CN114869920B - Huperzia serrata extract with acetylcholinesterase inhibition effect, and preparation method and application thereof - Google Patents
Huperzia serrata extract with acetylcholinesterase inhibition effect, and preparation method and application thereof Download PDFInfo
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- 241001090156 Huperzia serrata Species 0.000 title claims abstract description 110
- 239000000284 extract Substances 0.000 title claims abstract description 98
- 102000012440 Acetylcholinesterase Human genes 0.000 title claims abstract description 45
- 108010022752 Acetylcholinesterase Proteins 0.000 title claims abstract description 45
- 229940022698 acetylcholinesterase Drugs 0.000 title claims abstract description 45
- 230000005764 inhibitory process Effects 0.000 title claims abstract description 41
- 230000000694 effects Effects 0.000 title claims abstract description 32
- 238000002360 preparation method Methods 0.000 title claims abstract description 21
- 239000003960 organic solvent Substances 0.000 claims abstract description 100
- 239000011347 resin Substances 0.000 claims abstract description 60
- 229920005989 resin Polymers 0.000 claims abstract description 60
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 52
- 239000000741 silica gel Substances 0.000 claims abstract description 52
- 229910002027 silica gel Inorganic materials 0.000 claims abstract description 52
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims abstract description 42
- 238000011068 loading method Methods 0.000 claims abstract description 24
- 238000001035 drying Methods 0.000 claims abstract description 23
- 239000012535 impurity Substances 0.000 claims abstract description 22
- 238000010828 elution Methods 0.000 claims abstract description 21
- 239000004480 active ingredient Substances 0.000 claims abstract description 13
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 114
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 76
- 239000000243 solution Substances 0.000 claims description 55
- 238000000034 method Methods 0.000 claims description 16
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 12
- 239000007864 aqueous solution Substances 0.000 claims description 5
- 239000003480 eluent Substances 0.000 claims description 5
- 208000024827 Alzheimer disease Diseases 0.000 abstract description 11
- 206010039966 Senile dementia Diseases 0.000 abstract description 8
- 239000003814 drug Substances 0.000 abstract description 7
- 230000000052 comparative effect Effects 0.000 description 21
- 238000002386 leaching Methods 0.000 description 8
- 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
- 238000000605 extraction Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 206010042674 Swelling Diseases 0.000 description 3
- OIPILFWXSMYKGL-UHFFFAOYSA-N acetylcholine Chemical compound CC(=O)OCC[N+](C)(C)C OIPILFWXSMYKGL-UHFFFAOYSA-N 0.000 description 3
- 229960004373 acetylcholine Drugs 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 239000000544 cholinesterase inhibitor Substances 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 230000008961 swelling Effects 0.000 description 3
- 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 2
- 208000002193 Pain Diseases 0.000 description 2
- ZRJBHWIHUMBLCN-UHFFFAOYSA-N Shuangyiping Natural products N1C(=O)C=CC2=C1CC1C(=CC)C2(N)CC(C)=C1 ZRJBHWIHUMBLCN-UHFFFAOYSA-N 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 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 2
- 238000011534 incubation Methods 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 230000036407 pain Effects 0.000 description 2
- 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 2
- 238000012360 testing method Methods 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- SMBBQHHYSLHDHF-UHFFFAOYSA-M 2-acetyloxyethyl(trimethyl)azanium;iodide Chemical compound [I-].CC(=O)OCC[N+](C)(C)C SMBBQHHYSLHDHF-UHFFFAOYSA-M 0.000 description 1
- KIUMMUBSPKGMOY-UHFFFAOYSA-N 3,3'-Dithiobis(6-nitrobenzoic acid) Chemical compound C1=C([N+]([O-])=O)C(C(=O)O)=CC(SSC=2C=C(C(=CC=2)[N+]([O-])=O)C(O)=O)=C1 KIUMMUBSPKGMOY-UHFFFAOYSA-N 0.000 description 1
- 229940100578 Acetylcholinesterase inhibitor Drugs 0.000 description 1
- 206010007247 Carbuncle Diseases 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
- 208000034507 Haematemesis Diseases 0.000 description 1
- 208000032843 Hemorrhage Diseases 0.000 description 1
- 241001504070 Huperzia Species 0.000 description 1
- 206010061245 Internal injury Diseases 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
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 239000012916 chromogenic reagent Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 210000000225 synapse Anatomy 0.000 description 1
- 230000008736 traumatic injury Effects 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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- 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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- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- 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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- A61K2236/50—Methods involving additional extraction steps
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Abstract
The invention discloses a huperzia serrata extract with an acetylcholinesterase inhibition effect, a preparation method and application thereof. The preparation method comprises the following steps: (1) Extracting herba Lycopodii Serrati with organic solvent, concentrating the extractive solution, and removing organic solvent to obtain herba Lycopodii Serrati organic solvent extract; (2) Loading the huperzia serrata organic solvent extract on a macroporous resin column, eluting with 30-40% ethanol water solution to remove impurities; eluting with 60-80% ethanol water solution, collecting eluate eluted with 60-80% ethanol water solution, concentrating, and drying to obtain macroporous resin eluate of herba Lycopodii Serrati; (3) And (3) loading the huperzia serrata macroporous resin elution part on a silica gel column to enrich active ingredients, thus obtaining the huperzia serrata extract. The huperzia serrata extract has excellent acetylcholinesterase inhibition effect; therefore, the huperzia serrata extract has wide application prospect in developing medicaments for treating senile dementia.
Description
Technical Field
The invention relates to the technical field of natural pharmaceutical chemistry, in particular to a huperzia serrata extract with an acetylcholinesterase inhibition effect, and a preparation method and application thereof.
Background
Huperzia serrata is a huperzia serrata of huperzia genus of huperziaceae family, and is prepared from whole herb; it has effects of dispelling blood stasis, stopping bleeding, clearing away heat and toxic materials, and relieving swelling and pain; is used for treating traumatic injury, swelling and pain due to blood stasis, and hematemesis due to internal injury; it is indicated for carbuncle furuncle, toxic swelling, snake bite, burn and scald.
Alzheimer's Disease (AD) is a degenerative disease of the nervous system that develops with the underlying progression; the patient before 65 years old is called Alzheimer's disease; senile dementia is a disease occurring after 65 years of age. Acetylcholinesterase inhibitors, also known as anticholinesterase drugs, are reversible inhibitors of acetylcholinesterase, which allow the accumulation of acetylcholine (ACh) at the synapses, prolonging and increasing the action of acetylcholine; is widely used for resisting senile dementia clinically.
Therefore, the development of a drug having an inhibitory effect on acetylcholinesterase is one of the directions of developing a drug for preventing or treating senile dementia. The prior art separates the huperzine A from huperzia serrata, and researches show that the huperzine A has obvious inhibition effect on acetylcholinesterase and obvious curative effect on senile dementia.
However, there are few reports in the prior art on whether other active ingredients in huperzia serrata have an inhibitory effect on acetylcholinesterase. Therefore, the extract or the monomer compound with the inhibition effect on acetylcholinesterase is further developed by taking the Chinese medicine huperzia serrata as a raw material, so that more choices are provided for developing the medicine for preventing or treating senile dementia, and the application value is important.
Disclosure of Invention
In order to overcome at least one technical problem existing in the prior art, the invention firstly provides a huperzia serrata extract with acetylcholinesterase inhibition effect; research shows that the huperzia serrata extract prepared by the method has excellent acetylcholinesterase inhibition effect
The invention aims to solve the technical problems, and is realized by the following technical scheme:
a preparation method of a huperzia serrata extract with acetylcholinesterase inhibition effect, which comprises the following steps:
(1) Extracting herba Lycopodii Serrati with organic solvent, concentrating the extractive solution, and removing organic solvent to obtain herba Lycopodii Serrati organic solvent extract;
(2) Loading the huperzia serrata organic solvent extract on a macroporous resin column, eluting with 30-40% ethanol water solution to remove impurities; eluting with 60-80% ethanol water solution, collecting eluate eluted with 60-80% ethanol water solution, concentrating, and drying to obtain macroporous resin eluate of herba Lycopodii Serrati;
(3) And (3) loading the huperzia serrata macroporous resin elution part on a silica gel column to enrich active ingredients, thus obtaining the huperzia serrata extract.
A great number of experimental researches show that the huperzia serrata is extracted by an organic solvent and then enriched by a macroporous resin column and a silica gel column to obtain a great amount of active ingredients with the acetylcholinesterase inhibition effect; so that the prepared huperzia serrata extract has excellent acetylcholinesterase inhibition effect.
The inventor needs to emphasize that the step of enriching the active ingredients by the macroporous resin column and the silica gel column is indispensable, and a large amount of active ingredients with acetylcholinesterase inhibition effect can be obtained by the step of simultaneously enriching by the macroporous resin column and the silica gel column; the absence of any step results in no preparation of huperzia serrata extract with excellent acetylcholinesterase inhibition.
Preferably, the organic solvent in the step (1) is an ethanol aqueous solution with the volume fraction of 70-95%.
Preferably, the dosage ratio of the huperzia serrata to the organic solvent in the step (1) is 1 g:8-20 mL.
Most preferably, the dosage ratio of huperzia serrata to organic solvent in step (1) is 1g:12ml.
Preferably, the extraction in the step (1) means extraction by a cold leaching method, and the extraction time is 3-10 d.
Most preferably, the extraction time is 7d.
Preferably, the macroporous resin column in the step (2) refers to a D101 macroporous resin column.
Preferably, in the step (2), eluting with an ethanol aqueous solution with the volume fraction of 30-40% and the volume of 3-5 times of the column volume to remove impurities; eluting with 60-80% ethanol water solution with 5-8 times of column volume, collecting eluate eluted with 60-80% ethanol water solution, concentrating, and drying to obtain the macroporous resin eluate of Huperzia serrata.
Further preferably, in the step (2), eluting with 4 times of ethanol aqueous solution with the volume fraction of 40% of the column volume to remove impurities; eluting with 70% ethanol water solution with 6 times of column volume, collecting eluate eluted with 70% ethanol water solution, concentrating, and drying to obtain macroporous resin eluate of herba Lycopodii Serrati.
Preferably, in the step (3), the specific method for enriching the active ingredients by loading the eluted part of the huperzia serrata macroporous resin on a silica gel column is as follows:
loading the macroporous resin elution part of the huperzia serrata on a silica gel column, eluting with a mixed organic solvent consisting of chloroform and ethyl acetate with the volume ratio of 91-93:9-7 to remove impurities; then eluting with mixed organic solvent comprising chloroform and ethyl acetate in the volume ratio of 85-87:15-13, collecting eluent eluted with mixed organic solvent comprising chloroform and ethyl acetate in the volume ratio of 85-87:15-13, concentrating and drying to obtain the Huperzia serrata extract.
Further preferably, in the step (3), the specific method for enriching the active ingredients by loading the eluted part of the huperzia serrata macroporous resin on a silica gel column is as follows:
loading the eluted part of the huperzia serrata macroporous resin on a silica gel column, eluting with a mixed organic solvent consisting of chloroform and ethyl acetate with the volume ratio of 92:8, wherein the volume ratio of the column is 2-4 times, and removing impurities; then eluting with mixed organic solvent composed of chloroform and ethyl acetate with the volume ratio of 86:14 by using 4-6 times of column volume, collecting eluent eluted by mixed organic solvent composed of chloroform and ethyl acetate with the volume ratio of 86:14, concentrating and drying to obtain the Huperzia serrata extract.
The inventor finds that after the huperzia serrata organic solvent extract is eluted by macroporous resin, the elution condition of the silica gel column plays a decisive role in preparing the huperzia serrata extract with excellent acetylcholinesterase inhibition effect; proved by a great deal of experimental researches, the huperzia serrata extract prepared under the condition of silica gel column elution has excellent acetylcholinesterase inhibition effect; however, the huperzia serrata extract having excellent acetylcholinesterase inhibition was not prepared under other silica gel column elution conditions.
The invention also provides the huperzia serrata extract with the acetylcholinesterase inhibition effect, which is prepared by the preparation method.
The invention also provides an application of the huperzia serrata extract with the acetylcholinesterase inhibition effect in preparing an acetylcholinesterase inhibitor.
The invention also provides an application of the huperzia serrata extract with the acetylcholinesterase inhibition effect in preparing medicines for preventing or treating senile dementia.
The beneficial effects are that: the invention provides a brand-new preparation method of a huperzia serrata extract, and the huperzia serrata extract prepared by the method has an excellent acetylcholinesterase inhibition effect. Therefore, the huperzia serrata extract has wide application prospect in developing medicaments for treating senile dementia.
Detailed Description
The present invention is further illustrated below with reference to specific examples, which are not intended to limit the invention in any way.
EXAMPLE 1 preparation of Huperzia serrata extract
(1) Extracting herba Lycopodii Serrati with organic solvent (95% ethanol water solution) at room temperature for 7 days by cold leaching method, concentrating the extractive solution to remove organic solvent to obtain herba Lycopodii Serrati organic solvent extract;
wherein the dosage ratio of the huperzia serrata to the organic solvent is 1g to 12mL;
(2) Loading the huperzia serrata organic solvent extract onto macroporous resin column (filled with D101 macroporous resin, wherein the weight of macroporous resin is 50 times of that of the huperzia serrata organic solvent extract), eluting with 4 times of ethanol water solution with the volume fraction of 40% to remove impurities; eluting with 70% ethanol water solution with 6 times of column volume, collecting eluate eluted with 70% ethanol water solution, concentrating, and drying to obtain macroporous resin eluate of herba Lycopodii Serrati;
(3) Loading the eluate on silica gel column (filled with 200-300 mesh silica gel, the weight of silica gel is 25 times of that of eluate), eluting with mixed organic solvent composed of chloroform and ethyl acetate with volume ratio of 92:8 (3 times of column volume); and eluting with mixed organic solvent composed of chloroform and ethyl acetate with volume ratio of 86:14, collecting eluate eluted from mixed organic solvent composed of chloroform and ethyl acetate with volume ratio of 86:14, concentrating, and drying to obtain the herba Lycopodii Serrati extract.
EXAMPLE 2 preparation of Huperzia serrata extract
(1) Extracting herba Lycopodii Serrati with organic solvent (70% ethanol water solution) at room temperature for 10 days by cold leaching method, concentrating the extractive solution to remove organic solvent to obtain herba Lycopodii Serrati organic solvent extract;
wherein the dosage ratio of the huperzia serrata to the organic solvent is 1g to 15mL;
(2) Loading the huperzia serrata organic solvent extract onto macroporous resin column (filled with D101 macroporous resin, wherein the weight of macroporous resin is 50 times of that of the huperzia serrata organic solvent extract), eluting with ethanol water solution with volume fraction of 30% and 5 times of column volume to remove impurities; eluting with 80% ethanol water solution with 5 times of column volume, collecting eluate eluted with 80% ethanol water solution, concentrating, and drying to obtain macroporous resin eluate of herba Lycopodii Serrati;
(3) Loading the eluate on silica gel column (filled with 200-300 mesh silica gel, the weight of silica gel is 25 times of that of eluate), eluting with mixed organic solvent composed of chloroform and ethyl acetate with volume ratio of 93:7 (2 times of column volume); and eluting with mixed organic solvent composed of chloroform and ethyl acetate with volume ratio of 85:15 by 4 times of column volume, collecting eluate eluted from mixed organic solvent composed of chloroform and ethyl acetate with volume ratio of 85:15, concentrating, and drying to obtain the Huperzia serrata extract.
EXAMPLE 3 preparation of Huperzia serrata extract
(1) Extracting herba Lycopodii Serrati with organic solvent (80% ethanol water solution) at room temperature for 5 days by cold leaching method, concentrating the extractive solution to remove organic solvent to obtain herba Lycopodii Serrati organic solvent extract;
wherein, the dosage ratio of the huperzia serrata to the organic solvent is 1g to 10mL;
(2) Loading the huperzia serrata organic solvent extract onto macroporous resin column (filled with D101 macroporous resin, wherein the weight of macroporous resin is 50 times of that of the huperzia serrata organic solvent extract), eluting with 3 times of ethanol water solution with volume fraction of 30% to remove impurities; eluting with ethanol water solution with volume fraction of 60% and volume fraction of 8 times column volume, collecting eluate eluted from ethanol water solution with volume fraction of 60%, concentrating, and drying to obtain macroporous resin eluate of herba Lycopodii Serrati;
(3) Loading the eluate on silica gel column (filled with 200-300 mesh silica gel, the weight of silica gel is 25 times of that of eluate), eluting with mixed organic solvent composed of chloroform and ethyl acetate with volume ratio of 91:9 (4 times of column volume); and eluting with mixed organic solvent composed of chloroform and ethyl acetate with volume ratio of 87:13 by 4 times of column volume, collecting eluate eluted from mixed organic solvent composed of chloroform and ethyl acetate with volume ratio of 87:13, concentrating, and drying to obtain the Huperzia serrata extract.
Comparative example 1 preparation of Huperzia serrata extract
(1) Extracting herba Lycopodii Serrati with organic solvent (95% ethanol water solution) at room temperature for 7 days by cold leaching method, concentrating the extractive solution to remove organic solvent to obtain herba Lycopodii Serrati organic solvent extract;
wherein the dosage ratio of the huperzia serrata to the organic solvent is 1g to 12mL;
(2) Loading the huperzia serrata organic solvent extract onto macroporous resin column (filled with D101 macroporous resin, wherein the weight of macroporous resin is 50 times of that of the huperzia serrata organic solvent extract), eluting with 4 times of ethanol water solution with the volume fraction of 40% to remove impurities; eluting with 70% ethanol water solution with 6 times of column volume, collecting eluate eluted with 70% ethanol water solution, concentrating, and drying to obtain macroporous resin eluate of herba Lycopodii Serrati; i.e. the huperzia serrata extract.
Comparative example 1 differs from example 1 in that comparative example 1 was prepared by eluting only with macroporous resin column to prepare a huperzia serrata extract; in example 1, the huperzia serrata extract was prepared by eluting with a macroporous resin column and then eluting with a silica gel column.
Comparative example 2 preparation of Huperzia serrata extract
(1) Extracting herba Lycopodii Serrati with organic solvent (95% ethanol water solution) at room temperature for 7 days by cold leaching method, concentrating the extractive solution to remove organic solvent to obtain herba Lycopodii Serrati organic solvent extract;
wherein the dosage ratio of the huperzia serrata to the organic solvent is 1g to 12mL;
(2) Loading the organic solvent extract of herba Lycopodii Serrati onto silica gel column (filled with 200-300 mesh silica gel, the weight of silica gel is 25 times of that of the organic solvent extract of herba Lycopodii Serrati), eluting with mixed organic solvent composed of chloroform and ethyl acetate with volume ratio of 92:8 of 3 times of column volume to remove impurities; and eluting with mixed organic solvent composed of chloroform and ethyl acetate with volume ratio of 86:14, collecting eluate eluted from mixed organic solvent composed of chloroform and ethyl acetate with volume ratio of 86:14, concentrating, and drying to obtain the herba Lycopodii Serrati extract.
Comparative example 2 differs from example 1 in that comparative example 2 was directly eluted with a silica gel column without macroporous resin to prepare a huperzia serrata extract; in example 1, the huperzia serrata extract was prepared by eluting with macroporous resin column and then eluting with silica gel column.
Comparative example 3 preparation of Huperzia serrata extract
(1) Extracting herba Lycopodii Serrati with organic solvent (95% ethanol water solution) at room temperature for 7 days by cold leaching method, concentrating the extractive solution to remove organic solvent to obtain herba Lycopodii Serrati organic solvent extract;
wherein the dosage ratio of the huperzia serrata to the organic solvent is 1g to 12mL;
(2) Loading the huperzia serrata organic solvent extract onto macroporous resin column (filled with D101 macroporous resin, wherein the weight of macroporous resin is 50 times of that of the huperzia serrata organic solvent extract), eluting with 4 times of ethanol water solution with the volume fraction of 40% to remove impurities; eluting with 70% ethanol water solution with 6 times of column volume, collecting eluate eluted with 70% ethanol water solution, concentrating, and drying to obtain macroporous resin eluate of herba Lycopodii Serrati;
(3) Loading the eluate on silica gel column (filled with 200-300 mesh silica gel, the weight of silica gel is 25 times of that of eluate), eluting with mixed organic solvent composed of chloroform and ethyl acetate with volume ratio of 99:1 (3 times of column volume); and eluting with mixed organic solvent composed of chloroform and ethyl acetate with volume ratio of 95:5 (5 times of column volume), collecting eluate eluted from mixed organic solvent composed of chloroform and ethyl acetate with volume ratio of 95:5, concentrating, and drying to obtain the herba Lycopodii Serrati extract.
Comparative example 3 is different from example 1 in that the silica gel column elution conditions in comparative example 3 are different, and the silica gel column elution conditions in comparative example 3 are: eluting with a mixed organic solvent consisting of chloroform and ethyl acetate in a volume ratio of 99:1 to remove impurities; then eluting with a mixed organic solvent composed of chloroform and ethyl acetate in a volume ratio of 95:5. The conditions for the silica gel column of example 1 were: eluting with a mixed organic solvent consisting of chloroform and ethyl acetate in a volume ratio of 92:8 to remove impurities; then eluting with a mixed organic solvent composed of chloroform and ethyl acetate in a volume ratio of 86:14.
Comparative example 4 preparation of Huperzia serrata extract
(1) Extracting herba Lycopodii Serrati with organic solvent (95% ethanol water solution) at room temperature for 7 days by cold leaching method, concentrating the extractive solution to remove organic solvent to obtain herba Lycopodii Serrati organic solvent extract;
wherein the dosage ratio of the huperzia serrata to the organic solvent is 1g to 12mL;
(2) Loading the huperzia serrata organic solvent extract onto macroporous resin column (filled with D101 macroporous resin, wherein the weight of macroporous resin is 50 times of that of the huperzia serrata organic solvent extract), eluting with 4 times of ethanol water solution with the volume fraction of 40% to remove impurities; eluting with 70% ethanol water solution with 6 times of column volume, collecting eluate eluted with 70% ethanol water solution, concentrating, and drying to obtain macroporous resin eluate of herba Lycopodii Serrati;
(3) Loading the eluate on silica gel column (filled with 200-300 mesh silica gel, the weight of silica gel is 25 times of that of eluate), eluting with mixed organic solvent composed of chloroform and ethyl acetate with volume ratio of 3 times of column volume of 85:15; and eluting with mixed organic solvent composed of chloroform and ethyl acetate with a volume ratio of 80:20 (5 times of column volume), collecting eluate eluted from mixed organic solvent composed of chloroform and ethyl acetate with a volume ratio of 80:20, concentrating, and drying to obtain the Huperzia serrata extract.
Comparative example 4 is different from example 1 in that the silica gel column elution conditions in comparative example 4 are different, and the silica gel column elution conditions in comparative example 4 are: eluting with a mixed organic solvent consisting of chloroform and ethyl acetate in a volume ratio of 85:15 to remove impurities; then eluting with a mixed organic solvent composed of chloroform and ethyl acetate in a volume ratio of 80:20. The conditions for the silica gel column of example 1 were: eluting with a mixed organic solvent consisting of chloroform and ethyl acetate in a volume ratio of 92:8 to remove impurities; then eluting with a mixed organic solvent composed of chloroform and ethyl acetate in a volume ratio of 86:14.
Experimental example
Dissolving the huperzia serrata extracts prepared in examples 1 to 3 and comparative examples 1 to 4 with DMSO to prepare a solution to be measured of 2 mg/mL; the extracts of Huperzia serrata prepared in examples 1 to 3 and comparative examples 1 to 4 were used for testing the inhibition rate of acetylcholinesterase.
Taking 96-well plates, and adding 40 mu L of Tris-HCl buffer solution with the pH value of 8.0 and 10 mu L of acetylcholinesterase solution (prepared by using Tris-HCl buffer solution with the pH value of 8.0) into each well respectively, wherein the concentration is 1000U/mL; the blank control group uses an equivalent amount of Tris-HCl buffer solution with the pH value of 8.0 to replace the solution to be tested; then incubating in a shaker at 37 ℃ for 10min; then 20. Mu.L of the chromogenic reagent DTNB solution (prepared with Tris-HCl buffer at pH 8.0 at a concentration of 0.1 mM) and 10. Mu.L of the substrate acetylcholine iodide solution (prepared with Tris-HCl buffer at pH 8.0 at a concentration of 0.2 mM) were added, followed by further incubation in a shaker at 37℃for 10min; after the incubation is finished, measuring the absorbance at 412nm by using an enzyme-labeled instrument; calculating the inhibition rate of the solution to be tested on acetylcholinesterase; the test results are shown in Table 1.
TABLE 1 inhibition of acetylcholinesterase by Huperzia serrata extract of the present invention
As can be seen from the experimental data in Table 1, the inhibition rate of acetylcholinesterase of the Huperzia serrata extracts prepared in examples 1 to 3 reaches more than 88% at the concentration of 2 mg/mL. This illustrates: the huperzia serrata extract prepared by the method has excellent acetylcholinesterase inhibition effect.
As can be seen from the experimental data in Table 1, the inhibition rate of acetylcholinesterase of the Huperzia serrata extract prepared in example 1 is far higher than that of the Huperzia serrata extract prepared in comparative examples 1 and 2. This illustrates: the step of enriching active ingredients by a macroporous resin column and a silica gel column is indispensable, and a large amount of active ingredients with acetylcholinesterase inhibition effect can be obtained by the step of simultaneously enriching the active ingredients by the macroporous resin column and the silica gel column; the absence of any step results in no preparation of huperzia serrata extract with excellent acetylcholinesterase inhibition.
As can be seen from the experimental data of table 1, the extracts of huperzia serrata prepared in comparative examples 3 and 4 did not significantly increase the inhibition rate of acetylcholinesterase as compared with comparative example 1; the inhibition rate of acetylcholinesterase of the huperzia serrata extract prepared in the example 1 is greatly improved compared with that of the huperzia serrata extract prepared in the comparative example 1; the inhibition rate of acetylcholinesterase is far higher than that of the Huperzia serrata extracts prepared in comparative examples 3 and 4. This illustrates: after the huperzia serrata organic solvent extract is eluted by macroporous resin, the eluting condition of the silica gel column plays a decisive role in whether the huperzia serrata extract with excellent acetylcholinesterase inhibition effect can be prepared; the above studies indicate that: after the huperzia serrata organic solvent extract is eluted by macroporous resin, not all the huperzia serrata extracts prepared under any silica gel column elution conditions can further greatly improve the inhibition effect of the huperzia serrata on acetylcholinesterase, but not all the huperzia serrata extracts prepared under any silica gel column elution conditions have excellent acetylcholinesterase inhibition effect; only the huperzia serrata extract prepared under the silica gel column elution condition can further greatly improve the inhibition effect of the huperzia serrata extract on acetylcholinesterase, and only the huperzia serrata extract prepared under the silica gel column elution condition has excellent acetylcholinesterase inhibition effect; the huperzia serrata extract prepared under the condition of other silica gel column elution can not further greatly improve the inhibition effect of the huperzia serrata extract on acetylcholinesterase, and does not have excellent acetylcholinesterase inhibition effect.
Claims (5)
1. A preparation method of a huperzia serrata extract with an acetylcholinesterase inhibition effect, which is characterized by comprising the following steps:
(1) Extracting herba Lycopodii Serrati with organic solvent, concentrating the extractive solution, and removing organic solvent to obtain herba Lycopodii Serrati organic solvent extract;
(2) Loading the huperzia serrata organic solvent extract on a macroporous resin column, eluting with 30-40% ethanol water solution by volume fraction to remove impurities; eluting with 60-80% ethanol water solution, collecting eluate eluted from 60-80% ethanol water solution, concentrating and drying to obtain a macroporous resin elution part of huperzia serrata;
(3) Loading the eluate on silica gel column to enrich effective components to obtain herba Lycopodii Serrati extract;
the specific method for enriching the active ingredients on the silica gel column at the elution part of the huperzia serrata macroporous resin in the step (3) is as follows:
loading the macroporous resin elution part of the huperzia serrata on a silica gel column, eluting with a mixed organic solvent consisting of chloroform and ethyl acetate in a volume ratio of 91-93:9-7 to remove impurities; eluting with a mixed organic solvent composed of chloroform and ethyl acetate in a volume ratio of 85-87:15-13, collecting eluent eluted from the mixed organic solvent composed of chloroform and ethyl acetate in a volume ratio of 85-87:15-13, concentrating and drying to obtain the Huperzia serrata extract;
the organic solvent in the step (1) is ethanol aqueous solution with the volume fraction of 70-95%;
the macroporous resin column in the step (2) refers to a D101 macroporous resin column.
2. The preparation method of claim 1, wherein in the step (2), eluting with an aqueous ethanol solution with a volume fraction of 30-40% and 3-5 times of the column volume to remove impurities; and eluting with ethanol water solution with the volume fraction of 60-80% and 5-8 times of the column volume, collecting eluent eluted from the ethanol water solution with the volume fraction of 60-80%, concentrating and drying to obtain the huperzia serrata macroporous resin elution part.
3. The method according to claim 1, wherein in the step (2), eluting with an aqueous solution of ethanol having a volume fraction of 40% which is 4 times the column volume to remove impurities; eluting with 70% ethanol water solution with 6 times of column volume, collecting eluate eluted with 70% ethanol water solution, concentrating, and drying to obtain macroporous resin eluate of herba Lycopodii Serrati.
4. The preparation method of claim 1, wherein the specific method for enriching the active ingredients on the silica gel column at the elution part of the huperzia serrata macroporous resin in the step (3) is as follows:
loading the macroporous resin elution part of the huperzia serrata on a silica gel column, eluting with a mixed organic solvent composed of chloroform and ethyl acetate with the volume ratio of 92:8, wherein the volume ratio of the column is 2-4 times, and removing impurities; and eluting with a mixed organic solvent consisting of chloroform and ethyl acetate with the volume ratio of 86:14, collecting the eluent eluted by the mixed organic solvent consisting of chloroform and ethyl acetate with the volume ratio of 86:14, concentrating and drying to obtain the Huperzia serrata extract.
5. The huperzia serrata extract with acetylcholinesterase inhibition effect prepared by the preparation method of any one of claims 1 to 4.
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