点强效结合为作用特征,同时钳蝎碱甲和钳蝎碱乙具有金属离子螯合能力,本发明全蝎提取物可作为治疗阿尔茨海默病等神经退行性疾病的药物。(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 shown in the specification： 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.)

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 alkaloid A extracted by the method of claim 1, has the structure of formula (I):

3. the scorpion base B extracted by the method of claim 1, which has the structure of formula (II):

4. 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.

5. Comprises the scorpion extract containing scorpion alkaloid A and scorpion alkaloid B extracted by the method of claim 1.

6. The scorpion extract of claim 5, is applied to the preparation of anti-Alzheimer's disease drugs, drugs for inhibiting acetylcholinesterase and butyrylcholinesterase and metal ion chelating drugs.

7. A pharmaceutical composition, which comprises the scorpion alkaloid A and/or scorpion alkaloid B extracted by the method of claim 1 or pharmaceutically acceptable salt thereof, and pharmaceutically acceptable carriers and/or excipients.

8. The use of the pharmaceutical composition of claim 7 for the preparation of an anti-alzheimer's disease medicament, a medicament for inhibiting acetylcholinesterase and butyrylcholinesterase, and a metal ion chelating medicament.

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.

However, recent studies show that the Peripheral Active Site (PAS) of the acetylcholinesterase inhibitor has a close relationship with the hydrolysis of acetylcholine and can inhibit β amyloid aggregation.

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⁺Thus, reduction of the concentration of ions in the brain by metal ion chelation has also become one of the important strategies for the treatment of AD and also for the treatment of other neurodegenerative diseases (see Rapeng, et al. transition metal ions and neurodegenerative diseases university Chemistry, 2006,21 (6): 32-35; and Cristina rodi guez-rodi guez, et al. the art of building of architectural multiple functional-binding ligands from basic molecules research, 2012,256: 2308-.

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