CN114848689B - Herba Lycopodii Serrati effective component, its preparation method and application in preparing medicine for preventing or treating senile dementia - Google Patents
Herba Lycopodii Serrati effective component, its preparation method and application in preparing medicine for preventing or treating senile dementia Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 22
- 208000024827 Alzheimer disease Diseases 0.000 title claims abstract description 14
- 206010039966 Senile dementia Diseases 0.000 title claims abstract description 11
- 239000003960 organic solvent Substances 0.000 claims abstract description 136
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 98
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 78
- 238000000034 method Methods 0.000 claims abstract description 14
- 239000002253 acid Substances 0.000 claims abstract description 12
- 239000000725 suspension Substances 0.000 claims abstract description 12
- 238000000605 extraction Methods 0.000 claims abstract description 10
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- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 105
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 28
- 238000002791 soaking Methods 0.000 claims description 10
- 238000001035 drying Methods 0.000 claims description 9
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims description 7
- 239000000544 cholinesterase inhibitor Substances 0.000 claims description 4
- 239000012046 mixed solvent Substances 0.000 claims description 2
- 241001090156 Huperzia serrata Species 0.000 claims 6
- 240000005790 Oroxylum indicum Species 0.000 claims 3
- 235000012920 Oroxylum indicum Nutrition 0.000 claims 3
- 229940100578 Acetylcholinesterase inhibitor Drugs 0.000 claims 1
- ZQPQGKQTIZYFEF-WCVJEAGWSA-N Huperzine Natural products C1([C@H]2[C@H](O)C(=O)N[C@H]2[C@@H](O)C=2C=CC=CC=2)=CC=CC=C1 ZQPQGKQTIZYFEF-WCVJEAGWSA-N 0.000 claims 1
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- 238000011161 development Methods 0.000 abstract description 5
- 229940079593 drug Drugs 0.000 abstract description 5
- 230000002265 prevention Effects 0.000 abstract description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 18
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 18
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 5
- 239000007853 buffer solution Substances 0.000 description 5
- 229960004373 acetylcholine Drugs 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- OIPILFWXSMYKGL-UHFFFAOYSA-N acetylcholine Chemical compound CC(=O)OCC[N+](C)(C)C OIPILFWXSMYKGL-UHFFFAOYSA-N 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
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- NTBLZMAMTZXLBP-UHFFFAOYSA-M 2-acetylsulfanylethyl(trimethyl)azanium;iodide Chemical compound [I-].CC(=O)SCC[N+](C)(C)C NTBLZMAMTZXLBP-UHFFFAOYSA-M 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 206010007247 Carbuncle Diseases 0.000 description 1
- 240000006432 Carica papaya Species 0.000 description 1
- 235000009467 Carica papaya Nutrition 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 206010017553 Furuncle Diseases 0.000 description 1
- ZRJBHWIHUMBLCN-SEQYCRGISA-N Huperzine A Natural products N1C(=O)C=CC2=C1C[C@H]1/C(=C/C)[C@]2(N)CC(C)=C1 ZRJBHWIHUMBLCN-SEQYCRGISA-N 0.000 description 1
- 244000184734 Pyrus japonica Species 0.000 description 1
- ZRJBHWIHUMBLCN-UHFFFAOYSA-N Shuangyiping Natural products N1C(=O)C=CC2=C1CC1C(=CC)C2(N)CC(C)=C1 ZRJBHWIHUMBLCN-UHFFFAOYSA-N 0.000 description 1
- 208000004078 Snake Bites Diseases 0.000 description 1
- 206010053615 Thermal burn Diseases 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- -1 acetylcholine ester Chemical class 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000001784 detoxification Methods 0.000 description 1
- 230000003937 effect on alzheimer disease Effects 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- ZRJBHWIHUMBLCN-YQEJDHNASA-N huperzine A Chemical compound N1C(=O)C=CC2=C1C[C@H]1\C(=C/C)[C@]2(N)CC(C)=C1 ZRJBHWIHUMBLCN-YQEJDHNASA-N 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 230000004770 neurodegeneration Effects 0.000 description 1
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- 230000007096 poisonous effect Effects 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- ZRJBHWIHUMBLCN-BMIGLBTASA-N rac-huperzine A Natural products N1C(=O)C=CC2=C1C[C@@H]1C(=CC)[C@@]2(N)CC(C)=C1 ZRJBHWIHUMBLCN-BMIGLBTASA-N 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000006965 reversible inhibition Effects 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 210000000225 synapse Anatomy 0.000 description 1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/11—Pteridophyta or Filicophyta (ferns)
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- 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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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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Abstract
本发明公开了一种千层塔有效部位及其制备方法与在制备具有预防或治疗老年痴呆作用的药物中的应用。所述的制备方法，包含如下步骤：(1)取中药千层塔，用乙醇提取，将提取液浓缩去除乙醇后得千层塔总浸膏；(2)将千层塔总浸膏加水混悬，然后加入酸调节整个混悬体系的pH值至2～3，静置后加入有机溶剂A进行萃取得有机溶剂A层和酸水层；(3)取酸水层，向酸水层中加入碱，调节酸水层的pH值至9～10，静置后加入有机溶剂B进行萃取得有机溶剂B层和碱水层；取有机溶剂B层浓缩干燥后得千层塔有效部位。由该方法制备得到的千层塔有效部位具有优异的乙酰胆碱酯酶抑制作用；因此，将其用于开发预防和治疗老年痴呆的药物具有广阔的应用前景。The invention discloses an effective part of melaleuca, its preparation method and its application in the preparation of medicaments capable of preventing or treating senile dementia. The preparation method comprises the following steps: (1) taking the traditional Chinese medicine Melaleuca pagoda, extracting with ethanol, concentrating the extract to remove ethanol to obtain the total extract of Melaleuca pagoda; (2) adding water to mix the total extract of Melaleuca pagoda Then add acid to adjust the pH value of the whole suspension system to 2-3, add organic solvent A after standing to extract organic solvent A layer and acid water layer; (3) take acid water layer, add to acid water layer Add alkali to adjust the pH value of the acidic water layer to 9-10. After standing still, add organic solvent B for extraction to obtain organic solvent B layer and alkaline water layer; take organic solvent B layer and concentrate and dry to obtain the effective part of the Melaleuca tower. The effective part of the melaleuca prepared by the method has an excellent inhibitory effect on acetylcholinesterase; therefore, it has broad application prospects in the development of drugs for the prevention and treatment of senile dementia.
Description
技术领域technical field
本发明涉及天然药物化学技术领域，具体涉及一种千层塔有效部位及其制备方法与在制备具有预防或治疗老年痴呆作用的药物中的应用。The present invention relates to the technical field of natural medicine chemistry, in particular to an effective part of Melaleuca japonica and its preparation method and its application in the preparation of medicaments capable of preventing or treating senile dementia.
背景技术Background technique
千层塔为石杉科石杉属植物蛇足石杉，以全草入药；其具有散瘀止血、清热解毒以及消肿止痛之功效；主治跌打损伤、瘀血肿痛、内伤吐血；外用治痈疖肿毒、毒蛇咬伤以及烧烫伤。Melaleuca pagoda is Hupermus serrata, a plant of the genus Huperzinaceae, and the whole herb is used as medicine; it has the effects of dispelling blood stasis and stopping bleeding, clearing away heat and detoxification, and reducing swelling and pain; Carbuncle furuncle swollen poison, poisonous snake bites and burns and scalds.
阿尔茨海默病(AD)是一种起病隐匿的进行性发展的神经系统退行性疾病；65岁以前发病者，称早老性痴呆；65岁以后发病者称老年性痴呆。乙酰胆碱酯酶抑制剂，又称为抗胆碱酯酶药，是通过对乙酰胆碱酯酶的可逆性抑制，达到使乙酰胆碱(ACh)在突触处的积累，延长并且增加了乙酰胆碱的作用；在临床上广泛用于抗老年性痴呆。Alzheimer's disease (AD) is a neurodegenerative disease with insidious onset and progressive development; those who develop before the age of 65 are called Alzheimer's disease; those who develop after the age of 65 are called senile dementia. Acetylcholinesterase inhibitors, also known as anticholinesterase drugs, achieve the accumulation of acetylcholine (ACh) at the synapse through the reversible inhibition of acetylcholinesterase, prolonging and increasing the effect of acetylcholine; in clinical It is widely used in anti-senile dementia.
因此，开发对乙酰胆碱酯酶具有抑制作用的药物是开发预防或治疗老年痴呆作用的药物的方向之一。现有技术从千层塔中分离得到了单体化合物石杉碱甲，研究表明，其对乙酰胆碱酯酶具有明显的抑制作用，对老年痴呆症有显著疗效。Therefore, the development of drugs that inhibit acetylcholinesterase is one of the directions for the development of drugs for the prevention or treatment of senile dementia. In the prior art, the monomer compound huperzine A was isolated from the melaleuca tower. Studies have shown that it has an obvious inhibitory effect on acetylcholinesterase and has a significant curative effect on Alzheimer's disease.
然而，千层塔中是否还有其它有效成分具有乙酰胆碱酯酶具有抑制作用，现有技术的报道较少。因此，以中药千层塔为原料，进一步开发对乙酰胆碱酯酶具有抑制作用的有效部位或单体化合物，为开发预防或治疗老年痴呆的药物提供更多的选择，具有重要的应用价值。However, there are few reports in the prior art as to whether there are other active ingredients in Melaleuca that have an inhibitory effect on acetylcholinesterase. Therefore, it is of great application value to further develop effective parts or monomeric compounds that have an inhibitory effect on acetylcholinesterase using the traditional Chinese medicine Melaleuca papaya as a raw material, so as to provide more choices for the development of drugs for the prevention or treatment of senile dementia.
发明内容Contents of the invention
为了克服现有技术中存在的至少之一的技术问题，本发明首先提供了一种千层塔有效部位的制备方法。研究表明，由本发明所述方法制备得到的千层塔有效部位具有优异的乙酰胆碱酯酶抑制作用。In order to overcome at least one of the technical problems existing in the prior art, the present invention firstly provides a method for preparing the effective part of the Melaleuca Tower. Studies have shown that the effective part of the melaleuca prepared by the method of the invention has excellent inhibitory effect on acetylcholinesterase.
本发明所要解决上述技术问题，通过以下技术方案予以实现：The above-mentioned technical problems to be solved by the present invention are realized through the following technical solutions:
一种千层塔有效部位的制备方法，其特征在于，包含如下步骤：A method for preparing an effective part of a Melaleuca tower, characterized in that it comprises the following steps:
(1)取中药千层塔，用乙醇提取，将提取液浓缩去除乙醇后得千层塔总浸膏；(1) Get the Chinese medicine Melaleuca pagoda, extract with ethanol, and obtain the total extract of Melaleuca pagoda after concentrating the extract and removing ethanol;
(2)将千层塔总浸膏加水混悬，然后加入酸调节整个混悬体系的pH值至2～3，静置后加入有机溶剂A进行萃取得有机溶剂A层和酸水层；(2) Suspend the total extract of Melaleuca pagoda with water, then add acid to adjust the pH value of the entire suspension system to 2-3, add organic solvent A after standing to extract to obtain an organic solvent A layer and an acidic water layer;
(3)取酸水层，向酸水层中加入碱，调节酸水层的pH值至9～10，静置后加入有机溶剂B进行萃取得有机溶剂B层和碱水层；取有机溶剂B层浓缩干燥后得千层塔有效部位。(3) Get the acidic water layer, add alkali in the acidic water layer, adjust the pH value of the acidic water layer to 9～10, add organic solvent B after standing and extract to obtain the organic solvent B layer and the alkaline water layer; take the organic solvent After the B layer is concentrated and dried, the effective part of the Melaleuca tower is obtained.
优选地，步骤(1)中的乙醇是指体积分数为70～95％的乙醇水溶液。Preferably, the ethanol in step (1) refers to an aqueous ethanol solution with a volume fraction of 70-95%.
优选地，步骤(1)中千层塔与乙醇的用量比为1g:8～20mL。Preferably, the amount ratio of Melaleuca tower to ethanol in step (1) is 1g:8-20mL.
最优选地，步骤(1)中千层塔与乙醇的用量比为1g:12mL。Most preferably, in step (1), the consumption ratio of Melaleuca tower and ethanol is 1g:12mL.
优选地，步骤(1)中所述的提取是指采用冷浸法提取，提取时间为3～10d。Preferably, the extraction described in step (1) refers to extraction by cold soaking, and the extraction time is 3-10 days.
最优选地，所述的提取时间为7d。Most preferably, the extraction time is 7 days.
优选地，步骤(2)中千层塔总浸膏与水的用量比为1g:15～30mL。Preferably, in step (2), the dosage ratio of the total extract of Melaleuca to water is 1g:15-30mL.
最优选地，步骤(2)中千层塔总浸膏与水的用量比为1g:20mL。Most preferably, the amount ratio of the total extract of Melaleuca to water in step (2) is 1g:20mL.
优选地，步骤(2)中有机溶剂A为二氯甲烷；步骤(3)中有机溶剂B为二氯甲烷。Preferably, the organic solvent A in the step (2) is dichloromethane; the organic solvent B in the step (3) is dichloromethane.
本发明提供了一种全新的千层塔有效部位的制备方法；发明人在大量的实验中发现，该方法步骤(2)和(3)的pH值控制以及有机溶剂A和有机溶剂B的选择是一个整体的方案，其共同对于制备得到的千层塔有效部位是否具有优异的乙酰胆碱酯酶抑制作用起着决定性作用。The present invention provides a kind of brand-new preparation method of effective part of Melaleuca tower; The inventor finds in a large number of experiments, the pH value control of this method step (2) and (3) and the selection of organic solvent A and organic solvent B It is an overall scheme, which together play a decisive role in whether the prepared effective part of Melaleuca has excellent inhibitory effect on acetylcholinesterase.
发明人在大量的研究中发现：当步骤(2)中的pH值控制在2～3，步骤(3)中的pH值控制在9～10，有机溶剂A和B同时选用二氯甲烷时制备得到的千层塔有效部位具有优异的乙酰胆碱酯酶抑制作用。若步骤(2)和(3)的pH值在其它范围，或有机溶剂A和B选用其它有机溶剂制备得到的千层塔有效部位其对乙酰胆碱酯酶抑制作用要显著小于本发明，甚至制备不到具有乙酰胆碱酯酶抑制作用的有效部位。The inventor found in a large number of studies: when the pH value in step (2) is controlled at 2 to 3, the pH value in step (3) is controlled at 9 to 10, and organic solvents A and B are prepared when dichloromethane is selected for use at the same time The obtained effective part of Melaleuca has excellent inhibitory effect on acetylcholinesterase. If the pH value of steps (2) and (3) is in other ranges, or organic solvent A and B select the effective part of the Melaleuca tower prepared by other organic solvents, its inhibitory effect on acetylcholinesterase will be significantly less than the present invention, and even the preparation cannot To the effective site with acetylcholinesterase inhibitory effect.
优选地，步骤(2)中有机溶剂A为二氯甲烷；步骤(3)中有机溶剂B为由二氯甲烷和乙醚组成的混合溶剂。Preferably, the organic solvent A in the step (2) is dichloromethane; the organic solvent B in the step (3) is a mixed solvent consisting of dichloromethane and ether.
发明人在进一步研究中惊奇的发现：当步骤(2)中的pH值控制在2～3，步骤(3)中的pH值控制在9～10，有机溶剂A选用二氯甲烷，有机溶剂B选用二氯甲烷和乙醚的组合时制备得到的千层塔有效部位具有更加优异的乙酰胆碱酯酶抑制作用；其对乙酰胆碱酯酶的抑制作用与有机溶剂B仅仅选用二氯甲烷时制备得到的千层塔有效部相比，得到了进一步地大幅提高。The contriver surprisingly finds in further research: when the pH value in step (2) is controlled at 2～3, the pH value in step (3) is controlled at 9～10, organic solvent A selects methylene chloride for use, organic solvent B The effective part of the Melaleuca tower prepared when selecting the combination of dichloromethane and ether has more excellent acetylcholinesterase inhibitory action; Compared with the effective part of the tower, it has been further greatly improved.
其中，二氯甲烷和乙醚的体积比为4～6:1；最优选地，二氯甲烷和乙醚的体积比为5:1。Wherein, the volume ratio of dichloromethane and diethyl ether is 4-6:1; most preferably, the volume ratio of dichloromethane and diethyl ether is 5:1.
优选地，步骤(2)中有机溶剂A的体积用量与水的体积用量比为1～2:1；步骤(3)中有机溶剂B的体积与酸水层的体积比为1～2:1。Preferably, the volume ratio of organic solvent A to water in step (2) is 1 to 2:1; the volume ratio of organic solvent B to acidic water layer in step (3) is 1 to 2:1 .
优选地，步骤(2)中的酸为浓盐酸；步骤(3)中的碱为氨水。Preferably, the acid in step (2) is concentrated hydrochloric acid; the base in step (3) is ammonia water.
本发明还提供了一种由上述制备方法制备得到的千层塔有效部位。The present invention also provides an effective part of the Melaleuca tower prepared by the above preparation method.
本发明还提供了一种上述千层塔有效部位在制备乙酰胆碱酯酶抑制剂中的应用。The present invention also provides an application of the above-mentioned effective fraction of Melaleuca in the preparation of acetylcholinesterase inhibitors.
本发明还提供了一种上述千层塔有效部位在制备具有预防或治疗老年痴呆作用的药物中的应用。The present invention also provides an application of the above-mentioned effective part of Melaleuca in the preparation of a medicament for preventing or treating senile dementia.
有益效果：本发明提供了一种全新的千层塔有效部位的制备方法，由该方法制备得到的千层塔有效部位具有优异的乙酰胆碱酯酶抑制作用。因此，将该千层塔有效部位用于开发预防和治疗老年痴呆的药物具有广阔的应用前景。Beneficial effects: the present invention provides a new method for preparing the effective part of Melaleuca, and the effective part of Melaleuca prepared by the method has excellent inhibitory effect on acetylcholinesterase. Therefore, it has a broad application prospect to use the effective part of Melaleuca to develop drugs for preventing and treating senile dementia.
具体实施方式Detailed ways
以下结合具体实施例来进一步解释本发明，但实施例对本发明并不做任何形式的限定。The present invention is further explained below in conjunction with specific examples, but the examples do not limit the present invention in any form.
实施例1千层塔有效部位的制备The preparation of embodiment 1 Melaleuca tower effective part
(1)取中药千层塔，用乙醇(采用体积分数为95％的乙醇水溶液)采用冷浸法在室温下提取7d，将提取液浓缩去除乙醇后得千层塔总浸膏；(1) Take the traditional Chinese medicine Melaleuca Pagoda, use ethanol (adopting a 95% ethanol water solution by volume fraction) to extract at room temperature for 7 days by cold soaking, and concentrate the extract to remove ethanol to obtain the total extract of the Melaleuca Pagoda;
其中，千层塔与乙醇的用量比为1g:12mL；Wherein, the consumption ratio of Melaleuca tower and ethanol is 1g:12mL;
(2)将千层塔总浸膏加水混悬，然后加入浓盐酸调节整个混悬体系的pH值至2.5，静置10h后加入有机溶剂A进行萃取得有机溶剂A层和酸水层；(2) Suspend the total extract of Melaleuca pagoda with water, then add concentrated hydrochloric acid to adjust the pH value of the entire suspension system to 2.5, add organic solvent A after standing for 10 hours to extract to obtain an organic solvent A layer and an acidic water layer;
其中，千层塔总浸膏与水的用量比为1g:20mL；有机溶剂A的体积用量与水的体积用量比为2:1；所述的有机溶剂A选用二氯甲烷；Wherein, the consumption ratio of Melaleuca total extract and water is 1g:20mL; The volume consumption ratio of organic solvent A and water is 2:1; Described organic solvent A selects methylene chloride for use;
(3)取酸水层，向酸水层中加入氨水，调节酸水层的pH值至9.5，静置后加入有机溶剂B进行萃取得有机溶剂B层和碱水层；取有机溶剂B层浓缩干燥后得千层塔有效部位；(3) Get the acidic water layer, add ammoniacal liquor in the acidic water layer, adjust the pH value of the acidic water layer to 9.5, add organic solvent B after standing and extract to obtain the organic solvent B layer and the alkaline water layer; take the organic solvent B layer After concentration and drying, the effective part of Melaleuca tower is obtained;
其中，有机溶剂B的体积与酸水层的体积比为2:1；所述的有机溶剂B选用二氯甲烷。Wherein, the volume ratio of the volume of the organic solvent B to the acidic water layer is 2:1; the organic solvent B is dichloromethane.
实施例2千层塔有效部位的制备The preparation of the effective part of embodiment 2 Melaleuca tower
(1)取中药千层塔，用乙醇(采用体积分数为70％的乙醇水溶液)采用冷浸法在室温下提取10d，将提取液浓缩去除乙醇后得千层塔总浸膏；(1) Take the traditional Chinese medicine Melaleuca Pagoda, use ethanol (using a 70% ethanol aqueous solution by volume fraction) to extract at room temperature for 10 days by cold soaking, and concentrate the extract to remove ethanol to obtain the total extract of the Melaleuca Pagoda;
其中，千层塔与乙醇的用量比为1g:20mL；Wherein, the consumption ratio of Melaleuca tower and ethanol is 1g:20mL;
(2)将千层塔总浸膏加水混悬，然后加入浓盐酸调节整个混悬体系的pH值至2，静置10h后加入有机溶剂A进行萃取得有机溶剂A层和酸水层；(2) Suspend the total extract of Melaleuca pagoda with water, then add concentrated hydrochloric acid to adjust the pH value of the entire suspension system to 2, add organic solvent A after standing for 10 hours to extract to obtain an organic solvent A layer and an acidic water layer;
其中，千层塔总浸膏与水的用量比为1g:15mL；有机溶剂A的体积用量与水的体积用量比为2:1；所述的有机溶剂A选用二氯甲烷；Wherein, the consumption ratio of the total extract of Melaleuca pagoda and water is 1g:15mL; the volume consumption ratio of organic solvent A and water is 2:1; described organic solvent A selects dichloromethane for use;
(3)取酸水层，向酸水层中加入氨水，调节酸水层的pH值至9，静置后加入有机溶剂B进行萃取得有机溶剂B层和碱水层；取有机溶剂B层浓缩干燥后得千层塔有效部位；(3) Get the acidic water layer, add ammoniacal liquor in the acidic water layer, adjust the pH value of the acidic water layer to 9, add organic solvent B after standing and extract to obtain the organic solvent B layer and the alkaline water layer; take the organic solvent B layer After concentration and drying, the effective part of Melaleuca tower is obtained;
其中，有机溶剂B的体积与酸水层的体积比为2:1；所述的有机溶剂B选用二氯甲烷。Wherein, the volume ratio of the volume of the organic solvent B to the acidic water layer is 2:1; the organic solvent B is dichloromethane.
实施例3千层塔有效部位的制备The preparation of the effective part of embodiment 3 Melaleuca tower
(1)取中药千层塔，用乙醇(采用体积分数为80％的乙醇水溶液)采用冷浸法在室温下提取5d，将提取液浓缩去除乙醇后得千层塔总浸膏；(1) Take the Chinese medicine Melaleuca Pagoda, use ethanol (adopting a volume fraction of 80% ethanol aqueous solution) to extract at room temperature for 5 days by cold soaking, and concentrate the extract to remove ethanol to obtain the total extract of the Melaleuca Pagoda;
其中，千层塔与乙醇的用量比为1g:8mL；Wherein, the consumption ratio of Melaleuca tower and ethanol is 1g:8mL;
(2)将千层塔总浸膏加水混悬，然后加入浓盐酸调节整个混悬体系的pH值至3，静置10h后加入有机溶剂A进行萃取得有机溶剂A层和酸水层；(2) Suspend the total extract of Melaleuca pagoda with water, then add concentrated hydrochloric acid to adjust the pH value of the entire suspension system to 3, add organic solvent A after standing for 10 hours to extract to obtain an organic solvent A layer and an acidic water layer;
其中，千层塔总浸膏与水的用量比为1g:30mL；有机溶剂A的体积用量与水的体积用量比为1:1；所述的有机溶剂A选用二氯甲烷；Wherein, the consumption ratio of the total extract of Melaleuca pagoda and water is 1g:30mL; the volume consumption ratio of organic solvent A and water is 1:1; described organic solvent A selects dichloromethane for use;
(3)取酸水层，向酸水层中加入氨水，调节酸水层的pH值至10，静置后加入有机溶剂B进行萃取得有机溶剂B层和碱水层；取有机溶剂B层浓缩干燥后得千层塔有效部位；(3) Get the acidic water layer, add ammoniacal liquor in the acidic water layer, adjust the pH value of the acidic water layer to 10, add organic solvent B after standing and extract to obtain the organic solvent B layer and the alkaline water layer; take the organic solvent B layer After concentration and drying, the effective part of Melaleuca tower is obtained;
其中，有机溶剂B的体积与酸水层的体积比为1:1；所述的有机溶剂B选用二氯甲烷。Wherein, the volume ratio of the volume of the organic solvent B to the acidic water layer is 1:1; the organic solvent B is dichloromethane.
实施例4千层塔有效部位的制备The preparation of the effective part of embodiment 4 Melaleuca tower
(1)取中药千层塔，用乙醇(采用体积分数为95％的乙醇水溶液)采用冷浸法在室温下提取7d，将提取液浓缩去除乙醇后得千层塔总浸膏；(1) Take the traditional Chinese medicine Melaleuca Pagoda, use ethanol (adopting a 95% ethanol water solution by volume fraction) to extract at room temperature for 7 days by cold soaking, and concentrate the extract to remove ethanol to obtain the total extract of the Melaleuca Pagoda;
其中，千层塔与乙醇的用量比为1g:12mL；Wherein, the consumption ratio of Melaleuca tower and ethanol is 1g:12mL;
(2)将千层塔总浸膏加水混悬，然后加入浓盐酸调节整个混悬体系的pH值至2.5，静置10h后加入有机溶剂A进行萃取得有机溶剂A层和酸水层；(2) Suspend the total extract of Melaleuca pagoda with water, then add concentrated hydrochloric acid to adjust the pH value of the entire suspension system to 2.5, add organic solvent A after standing for 10 hours to extract to obtain an organic solvent A layer and an acidic water layer;
其中，千层塔总浸膏与水的用量比为1g:20mL；有机溶剂A的体积用量与水的体积用量比为2:1；所述的有机溶剂A选用二氯甲烷；Wherein, the consumption ratio of Melaleuca total extract and water is 1g:20mL; The volume consumption ratio of organic solvent A and water is 2:1; Described organic solvent A selects methylene chloride for use;
(3)取酸水层，向酸水层中加入氨水，调节酸水层的pH值至9.5，静置后加入有机溶剂B进行萃取得有机溶剂B层和碱水层；取有机溶剂B层浓缩干燥后得千层塔有效部位；(3) Get the acidic water layer, add ammoniacal liquor in the acidic water layer, adjust the pH value of the acidic water layer to 9.5, add organic solvent B after standing and extract to obtain the organic solvent B layer and the alkaline water layer; take the organic solvent B layer After concentration and drying, the effective part of Melaleuca tower is obtained;
其中，有机溶剂B的体积与酸水层的体积比为2:1；所述的有机溶剂B选用由体积比为5:1的二氯甲烷和乙醚组成的混合有机溶剂。Wherein, the volume ratio of the volume of the organic solvent B to the acidic water layer is 2:1; the organic solvent B is a mixed organic solvent composed of dichloromethane and ether with a volume ratio of 5:1.
对比例1千层塔有效部位的制备The preparation of the effective part of comparative example 1 Melaleuca tower
(1)取中药千层塔，用乙醇(采用体积分数为95％的乙醇水溶液)采用冷浸法在室温下提取7d，将提取液浓缩去除乙醇后得千层塔总浸膏；(1) Take the traditional Chinese medicine Melaleuca Pagoda, use ethanol (adopting a 95% ethanol water solution by volume fraction) to extract at room temperature for 7 days by cold soaking, and concentrate the extract to remove ethanol to obtain the total extract of the Melaleuca Pagoda;
其中，千层塔与乙醇的用量比为1g:12mL；Wherein, the consumption ratio of Melaleuca tower and ethanol is 1g:12mL;
(2)将千层塔总浸膏加水混悬，然后加入浓盐酸调节整个混悬体系的pH值至4，静置10h后加入有机溶剂A进行萃取得有机溶剂A层和酸水层；(2) Suspend the total extract of Melaleuca pagoda with water, then add concentrated hydrochloric acid to adjust the pH value of the entire suspension system to 4, add organic solvent A after standing for 10 hours to extract to obtain an organic solvent A layer and an acidic water layer;
其中，千层塔总浸膏与水的用量比为1g:20mL；有机溶剂A的体积用量与水的体积用量比为2:1；所述的有机溶剂A选用氯仿；Wherein, the consumption ratio of the total extract of Melaleuca pagoda and water is 1g:20mL; the volume consumption ratio of organic solvent A and water is 2:1; described organic solvent A selects chloroform for use;
(3)取酸水层，向酸水层中加入氨水，调节酸水层的pH值至11，静置后加入有机溶剂B进行萃取得有机溶剂B层和碱水层；取有机溶剂B层浓缩干燥后得千层塔有效部位；(3) Get the acidic water layer, add ammoniacal liquor in the acidic water layer, adjust the pH value of the acidic water layer to 11, add organic solvent B after standing and extract to obtain the organic solvent B layer and the alkaline water layer; take the organic solvent B layer After concentration and drying, the effective part of Melaleuca tower is obtained;
其中，有机溶剂B的体积与酸水层的体积比为2:1；所述的有机溶剂B选用氯仿。Wherein, the volume ratio of the volume of organic solvent B to the acidic water layer is 2:1; described organic solvent B selects chloroform for use.
对比例1与实施例1的区别在于，步骤(2)和(3)中的pH值不同，以及有机溶剂A和B不同；对比例1步骤(2)中的pH值控制在4，步骤(3)中的pH值控制在11，有机溶剂A和B同时选用氯仿；而实施例1步骤(2)中的pH值控制在2.5，步骤(3)中的pH值控制在9.5，有机溶剂A和B同时选用二氯甲烷。The difference between Comparative Example 1 and Example 1 is that the pH value in steps (2) and (3) is different, and the organic solvent A and B are different; the pH value in Comparative Example 1 step (2) is controlled at 4, and the step ( 3) The pH value in the middle is controlled at 11, and organic solvent A and B select chloroform for use simultaneously; And the pH value in embodiment 1 step (2) is controlled at 2.5, and the pH value in step (3) is controlled at 9.5, and organic solvent A And B selects dichloromethane simultaneously.
对比例2千层塔有效部位的制备Preparation of the effective part of comparative example 2 Melaleuca tower
(1)取中药千层塔，用乙醇(采用体积分数为95％的乙醇水溶液)采用冷浸法在室温下提取7d，将提取液浓缩去除乙醇后得千层塔总浸膏；(1) Take the traditional Chinese medicine Melaleuca Pagoda, use ethanol (adopting a 95% ethanol water solution by volume fraction) to extract at room temperature for 7 days by cold soaking, and concentrate the extract to remove ethanol to obtain the total extract of the Melaleuca Pagoda;
其中，千层塔与乙醇的用量比为1g:12mL；Wherein, the consumption ratio of Melaleuca tower and ethanol is 1g:12mL;
(2)将千层塔总浸膏加水混悬，然后加入浓盐酸调节整个混悬体系的pH值至4，静置10h后加入有机溶剂A进行萃取得有机溶剂A层和酸水层；(2) Suspend the total extract of Melaleuca pagoda with water, then add concentrated hydrochloric acid to adjust the pH value of the entire suspension system to 4, add organic solvent A after standing for 10 hours to extract to obtain an organic solvent A layer and an acidic water layer;
其中，千层塔总浸膏与水的用量比为1g:20mL；有机溶剂A的体积用量与水的体积用量比为2:1；所述的有机溶剂A选用乙酸乙酯；Wherein, the consumption ratio of the total extract of Melaleuca pagoda and water is 1g:20mL; the volume consumption ratio of organic solvent A and water is 2:1; described organic solvent A selects ethyl acetate for use;
(3)取酸水层，向酸水层中加入氨水，调节酸水层的pH值至11，静置后加入有机溶剂B进行萃取得有机溶剂B层和碱水层；取有机溶剂B层浓缩干燥后得千层塔有效部位；(3) Get the acidic water layer, add ammoniacal liquor in the acidic water layer, adjust the pH value of the acidic water layer to 11, add organic solvent B after standing and extract to obtain the organic solvent B layer and the alkaline water layer; take the organic solvent B layer After concentration and drying, the effective part of Melaleuca tower is obtained;
其中，有机溶剂B的体积与酸水层的体积比为2:1；所述的有机溶剂B选用乙酸乙酯。Wherein, the volume ratio of the volume of the organic solvent B to the acidic water layer is 2:1; the organic solvent B is ethyl acetate.
对比例1与实施例1的区别在于，步骤(2)和(3)中的pH值不同，以及有机溶剂A和B不同；对比例1步骤(2)中的pH值控制在4，步骤(3)中的pH值控制在11，有机溶剂A和B同时选用乙酸乙酯；而实施例1步骤(2)中的pH值控制在2.5，步骤(3)中的pH值控制在9.5，有机溶剂A和B同时选用乙酸乙酯。The difference between Comparative Example 1 and Example 1 is that the pH value in steps (2) and (3) is different, and the organic solvent A and B are different; the pH value in Comparative Example 1 step (2) is controlled at 4, and the step ( 3) The pH value in the middle is controlled at 11, and organic solvent A and B select ethyl acetate for use simultaneously; And the pH value in the embodiment 1 step (2) is controlled at 2.5, and the pH value in the step (3) is controlled at 9.5, organic Solvent A and B select ethyl acetate simultaneously.
对比例3千层塔有效部位的制备Preparation of the effective part of comparative example 3 Melaleuca tower
(1)取中药千层塔，用乙醇(采用体积分数为95％的乙醇水溶液)采用冷浸法在室温下提取7d，将提取液浓缩去除乙醇后得千层塔总浸膏；(1) Take the traditional Chinese medicine Melaleuca Pagoda, use ethanol (adopting a 95% ethanol water solution by volume fraction) to extract at room temperature for 7 days by cold soaking, and concentrate the extract to remove ethanol to obtain the total extract of the Melaleuca Pagoda;
其中，千层塔与乙醇的用量比为1g:12mL；Wherein, the consumption ratio of Melaleuca tower and ethanol is 1g:12mL;
(2)将千层塔总浸膏加水混悬，然后加入浓盐酸调节整个混悬体系的pH值至2.5，静置10h后加入有机溶剂A进行萃取得有机溶剂A层和酸水层；(2) Suspend the total extract of Melaleuca pagoda with water, then add concentrated hydrochloric acid to adjust the pH value of the entire suspension system to 2.5, add organic solvent A after standing for 10 hours to extract to obtain an organic solvent A layer and an acidic water layer;
其中，千层塔总浸膏与水的用量比为1g:20mL；有机溶剂A的体积用量与水的体积用量比为2:1；所述的有机溶剂A选用二氯甲烷；Wherein, the consumption ratio of Melaleuca total extract and water is 1g:20mL; The volume consumption ratio of organic solvent A and water is 2:1; Described organic solvent A selects methylene chloride for use;
(3)取酸水层，向酸水层中加入氨水，调节酸水层的pH值至9.5，静置后加入有机溶剂B进行萃取得有机溶剂B层和碱水层；取有机溶剂B层浓缩干燥后得千层塔有效部位；(3) Get the acidic water layer, add ammoniacal liquor in the acidic water layer, adjust the pH value of the acidic water layer to 9.5, add organic solvent B after standing and extract to obtain the organic solvent B layer and the alkaline water layer; take the organic solvent B layer After concentration and drying, the effective part of Melaleuca tower is obtained;
其中，有机溶剂B的体积与酸水层的体积比为2:1；所述的有机溶剂B选用由体积比为5:1的二氯甲烷和乙酸乙酯组成的混合有机溶剂。Wherein, the volume ratio of the volume of the organic solvent B to the acidic water layer is 2:1; the organic solvent B is a mixed organic solvent composed of dichloromethane and ethyl acetate with a volume ratio of 5:1.
对比例3与实施例4的区别在于，有机溶剂B的选用不同，对比例3中有机溶剂B选用的是由二氯甲烷和乙酸乙酯组成的混合有机溶剂；而实施例4中有机溶剂B选用的是由二氯甲烷和乙醚组成的混合有机溶剂。The difference between comparative example 3 and embodiment 4 is that the selection of organic solvent B is different, and what organic solvent B selects in comparative example 3 is the mixed organic solvent that is made up of dichloromethane and ethyl acetate; And in embodiment 4, organic solvent B What is selected is a mixed organic solvent composed of dichloromethane and ether.
对比例4千层塔有效部位的制备The preparation of the effective part of comparative example 4 Melaleuca tower
(1)取中药千层塔，用乙醇(采用体积分数为95％的乙醇水溶液)采用冷浸法在室温下提取7d，将提取液浓缩去除乙醇后得千层塔总浸膏；(1) Take the traditional Chinese medicine Melaleuca Pagoda, use ethanol (adopting a 95% ethanol water solution by volume fraction) to extract at room temperature for 7 days by cold soaking, and concentrate the extract to remove ethanol to obtain the total extract of the Melaleuca Pagoda;
其中，千层塔与乙醇的用量比为1g:12mL；Wherein, the consumption ratio of Melaleuca tower and ethanol is 1g:12mL;
(2)将千层塔总浸膏加水混悬，然后加入浓盐酸调节整个混悬体系的pH值至2.5，静置10h后加入有机溶剂A进行萃取得有机溶剂A层和酸水层；(2) Suspend the total extract of Melaleuca pagoda with water, then add concentrated hydrochloric acid to adjust the pH value of the entire suspension system to 2.5, add organic solvent A after standing for 10 hours to extract to obtain an organic solvent A layer and an acidic water layer;
其中，千层塔总浸膏与水的用量比为1g:20mL；有机溶剂A的体积用量与水的体积用量比为2:1；所述的有机溶剂A选用二氯甲烷；Wherein, the consumption ratio of Melaleuca total extract and water is 1g:20mL; The volume consumption ratio of organic solvent A and water is 2:1; Described organic solvent A selects methylene chloride for use;
(3)取酸水层，向酸水层中加入氨水，调节酸水层的pH值至9.5，静置后加入有机溶剂B进行萃取得有机溶剂B层和碱水层；取有机溶剂B层浓缩干燥后得千层塔有效部位；(3) Get the acidic water layer, add ammoniacal liquor in the acidic water layer, adjust the pH value of the acidic water layer to 9.5, add organic solvent B after standing and extract to obtain the organic solvent B layer and the alkaline water layer; take the organic solvent B layer After concentration and drying, the effective part of Melaleuca tower is obtained;
其中，有机溶剂B的体积与酸水层的体积比为2:1；所述的有机溶剂B选用由体积比为5:1的二氯甲烷和环己烷组成的混合有机溶剂。Wherein, the volume ratio of the volume of the organic solvent B to the acidic water layer is 2:1; the organic solvent B is a mixed organic solvent composed of dichloromethane and cyclohexane with a volume ratio of 5:1.
对比例4与实施例4的区别在于，有机溶剂B的选用不同，对比例4中有机溶剂B选用的是由二氯甲烷和环己烷组成的混合有机溶剂；而实施例4中有机溶剂B选用的是由二氯甲烷和乙醚组成的混合有机溶剂。The difference between comparative example 4 and embodiment 4 is that the selection of organic solvent B is different, and what organic solvent B selects in comparative example 4 is the mixed organic solvent that is made up of dichloromethane and hexanaphthene; And in embodiment 4, organic solvent B What is selected is a mixed organic solvent composed of dichloromethane and ether.
实验例Experimental example
将实施例1～4以及对比例1～4制备得到的千层塔有效部位用DMSO溶解，制成2mg/mL的待测溶液；用于测试实施例1～4以及对比例1～4制备得到的千层塔有效部位对乙酰胆碱酯酶抑制率。Dissolve the effective parts of Melaleuca Tower prepared in Examples 1-4 and Comparative Examples 1-4 with DMSO to make a 2 mg/mL solution to be tested; used for testing Examples 1-4 and Comparative Examples 1-4 to obtain The inhibitory rate of the effective part of Melaleuca to acetylcholinesterase.
取96孔板，每孔分别加入待测溶液10μL、pH值为8.0的Tris-HCl缓冲溶液40μL以及乙酰胆碱酯酶溶液10μL(用pH值为8.0的Tris-HCl缓冲溶液配制，浓度为1000U/mL)；空白对照组用等量的pH值为8.0的Tris-HCl缓冲溶液替代待测溶液；然后于37℃下在摇床中孵育10min；接着加入显色剂DTNB溶液20μL(用pH值为8.0的Tris-HCl缓冲溶液配制，浓度为0.1mM)以及底物碘化乙酰硫代胆碱溶液10μL(用pH值为8.0的Tris-HCl缓冲溶液配制，浓度为0.2mM)，接着于37℃下在摇床中继续孵育10min；孵育结束后，用酶标仪测定412nm下的吸光度；计算待测溶液对于乙酰胆碱酯酶的抑制率；测试结果见表1。Take a 96-well plate, and add 10 μL of the solution to be tested, 40 μL of Tris-HCl buffer solution with a pH value of 8.0, and 10 μL of acetylcholinesterase solution (prepared with a Tris-HCl buffer solution with a pH value of 8.0, with a concentration of 1000 U/mL) in each well. ); the blank control group replaced the solution to be tested with an equivalent amount of Tris-HCl buffer solution with a pH value of 8.0; then incubated in a shaker at 37°C for 10 min; Tris-HCl buffer solution prepared with a concentration of 0.1mM) and 10 μL of the substrate acetylthiocholine iodide solution (prepared with a Tris-HCl buffer solution with a pH value of 8.0 and a concentration of 0.2mM), then at 37°C Continue to incubate in a shaker for 10 min; after the incubation, measure the absorbance at 412 nm with a microplate reader; calculate the inhibition rate of the solution to be tested for acetylcholinesterase; the test results are shown in Table 1.
表1.本发明千层塔有效部位对乙酰胆碱酯酶的抑制作用Table 1. The inhibitory effect of the effective part of Melaleuca tower of the present invention on acetylcholinesterase
从表1实验数据可以看出，实施例1～3制备得到的千层塔有效部位其在2mg/mL浓度时对乙酰胆碱酯酶抑制率达到了60％以上。这说明：采用本发明方法制备得到的千层塔有效部位具有优异的乙酰胆碱酯酶抑制作用。As can be seen from the experimental data in Table 1, the effective fraction of Melaleuca prepared in Examples 1 to 3 has an inhibitory rate of more than 60% to acetylcholinesterase at a concentration of 2 mg/mL. This shows that: the effective part of the Melaleuca prepared by the method of the present invention has excellent inhibitory effect on acetylcholinesterase.
从表1实验数据可以看出，实施例1制备得到的千层塔有效部位，其对乙酰胆碱酯酶抑制率远远高于对比例1和2制备得到的千层塔有效部位。而对比1和2与实施例1的区别在于，步骤(2)和(3)的pH值不同以及有机溶剂A和B不同；这说明：该方法步骤(2)和(3)的pH值控制以及有机溶剂A和有机溶剂B的选择是一个整体的方案，其共同对于制备得到的千层塔有效部位是否具有优异的乙酰胆碱酯酶抑制作用起着决定性作用；上述研究表明：只有当步骤(2)中的pH值控制在2～3，步骤(3)中的pH值控制在9～10，有机溶剂A和B同时选用二氯甲烷时制备得到的千层塔有效部位才具有优异的乙酰胆碱酯酶抑制作用；步骤(2)和(3)在其它pH值条件下以及有机溶剂A和B采用其它有机溶剂时制备得到的千层塔有效部位并不具有优异的乙酰胆碱酯酶抑制作用。As can be seen from the experimental data in Table 1, the effective part of the Melaleuca prepared in Example 1 has a much higher inhibitory rate to acetylcholinesterase than the effective part of the Melaleuca prepared in Comparative Examples 1 and 2. And the difference between contrast 1 and 2 and embodiment 1 is that the pH value of step (2) and (3) is different and organic solvent A and B are different; This illustrates: the pH value control of this method step (2) and (3) And the selection of organic solvent A and organic solvent B is an overall scheme, and whether it has excellent acetylcholinesterase inhibitory effect jointly for the effective part of the prepared Melaleuca tower; Above-mentioned research shows: only when step (2 ) in the pH value is controlled at 2～3, and the pH value in the step (3) is controlled at 9～10, and organic solvent A and B select the effective part of the Melaleuca tower that prepares to have excellent acetylcholine ester when selecting dichloromethane simultaneously Enzyme inhibition; steps (2) and (3) under other pH conditions and organic solvents A and B when other organic solvents are used to prepare the effective part of the Melaleuca tower does not have excellent acetylcholinesterase inhibition.
从表1实验数据可以看出，实施例4制备得到的千层塔有效部位其在2mg/mL浓度时对乙酰胆碱酯酶抑制率达到了91.8％；与实施例1制备得到的千层塔有效部位得到了进一步大幅地提高，具有十分优异的乙酰胆碱酯酶抑制作用。这说明：当步骤(2)中的pH值控制在2～3，步骤(3)中的pH值控制在9～10，有机溶剂A选用二氯甲烷，有机溶剂B选用二氯甲烷和乙醚的组合时制备得到的千层塔有效部位具有更加优异的乙酰胆碱酯酶抑制作用；其对乙酰胆碱酯酶抑制作用与有机溶剂B仅仅选用二氯甲烷时制备得到的千层塔有效部相比，得到了进一步地大幅提高。As can be seen from the experimental data in Table 1, the effective part of the Melaleuca prepared in Example 4 reached 91.8% at the time of 2 mg/mL concentration; the effective part of the Melaleuca prepared in Example 1 It has been further greatly improved, and has a very excellent inhibitory effect on acetylcholinesterase. This explanation: when the pH value in the step (2) is controlled at 2～3, the pH value in the step (3) is controlled at 9～10, organic solvent A selects dichloromethane for use, and organic solvent B selects dichloromethane and ether for use The effective part of the Melaleuca tower prepared during combination has more excellent acetylcholinesterase inhibitory effect; Compared with the effective part of the Melaleuca tower prepared when the organic solvent B only selects dichloromethane to the inhibitory effect of acetylcholinesterase, obtained significantly improved further.
从表1实验数据可以看出，对比例3和4制备得到的千层塔有效部位，其与实施例1相比，其对乙酰胆碱酯酶抑制作用并未得到明显的提高，甚至还有所降低；这说明：有机溶剂B必须选用由二氯甲烷和乙醚组成的组合时才能进一步大幅提高制备得到的千层塔有效部位对乙酰胆碱酯酶的抑制作用，才能使得制备得的千层塔有效部位具有更加优异的乙酰胆碱酯酶抑制作用；而有机溶剂B选用其它有机溶剂的组合，并不能提高或显著提高制备得到的千层塔有效部位对乙酰胆碱酯酶的抑制作用。As can be seen from the experimental data in Table 1, the effective parts of Melaleuca prepared in Comparative Examples 3 and 4, compared with Example 1, its inhibitory effect on acetylcholinesterase has not been significantly improved, or even decreased ; This explanation: organic solvent B must select the combination that can further greatly improve the inhibitory action of the prepared Melaleuca effective part to acetylcholinesterase when being selected from dichloromethane and ether, could make the prepared Melaleuca effective part have More excellent inhibitory effect on acetylcholinesterase; while organic solvent B selects the combination of other organic solvents, and can not improve or significantly improve the inhibitory effect on acetylcholinesterase of the effective part of the prepared Melaleuca tower.
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