CN107445952B - Method for extracting gladiolide and coptisine and application - Google Patents
Method for extracting gladiolide and coptisine and application Download PDFInfo
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- CN107445952B CN107445952B CN201710723225.3A CN201710723225A CN107445952B CN 107445952 B CN107445952 B CN 107445952B CN 201710723225 A CN201710723225 A CN 201710723225A CN 107445952 B CN107445952 B CN 107445952B
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- XDVZNDLANFJOQR-UHFFFAOYSA-N Coptisine Natural products O=Cc1c2OCOc2ccc1C=C3/NCCc4cc5OCOc5cc34 XDVZNDLANFJOQR-UHFFFAOYSA-N 0.000 title claims abstract description 33
- 238000000034 method Methods 0.000 title claims abstract description 20
- XYHOBCMEDLZUMP-UHFFFAOYSA-N coptisine Chemical compound C1=C2C=C(C3=C(C=C4OCOC4=C3)CC3)[N+]3=CC2=C2OCOC2=C1 XYHOBCMEDLZUMP-UHFFFAOYSA-N 0.000 title claims abstract description 14
- 238000000605 extraction Methods 0.000 claims abstract description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 66
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 51
- 239000012452 mother liquor Substances 0.000 claims description 37
- LUXPUVKJHVUJAV-UHFFFAOYSA-M coptisine, chloride Chemical compound [Cl-].C1=C2C=C(C3=C(C=C4OCOC4=C3)CC3)[N+]3=CC2=C2OCOC2=C1 LUXPUVKJHVUJAV-UHFFFAOYSA-M 0.000 claims description 35
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 27
- MXTLAHSTUOXGQF-UHFFFAOYSA-O Jatrorrhizine Chemical compound COC1=CC=C2C=C3C(C=C(C(=C4)O)OC)=C4CC[N+]3=CC2=C1OC MXTLAHSTUOXGQF-UHFFFAOYSA-O 0.000 claims description 27
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 27
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 25
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 25
- 239000004952 Polyamide Substances 0.000 claims description 24
- 229920002647 polyamide Polymers 0.000 claims description 24
- 238000011068 loading method Methods 0.000 claims description 22
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 21
- 239000000706 filtrate Substances 0.000 claims description 21
- 241000218202 Coptis Species 0.000 claims description 20
- 235000002991 Coptis groenlandica Nutrition 0.000 claims description 20
- WVTKBKWTSCPRNU-KYJUHHDHSA-N (+)-Tetrandrine Chemical compound C([C@H]1C=2C=C(C(=CC=2CCN1C)OC)O1)C(C=C2)=CC=C2OC(=C2)C(OC)=CC=C2C[C@@H]2N(C)CCC3=CC(OC)=C(OC)C1=C23 WVTKBKWTSCPRNU-KYJUHHDHSA-N 0.000 claims description 18
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 18
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 17
- 238000001914 filtration Methods 0.000 claims description 17
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 16
- 239000002244 precipitate Substances 0.000 claims description 16
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 15
- 239000000287 crude extract Substances 0.000 claims description 15
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 13
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 12
- 239000003795 chemical substances by application Substances 0.000 claims description 11
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 10
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 10
- 239000000463 material Substances 0.000 claims description 10
- 239000000203 mixture Substances 0.000 claims description 10
- 239000011347 resin Substances 0.000 claims description 10
- 229920005989 resin Polymers 0.000 claims description 10
- 239000004519 grease Substances 0.000 claims description 9
- WVTKBKWTSCPRNU-UHFFFAOYSA-N rac-Tetrandrin Natural products O1C(C(=CC=2CCN3C)OC)=CC=2C3CC(C=C2)=CC=C2OC(=C2)C(OC)=CC=C2CC2N(C)CCC3=CC(OC)=C(OC)C1=C23 WVTKBKWTSCPRNU-UHFFFAOYSA-N 0.000 claims description 9
- 238000001179 sorption measurement Methods 0.000 claims description 9
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Natural products CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 claims description 8
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims description 7
- 241000037740 Coptis chinensis Species 0.000 claims description 6
- QISXPYZVZJBNDM-UHFFFAOYSA-N berberine Natural products COc1ccc2C=C3N(Cc2c1OC)C=Cc4cc5OCOc5cc34 QISXPYZVZJBNDM-UHFFFAOYSA-N 0.000 claims description 6
- YBHILYKTIRIUTE-UHFFFAOYSA-N berberine Chemical compound C1=C2CC[N+]3=CC4=C(OC)C(OC)=CC=C4C=C3C2=CC2=C1OCO2 YBHILYKTIRIUTE-UHFFFAOYSA-N 0.000 claims description 6
- 229940093265 berberine Drugs 0.000 claims description 6
- 238000010828 elution Methods 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 6
- 239000003513 alkali Substances 0.000 claims description 5
- WDIHJSXYQDMJHN-UHFFFAOYSA-L barium chloride Chemical compound [Cl-].[Cl-].[Ba+2] WDIHJSXYQDMJHN-UHFFFAOYSA-L 0.000 claims description 5
- 229910001626 barium chloride Inorganic materials 0.000 claims description 5
- 239000013078 crystal Substances 0.000 claims description 5
- 238000002425 crystallisation Methods 0.000 claims description 5
- 230000008025 crystallization Effects 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 5
- 239000003208 petroleum Substances 0.000 claims description 5
- 238000010992 reflux Methods 0.000 claims description 5
- 239000000741 silica gel Substances 0.000 claims description 5
- 229910002027 silica gel Inorganic materials 0.000 claims description 5
- 238000010898 silica gel chromatography Methods 0.000 claims description 5
- 238000004809 thin layer chromatography Methods 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- NIQQIJXGUZVEBB-UHFFFAOYSA-N methanol;propan-2-one Chemical compound OC.CC(C)=O NIQQIJXGUZVEBB-UHFFFAOYSA-N 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 3
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 claims description 2
- OJVABJMSSDUECT-UHFFFAOYSA-L berberin sulfate Chemical compound [O-]S([O-])(=O)=O.C1=C2CC[N+]3=CC4=C(OC)C(OC)=CC=C4C=C3C2=CC2=C1OCO2.C1=C2CC[N+]3=CC4=C(OC)C(OC)=CC=C4C=C3C2=CC2=C1OCO2 OJVABJMSSDUECT-UHFFFAOYSA-L 0.000 claims description 2
- 238000011161 development Methods 0.000 claims description 2
- 229910017053 inorganic salt Inorganic materials 0.000 claims description 2
- 230000000704 physical effect Effects 0.000 claims description 2
- UEAPAHNNFSZHMW-UHFFFAOYSA-N stepahnine Natural products COC1=CC=CC(C2=C34)=C1CC3N(C)CCC4=CC1=C2OCO1 UEAPAHNNFSZHMW-UHFFFAOYSA-N 0.000 claims 1
- UEAPAHNNFSZHMW-CQSZACIVSA-N stephanine Chemical compound CN([C@@H]1CC2=C(C3=C11)C=CC=C2OC)CCC1=CC1=C3OCO1 UEAPAHNNFSZHMW-CQSZACIVSA-N 0.000 claims 1
- 238000005406 washing Methods 0.000 claims 1
- 239000003814 drug Substances 0.000 abstract description 5
- 102100033639 Acetylcholinesterase Human genes 0.000 description 14
- 108010022752 Acetylcholinesterase Proteins 0.000 description 14
- 229940022698 acetylcholinesterase Drugs 0.000 description 12
- 230000005764 inhibitory process Effects 0.000 description 10
- 230000000694 effects Effects 0.000 description 8
- 238000011160 research Methods 0.000 description 7
- 229930013930 alkaloid Natural products 0.000 description 4
- 230000002401 inhibitory effect Effects 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 229940100578 Acetylcholinesterase inhibitor Drugs 0.000 description 3
- 150000003797 alkaloid derivatives Chemical class 0.000 description 3
- 239000000544 cholinesterase inhibitor Substances 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 239000003960 organic solvent Substances 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- UICBHOXXGLYZJH-UHFFFAOYSA-N 5,6-dihydroisoquinolino[2,1-b]isoquinolin-7-ium Chemical compound C1=CC=C2CC[N+]3=CC4=CC=CC=C4C=C3C2=C1 UICBHOXXGLYZJH-UHFFFAOYSA-N 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 230000000144 pharmacologic effect Effects 0.000 description 2
- 235000017807 phytochemicals Nutrition 0.000 description 2
- 229930000223 plant secondary metabolite Natural products 0.000 description 2
- 239000013558 reference substance Substances 0.000 description 2
- 239000000126 substance Substances 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
- SZMVXHRECFRCKQ-UHFFFAOYSA-M 2-ethanethioyloxyethyl(trimethyl)azanium;iodide Chemical compound [I-].CC(=S)OCC[N+](C)(C)C SZMVXHRECFRCKQ-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
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 108010053652 Butyrylcholinesterase Proteins 0.000 description 1
- 102100032404 Cholinesterase Human genes 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000893536 Epimedium Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- PTPHDVKWAYIFRX-UHFFFAOYSA-N Palmatine Natural products C1C2=C(OC)C(OC)=CC=C2C=C2N1CCC1=C2C=C(OC)C(OC)=C1 PTPHDVKWAYIFRX-UHFFFAOYSA-N 0.000 description 1
- 206010039966 Senile dementia Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- FPJQGFLUORYYPE-UHFFFAOYSA-N epiberberine Chemical compound C1=C2C=C(C3=C(C=C(C(=C3)OC)OC)CC3)[N+]3=CC2=C2OCOC2=C1 FPJQGFLUORYYPE-UHFFFAOYSA-N 0.000 description 1
- 235000018905 epimedium Nutrition 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 229930013397 isoquinoline alkaloid Natural products 0.000 description 1
- 125000002183 isoquinolinyl group Chemical class C1(=NC=CC2=CC=CC=C12)* 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- QUCQEUCGKKTEBI-UHFFFAOYSA-N palmatine Chemical compound COC1=CC=C2C=C(C3=C(C=C(C(=C3)OC)OC)CC3)[N+]3=CC2=C1OC QUCQEUCGKKTEBI-UHFFFAOYSA-N 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 229930001510 protoberberine alkaloid Natural products 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000001953 recrystallisation Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- YLJREFDVOIBQDA-UHFFFAOYSA-N tacrine Chemical compound C1=CC=C2C(N)=C(CCCC3)C3=NC2=C1 YLJREFDVOIBQDA-UHFFFAOYSA-N 0.000 description 1
- 229960001685 tacrine Drugs 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D455/00—Heterocyclic compounds containing quinolizine ring systems, e.g. emetine alkaloids, protoberberine; Alkylenedioxy derivatives of dibenzo [a, g] quinolizines, e.g. berberine
- C07D455/03—Heterocyclic compounds containing quinolizine ring systems, e.g. emetine alkaloids, protoberberine; Alkylenedioxy derivatives of dibenzo [a, g] quinolizines, e.g. berberine containing quinolizine ring systems directly condensed with at least one six-membered carbocyclic ring, e.g. protoberberine; Alkylenedioxy derivatives of dibenzo [a, g] quinolizines, e.g. berberine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
- A61K31/4375—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having nitrogen as a ring heteroatom, e.g. quinolizines, naphthyridines, berberine, vincamine
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- Medicines Containing Plant Substances (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention belongs to the technical field of traditional Chinese medicine extraction, and discloses a method for extracting gladiodine and coptisine and application thereof, aiming at solving the technical problems of new technology and new application of coptisine and gladiodine extraction.
Description
Technical Field
The invention relates to the technical field of medicine production, in particular to a method for extracting gladiolide and coptisine and application thereof.
Background
The coptis chinensis is one of traditional Chinese medicinal materials, has a long history of medication, contains rich protoberberine type isoquinoline alkaloids, has various biological activities of resisting tumor, bacteria and HIV, reducing blood sugar and fat and the like, and has the characteristics of low toxicity, low cost and the like. In addition, early researches find that the rhizoma coptidis contains abundant protoberberine alkaloids such as jateorhizine, coptisine and epiberberine. Because the alkaloid has more isomers and similar structures, the separation and purification (except berberine and palmatine) are relatively difficult, the alkaloid is generally only used as a phytochemical reference substance in the market, the coptisine and glandian are separated and purified by adopting a traditional system separation method, various chromatographic materials and different solvent systems are adopted, and the chromatography is repeatedly carried out by using various chromatographic materials, so that a large amount of organic solvents are applied, the separation period is long, the organic solvents are large in dosage, the environment protection is not facilitated, the time and labor are wasted, the cost is high, the new prices of the coptisine and glandian are expensive, the alkaloid is generally only used as a phytochemical reference substance or a research reagent in scientific research in the market, and the later-stage pharmacological activity research and the application thereof are limited.
Disclosure of Invention
The invention aims to overcome the existing defects, provides a method for extracting gladiolide and coptisine, has high extraction efficiency and stable process, utilizes the property of coptisine sulfate that the coptisine sulfate is insoluble in water and alcohol, has relatively high solubility of other alkaloids, can separate coptisine by recrystallization, utilizes the reverse chromatography and hydrogen bond adsorption of polyamide to separate phenolic alkaloid from coptis, and can obtain gladiolide by further column chromatography, thereby greatly reducing the using amount of organic solvents, greatly reducing the price of the gladiolide and the coptisine, lightening the burden of people and effectively solving the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme: a method for extracting gladiolide and coptisine comprises the following steps:
1) crushing coptis chinensis or coptis chinensis fibrous roots to extract mother liquor: firstly, taking out one kilogram of coptis and crushing the coptis by using a medicinal material crusher or cutting and crushing fibrous roots of the coptis; adding concentrated hydrochloric acid 10-20ml to precipitate berberine, filtering with diatomite, and keeping mother liquor for use;
2) removing grease from the mother liquor; adding ammonia water into the mother liquor to adjust the pH value to 11, filtering, adding ether or petroleum ether into the filtrate for extraction, and removing low-grade grease;
3) and (3) crystallization: by utilizing the physical properties that coptisine has high solubility and relatively high content in ethanol, sulfate of coptisine is insoluble in water and ethanol, and the solubility of coptisine sulfate in ethanol is 1: 500, solubility of berberine sulfate in ethanol at room temperature is 1: 30, adding 90% sulfuric acid into the alkali solution, dropwise adding while stirring until a yellow precipitate is generated, ultrasonically dissolving the yellow precipitate at 50 ℃ by using 1L-2L of ethanol, dropwise adding 5-10ml of sulfuric acid, repeatedly recrystallizing for two to three times to obtain more than 80% coptisine sulfate precipitate, detecting the purity by HPLC (high performance liquid chromatography) to be more than 85%, then adding a barium chloride solution with the molar ratio of 1:1, refluxing for half an hour, filtering, concentrating, and recrystallizing by methanol acetone to obtain 800mg of coptisine hydrochloride crystals 400-800 mg;
4) concentration: using the residual mother liquor, adjusting the pH value of the filtrate to 10-11 by using 5-10% of sodium hydroxide, dissociating phenolic alkaloid, filtering by using a filter, adjusting the pH value of the filtrate to 7 by using 5-10% of dilute hydrochloric acid, loading the filtrate on macroporous resin D101, loading the filtrate on a column at a column volume speed of 0.5-2.5% per minute, eluting the filtrate by using 95% of ethanol, concentrating the filtrate to obtain a crude extract, and dissolving the crude extract by using 1mol/L of ammonia water to obtain a column loading solution;
5) column mounting: when the polyamide is loaded on the column, the polyamide is loaded on the polyamide column according to 0.5 to 1.0 percent/min of the volume of the column, and then 0.8 to 1.5mol/L ammonia water is used for elution;
6) eluting and collecting: eluting the column according to the volume of 0.5-1.0%/min, sequentially presenting A, B, C color bands from top to bottom when the components move down to three quarters of the bottom of the column, collecting a B color band, performing thin-layer identification by using a developing agent and 1 part of ammonia water in a double-tank development manner according to the volume ratio of 4:2:1:1 of toluene, ethyl acetate, acetic acid and methanol, wherein the components of the B color band are a mixture of jatrorrhizine, african tetrandrine and gladiodine, detecting the flow by using thin-layer chromatography, and combining the same flow;
7) preparing gladiobin: adjusting the pH value of the component of the B color band group to be 7, adding macroporous resin D101, performing column loading adsorption at the column volume speed of 0.5-2.5% per minute, eluting with water to remove inorganic salt, eluting with 95% ethanol, concentrating to obtain a crude extract, recrystallizing acetone and methanol to obtain jateorhizine, wherein the mother liquor is a mixture of jateorhizine, African tetrandrine and glatirnew, mixing the mother liquor with 100-200-mesh silica gel, performing 300-400-mesh silica gel column chromatography, eluting with chloroform and methanol according to the volume ratio of 10:1 to 20:1, developing with a developing agent of 4:2:1:1 and 1 part of ammonia water according to the volume ratio of toluene, ethyl acetate, acetic acid and methanol, performing thin-layer identification, combining the same Rf value to obtain glatirnew, and performing HPLC detection to obtain 95%;
as a preferred embodiment of the present invention, the remaining mother liquor in step 2) is allowed to stand for 6 to 12 hours after adjusting the pH to 10 to 11.
As a preferable embodiment of the present invention, the step 5) is performed by stopping the column feeding when the amount of the solution in the column is one third to one fourth of the volume of the column.
As a preferable technical scheme of the invention, in the steps 5) and 6), when the polyamide column is loaded on the column, the polyamide column is loaded according to 0.5-1.0%/min of the column volume, and then 0.8-1.5 mol/L ammonia water is used for elution;
compared with the prior art, the invention has the beneficial effects that: the method and the process for extracting glandine and coptisine have the advantages of simple and feasible process, high extraction efficiency and high repeatability, the acetylcholinesterase inhibition activity of coptisine and glandine is not reported in documents, and the activity screening of coptisine and glandine shows that the coptisine and glandine have high acetylcholinesterase inhibition activity, can reduce the price of glandine and coptisine, and provide a material basis for subsequent research.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The first embodiment is as follows:
a method for extracting gladiolide and coptisine comprises the following steps:
1) crushing coptis and extracting mother liquor: firstly, taking one kilogram of coptis or coptis root, crushing the coptis or coptis root by using a medicinal material crusher, adding 10ml of concentrated hydrochloric acid to precipitate berberine, filtering by using kieselguhr, and reserving mother liquor for later use;
2) removing grease; adding ammonia water into the mother liquor to adjust the pH value to 11, and then adding ether or petroleum ether for extraction to remove low-grade grease;
3) and (3) crystallization: adding 90% sulfuric acid into the residual alkali solution, dropwise adding while stirring until a yellow precipitate is generated, ultrasonically dissolving the yellow precipitate at 50 ℃ by using 2L of ethanol, dropwise adding 2-3 ml, repeatedly recrystallizing for two to three times to obtain more than 80% coptisine sulfate precipitate, dissolving the coptisine sulfate precipitate with the purity of more than 85% by HPLC (high performance liquid chromatography), then adding a barium chloride solution with the molar ratio of 1:1, refluxing for half an hour, filtering by using a filter, and recrystallizing by using methanol and acetone to obtain 400mg of coptisine hydrochloride crystals;
4) concentration: dissociating phenolic alkaloid from residual mother liquor by using 5% of sodium hydroxide, adjusting the pH value of filtrate to 10 by using the 5% of sodium hydroxide, dissociating the phenolic alkaloid from the residual mother liquor after adjusting the pH value to 10, standing the solution for 6 hours, filtering the solution by using a filter, adjusting the pH value of the filtrate to 7 by using 5% of dilute hydrochloric acid, loading macroporous resin D101 on the filtrate, performing column loading adsorption at the column volume speed of 0.5% per minute, eluting the filtrate by using 95% of ethanol, concentrating the filtrate to obtain crude extract, and dissolving the crude extract by using 1mol/L of ammonia water to obtain column loading liquid;
5) column mounting: when the polyamide is loaded on the column, the polyamide is loaded on the polyamide column according to 0.5%/min of the column volume, and the loading of the polyamide is stopped when the amount of the solution on the polyamide column is one third of the column volume, and then 0.8mol/L ammonia water is used for elution;
6) eluting and collecting: eluting the column according to 0.5%/min of column volume, sequentially presenting A, B, C color bands from top to bottom when the components move down to three quarters of the column bottom, collecting B color band, performing thin layer identification by developing toluene, ethyl acetate, acetic acid and methanol in a developing agent and 1 part of ammonia water at a volume ratio of 4:2:1:1, wherein the component of B color band is a mixture of jatrorrhizine, african tetrandrine and gladiozone, detecting fractions by thin layer chromatography, and combining the same fractions;
7) preparing gladiobin: adjusting the pH value of the component of the B color band to be 7, loading the component on macroporous resin D101, loading the component on a column at the column volume speed of 0.5 percent per minute for adsorption, eluting by using 95 percent ethanol, concentrating to obtain a crude extract, recrystallizing acetone and methanol to obtain jatrorrhizine, wherein the mother liquor is a mixture of jatrorrhizine, African tetrandrine and glatirnew, mixing the mother liquor with 100-mesh silica gel, carrying out 300-mesh silica gel column chromatography, eluting by using chloroform and methanol according to the volume ratio of 20:1, developing by using a developing agent and 1 part of ammonia water in a double-tank manner according to the volume ratio of 4:2:1:1 for thin-layer identification, combining the same Rf value to obtain 300mg of glatirnew, and detecting the purity of 95 percent by HPLC.
Example two:
a method for extracting gladiolide and coptisine comprises the following steps:
1) crushing coptis and extracting mother liquor: firstly, taking one kilogram of coptis or coptis root, crushing the coptis or coptis root by using a medicinal material crusher, adding 15ml of concentrated hydrochloric acid to precipitate berberine, filtering by using kieselguhr, and reserving mother liquor for later use;
2) removing grease; adding ammonia water into the mother liquor to adjust the pH value to 11, and then adding ether or petroleum ether for extraction to remove low-grade grease;
3) and (3) crystallization: adding 90% sulfuric acid into the residual alkali solution, dropwise adding while stirring until yellow precipitate is generated, ultrasonically dissolving the yellow precipitate with 1L of ethanol at 50 ℃, dropwise adding 10ml of sulfuric acid, repeatedly recrystallizing for two to three times to obtain more than 80% coptisine sulfate precipitate, dissolving with methanol or ethanol after HPLC detection, adding barium chloride solution with a molar ratio of 1:1, refluxing for half an hour, filtering by a filter, and recrystallizing with methanol acetone to obtain 500mg of coptisine hydrochloride crystals;
4) concentration: using the residual mother liquor, adjusting the pH value of the filtrate to 11 by using 8% sodium hydroxide, adjusting the pH value of the residual mother liquor to 10, dissociating phenolic alkaloid, placing the solution for 8 hours, filtering by using a filter, adjusting the pH value of the filtrate to 7 by using 6% dilute hydrochloric acid, feeding the filtrate to a macroporous resin D101, performing column loading adsorption at the column volume speed of 1% per minute, eluting by using 95% ethanol, concentrating to obtain a crude extract, and dissolving the crude extract by using 1mol/L ammonia water to obtain column loading liquid;
5) column mounting: when the polyamide is loaded on the column, the polyamide is loaded on the polyamide column according to 0.7% per minute of the column volume, the loading of the polyamide is stopped when the amount of the solution on the polyamide column is one third of the column volume, and then 1.0mol/L ammonia water is used for elution;
6) eluting and collecting: eluting the column according to 0.7%/min of column volume, sequentially presenting A, B, C color bands from top to bottom when the components move down to three quarters of the column bottom, collecting B color band, performing thin layer identification with developing agent containing jatrorrhizine, African tetrandrine, and glandine at volume ratio of toluene, ethyl acetate, isopropanol, methanol, and ammonia water of 6:3:1.5:2:0.5, detecting fractions with thin layer chromatography, and mixing the same fractions;
7) preparing gladiobin: adjusting the pH value of the component of the B color band to 7, loading the component on macroporous resin D101, loading the component on a column at the column volume speed of 1 percent per minute, eluting by using 95 percent ethanol, concentrating to obtain a crude extract, recrystallizing acetone and methanol to obtain jatrorrhizine, wherein the mother liquor is a mixture of jatrorrhizine, african tetrandrine and gladiolide, the mother liquor is mixed with 100-mesh silica gel, is subjected to 300-mesh 400-mesh silica gel column chromatography, is eluted by using chloroform and methanol according to the volume ratio of 10:1, is subjected to thin-layer identification by using a developing agent of toluene, ethyl acetate, acetic acid and methanol according to the volume ratio of 4:2:1:1, and is combined with the same Rf value to obtain 50mg of gladiolide, and the HPLC detection purity is 95 percent.
Example three:
a method for extracting gladiolide and coptisine comprises the following steps:
1) cutting coptis fibrous root to extract mother liquor: firstly, taking one kilogram of coptis or coptis root, crushing by using a medicinal material crusher, adding 10-20ml of concentrated hydrochloric acid to precipitate berberine, filtering by using kieselguhr, and reserving mother liquor for later use;
2) removing grease; adding ammonia water into the mother liquor to adjust the pH value to 11, and then adding ether or petroleum ether for extraction to remove low-grade grease;
3) and (3) crystallization: adding 90% sulfuric acid into the residual alkali solution, dropwise adding while stirring until a yellow precipitate is generated, ultrasonically dissolving the yellow precipitate with 1L ethanol at 50 ℃, dropwise adding 6ml concentrated sulfuric acid, repeatedly recrystallizing for two to three times, dropwise adding while stirring to obtain more than 80% coptisine sulfate precipitate, detecting the purity by HPLC to be more than 85%, then adding 200ml methanol or ethanol, then adding coptisine sulfate: barium chloride with about a molar ratio of 1:1, refluxing for half an hour, filtering by a filter, and recrystallizing with methanol acetone to obtain 600mg of coptisine hydrochloride crystals;
4) concentration: using the residual mother liquor, adjusting the pH value of the filtrate to 11 by using 10% sodium hydroxide, adjusting the pH value of the residual mother liquor to 10, dissociating phenolic alkaloid, placing the solution for 10 hours, filtering by using a filter, adjusting the pH value of the filtrate to 7 by using 8% dilute hydrochloric acid, feeding the filtrate to a macroporous resin D101, performing column adsorption at a column volume speed of 2.0% per minute, eluting by using 95% ethanol, concentrating to obtain a crude extract, and dissolving the crude extract by using 1mol/L ammonia water to obtain a column feeding solution;
5) column mounting: when the polyamide is loaded on the column, the polyamide is loaded on the polyamide column according to 0.8%/min of the column volume, the loading of the polyamide is stopped when the amount of the solution on the polyamide column is one third of the column volume, and then 1.3mol/L ammonia water is used for elution;
6) eluting and collecting: eluting the mixture on the column according to the volume of 0.8%/min, sequentially presenting A, B, C color bands from top to bottom when the components move down to the bottom of the column by three quarters, collecting a B color band, performing thin-layer identification by using a developing agent and 1 part of ammonia water in a volume ratio of toluene, ethyl acetate, acetic acid and methanol of 4:2:1:1, wherein the components of the B color band are a mixture of jatrorrhizine, african tetrandrine and gladiodine, detecting fractions by thin-layer chromatography, and combining the same fractions;
7) preparing gladiobin: adjusting the pH value of the component of the B color band to 7, loading the component on macroporous resin D101, loading the component on a column at the column volume speed of 2.0% per minute for adsorption, eluting by using 95% ethanol, concentrating to obtain a crude extract, recrystallizing acetone and methanol to obtain jatrorrhizine, wherein the mother liquor is a mixture of jatrorrhizine, African tetrandrine and glatirnew, mixing the mother liquor with 60-100 meshes of silica gel, carrying out 300-400 meshes of silica gel column chromatography, eluting by using chloroform and methanol according to the volume ratio of 20:1, then carrying out thin-layer identification by using toluene, ethyl acetate, acetic acid and methanol according to the volume ratio of 4:2:1:1 of developing agent and 1 part of ammonia water, combining the same Rf value to obtain 40mg of glatirnew, and detecting the purity of 95% by HPLC.
Example four, gladiolide and coptisine acetylcholinesterase inhibitory Activity
The principle is as follows: the product of acetylcholinesterase and thioacetylcholine iodide and 5, 5-dithio-bis-nitrobenzoic acid form a yellow substance, which can be used to determine the degree of inhibition of acetylcholinesterase by the sample to be tested. The specific method is shown in the literature: a comparison research on a micro-screening model of an acetylcholinesterase inhibitor, Sun Qian Yun Yangyo Pamei, China pharmacological report 2008, 24(10): 1387-13925. the in vitro AChE inhibitory activity is tested by adopting an Ellman method, acetylcholine iodide is used as a substrate, chemical-labeled dithionitrobenzoic acid is used as a color developing agent, and the AChE inhibitory activity of a sample is tested on a 96-well plate. Tacrine is selected as a positive control drug. The inhibition rates of three different concentrations of acetylcholinesterase are obtained.
See table one
Epimedium coptisine and gladioxin Single concentration inhibition of acetylcholinesterase and butyrylcholinesterase
Simple comparison from Table I, the instruments of glandinew and coptisineHas obvious effect of inhibiting acetylcholinesterase, especially the gladioxin in single concentration of 1X 10-6 mol/L-1The inhibition rate of the acetylcholinesterase exceeds 50 percent, which shows that the acetylcholinesterase inhibitor has better selectivity on the inhibition activity of two enzymes, and can be used as a medicament for treating senile dementia, and the action mechanism of the acetylcholinesterase inhibitor is mainly the inhibition of the acetylcholinesterase.
The method has the advantages of simple and easy process, good repeatability and high extraction efficiency, can greatly reduce the price of gladiolide and coptisine, provides a material basis for subsequent research, and increases the additional value of the coptis chinensis. The inhibition activity of coptisine and gladioxin on acetylcholinesterase is not reported in documents, and the activity screening of coptisine and gladioxin finds that the coptisine and gladioxin have the inhibition activity on acetylcholinesterase.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (3)
1. A method for extracting gladiolide and coptisine is characterized by comprising the following steps:
1) crushing coptis chinensis or coptis chinensis fibrous roots to extract mother liquor: firstly, taking out one kilogram of coptis and crushing the coptis by using a medicinal material crusher or cutting and crushing fibrous roots of the coptis; adding concentrated hydrochloric acid 10-20ml to precipitate berberine, filtering with diatomite, and keeping mother liquor for use;
2) removing grease from the mother liquor; adding ammonia water into the mother liquor to adjust the pH value to 11, filtering, adding diethyl ether or petroleum ether into the filtrate for extraction, and removing low-polarity oil;
3) and (3) crystallization: by utilizing the physical properties that coptisine has high solubility and relatively high content in ethanol, sulfate of coptisine is insoluble in water and ethanol, and the solubility of coptisine sulfate in ethanol is 1: 500, solubility of berberine sulfate in ethanol at room temperature is 1: 30, adding 90% sulfuric acid into the alkali solution, dropwise adding while stirring until a yellow precipitate is generated, ultrasonically dissolving the yellow precipitate at 50 ℃ by using 1L-2L of ethanol, dropwise adding 5-10ml of sulfuric acid, repeatedly recrystallizing for two to three times to obtain more than 80% coptisine sulfate precipitate, detecting the purity by HPLC (high performance liquid chromatography) to be more than 85%, then adding a barium chloride solution with the molar ratio of 1:1, refluxing for half an hour, filtering, concentrating, and recrystallizing by methanol acetone to obtain 400-800mg coptisine hydrochloride crystals;
4) concentration: using the residual mother liquor, adjusting the pH value of the filtrate to 10-11 by using 5-10% of sodium hydroxide, dissociating phenolic alkaloid, filtering by using a filter, adjusting the pH value of the filtrate to 7 by using 5-10% of dilute hydrochloric acid, adding macroporous resin D101, performing column loading adsorption at the column volume speed of 0.5-2.5% per minute, eluting by using 95% ethanol, concentrating to obtain a crude extract, and dissolving the crude extract by using 1mol/L ammonia water to obtain column loading liquid;
5) column mounting: when the polyamide is loaded on the column, the polyamide is loaded on the polyamide column according to 0.5 to 1.0 percent/min of the volume of the column, and then 0.8 to 1.5mol/L ammonia water is used for elution;
6) eluting and collecting: eluting the column according to the volume of 0.5-1.0%/min, sequentially presenting A, B, C color bands from top to bottom when the components move down to three quarters of the bottom of the column, collecting a B color band, performing thin-layer identification by using a developing agent and 1 part of ammonia water in a double-tank development manner according to the volume ratio of 4:2:1:1 of toluene, ethyl acetate, acetic acid and methanol, wherein the components of the B color band are a mixture of jatrorrhizine, african tetrandrine and gladiodine, detecting the flow by using thin-layer chromatography, and combining the same flow;
7) preparing gladiobin: adjusting the pH value of the component of the B color band to 7, loading the component on macroporous resin D101, loading the component on a column at the column volume speed of 0.5-2.5% per minute for adsorption, washing with water to remove inorganic salt, eluting with 95% ethanol, concentrating to obtain a crude extract, recrystallizing with acetone and methanol to obtain jateorhizine, wherein the mother liquor is a mixture of jateorhizine, african stephanine and glatirnew, mixing the mother liquor with 100-200-mesh silica gel, performing 300-400-mesh silica gel column chromatography, eluting with chloroform and methanol at the volume ratio of 10: 1-20: 1, developing with toluene, ethyl acetate, acetic acid and methanol at the volume ratio of 4:2:1:1 in a developing agent and 1 part of ammonia water, performing thin-layer identification, combining the same Rf value to obtain glatirnew, and detecting the purity of 95% by HPLC.
2. The method for extracting gladiolide and coptisine as claimed in claim 1, wherein: after the pH value of the residual mother liquor in the step 2) is adjusted to 11, the solution is required to be placed for 6-12 hours.
3. The method for extracting gladiolide and coptisine as claimed in claim 1, wherein: and (3) stopping the column feeding when the amount of the solution on the column is one third to one fourth of the volume of the column during the column feeding in the step 5).
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