CN108623524A - Imidazoles dimer alkaloid and its preparation method and application - Google Patents
Imidazoles dimer alkaloid and its preparation method and application Download PDFInfo
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- CN108623524A CN108623524A CN201810343896.1A CN201810343896A CN108623524A CN 108623524 A CN108623524 A CN 108623524A CN 201810343896 A CN201810343896 A CN 201810343896A CN 108623524 A CN108623524 A CN 108623524A
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- column chromatography
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- 229930013930 alkaloid Natural products 0.000 title claims abstract description 51
- -1 Imidazoles dimer alkaloid Chemical class 0.000 title claims abstract description 45
- 238000002360 preparation method Methods 0.000 title claims description 15
- 239000003814 drug Substances 0.000 claims abstract description 13
- 210000005036 nerve Anatomy 0.000 claims abstract description 6
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical group ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 69
- 238000010898 silica gel chromatography Methods 0.000 claims description 35
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 33
- 238000010828 elution Methods 0.000 claims description 33
- WGLUMOCWFMKWIL-UHFFFAOYSA-N dichloromethane;methanol Chemical compound OC.ClCCl WGLUMOCWFMKWIL-UHFFFAOYSA-N 0.000 claims description 30
- 244000269722 Thea sinensis Species 0.000 claims description 22
- 235000020279 black tea Nutrition 0.000 claims description 20
- 235000006468 Thea sinensis Nutrition 0.000 claims description 19
- 238000004440 column chromatography Methods 0.000 claims description 15
- 239000000284 extract Substances 0.000 claims description 15
- 239000000243 solution Substances 0.000 claims description 12
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 10
- 239000000843 powder Substances 0.000 claims description 9
- 239000007864 aqueous solution Substances 0.000 claims description 8
- XELZGAJCZANUQH-UHFFFAOYSA-N methyl 1-acetylthieno[3,2-c]pyrazole-5-carboxylate Chemical compound CC(=O)N1N=CC2=C1C=C(C(=O)OC)S2 XELZGAJCZANUQH-UHFFFAOYSA-N 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 6
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 6
- 238000001641 gel filtration chromatography Methods 0.000 claims description 6
- 239000000463 material Substances 0.000 claims description 6
- 238000000746 purification Methods 0.000 claims description 6
- FEWJPZIEWOKRBE-UHFFFAOYSA-N Tartaric acid Natural products [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O FEWJPZIEWOKRBE-UHFFFAOYSA-N 0.000 claims description 5
- 238000000605 extraction Methods 0.000 claims description 5
- 238000000926 separation method Methods 0.000 claims description 5
- 235000011121 sodium hydroxide Nutrition 0.000 claims description 5
- 239000000126 substance Substances 0.000 claims description 5
- 235000002906 tartaric acid Nutrition 0.000 claims description 5
- 239000011975 tartaric acid Substances 0.000 claims description 5
- 239000002552 dosage form Substances 0.000 claims description 4
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims description 4
- 230000000324 neuroprotective effect Effects 0.000 claims description 4
- 239000003513 alkali Substances 0.000 claims description 3
- 235000019270 ammonium chloride Nutrition 0.000 claims description 3
- 238000004090 dissolution Methods 0.000 claims description 3
- 229940079593 drug Drugs 0.000 claims description 3
- 239000003208 petroleum Substances 0.000 claims description 3
- 239000000049 pigment Substances 0.000 claims description 3
- 238000012545 processing Methods 0.000 claims description 3
- 239000002994 raw material Substances 0.000 claims description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 2
- 238000004458 analytical method Methods 0.000 claims description 2
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 claims 2
- 229910052710 silicon Inorganic materials 0.000 claims 2
- 239000010703 silicon Substances 0.000 claims 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims 1
- 239000000741 silica gel Substances 0.000 claims 1
- 229910002027 silica gel Inorganic materials 0.000 claims 1
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 14
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 10
- 238000005481 NMR spectroscopy Methods 0.000 description 9
- 239000013078 crystal Substances 0.000 description 9
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 6
- RYYVLZVUVIJVGH-UHFFFAOYSA-N caffeine Chemical compound CN1C(=O)N(C)C(=O)C2=C1N=CN2C RYYVLZVUVIJVGH-UHFFFAOYSA-N 0.000 description 6
- 238000004611 spectroscopical analysis Methods 0.000 description 6
- 150000001875 compounds Chemical class 0.000 description 5
- 150000002460 imidazoles Chemical class 0.000 description 5
- 239000011734 sodium Substances 0.000 description 4
- 235000013616 tea Nutrition 0.000 description 4
- 238000005160 1H NMR spectroscopy Methods 0.000 description 3
- 241001269238 Data Species 0.000 description 3
- LPHGQDQBBGAPDZ-UHFFFAOYSA-N Isocaffeine Natural products CN1C(=O)N(C)C(=O)C2=C1N(C)C=N2 LPHGQDQBBGAPDZ-UHFFFAOYSA-N 0.000 description 3
- VJEONQKOZGKCAK-UHFFFAOYSA-N caffeine Natural products CN1C(=O)N(C)C(=O)C2=C1C=CN2C VJEONQKOZGKCAK-UHFFFAOYSA-N 0.000 description 3
- 229960001948 caffeine Drugs 0.000 description 3
- 238000005100 correlation spectroscopy Methods 0.000 description 3
- 230000008878 coupling Effects 0.000 description 3
- 238000010168 coupling process Methods 0.000 description 3
- 238000005859 coupling reaction Methods 0.000 description 3
- 230000006378 damage Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000003919 heteronuclear multiple bond coherence Methods 0.000 description 3
- 238000003929 heteronuclear multiple quantum coherence Methods 0.000 description 3
- 238000002114 high-resolution electrospray ionisation mass spectrometry Methods 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 210000002569 neuron Anatomy 0.000 description 3
- 230000001681 protective effect Effects 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 230000003595 spectral effect Effects 0.000 description 3
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- OKKJLVBELUTLKV-MZCSYVLQSA-N Deuterated methanol Chemical group [2H]OC([2H])([2H])[2H] OKKJLVBELUTLKV-MZCSYVLQSA-N 0.000 description 2
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical group [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 2
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical group [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 2
- 150000003797 alkaloid derivatives Chemical class 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- NEHMKBQYUWJMIP-UHFFFAOYSA-N chloromethane Chemical class ClC NEHMKBQYUWJMIP-UHFFFAOYSA-N 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000000546 pharmaceutical excipient Substances 0.000 description 2
- 230000000144 pharmacologic effect Effects 0.000 description 2
- 239000006187 pill Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000003826 tablet Substances 0.000 description 2
- YAPQBXQYLJRXSA-UHFFFAOYSA-N theobromine Chemical compound CN1C(=O)NC(=O)C2=C1N=CN2C YAPQBXQYLJRXSA-UHFFFAOYSA-N 0.000 description 2
- ZFXYFBGIUFBOJW-UHFFFAOYSA-N theophylline Chemical compound O=C1N(C)C(=O)N(C)C2=C1NC=N2 ZFXYFBGIUFBOJW-UHFFFAOYSA-N 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 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 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
- 206010028980 Neoplasm Diseases 0.000 description 1
- 206010029260 Neuroblastoma Diseases 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
- 244000062793 Sorghum vulgare Species 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000000879 anti-atherosclerotic effect Effects 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000000675 anti-caries Effects 0.000 description 1
- 230000036528 appetite Effects 0.000 description 1
- 235000019789 appetite Nutrition 0.000 description 1
- 208000006673 asthma Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000000496 cardiotonic agent Substances 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000005779 cell damage Effects 0.000 description 1
- 208000037887 cell injury Diseases 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 125000003963 dichloro group Chemical group Cl* 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 230000007760 free radical scavenging Effects 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000008187 granular material Substances 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
- 230000006872 improvement Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000002932 luster Substances 0.000 description 1
- 235000019713 millet Nutrition 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 150000003212 purines Chemical class 0.000 description 1
- 150000003230 pyrimidines Chemical class 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
- 230000005855 radiation Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229960004559 theobromine Drugs 0.000 description 1
- 229960000278 theophylline Drugs 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
- C07D233/54—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
- C07D233/66—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D233/88—Nitrogen atoms, e.g. allantoin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Neurology (AREA)
- Neurosurgery (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a kind of imidazoles dimer alkaloid that can be used in preparing nerve protection medicine, the representation shown in formula I or formula II or formula III：
Description
Technical field
The present invention relates to natural product field, relate in particular to a kind of imidazoles dimer alkaloid and preparation method thereof and
Using.
Background technology
Contain abundant alkaloid in tealeaves, accounts for the 3%-5% of dry matter of tea.Wherein, it is largely purines biology
Alkali and a small amount of pyrimidine Alkaloid.Wherein caffeine accounts for 3%-4%, and theobromine accounts for 0.15%-0.2%, and theophylline accounts for
0.02%-0.04%.Studies have shown that caffeine is taste substance important in tealeaves.Moreover, caffeine also has cardiac stimulant, profit
The pharmacological functions such as urinate, detoxify, relievining asthma.
Black tea is full fermentation tea, and the processing of Keemun black tea mainly has the processes such as wither, rub, fermenting, drying, ultimately forms
Its dry tea color and luster and the millet paste that brews are using red as homophony.Research confirms that black tea has good free-radical scavenging activity, may be used also
To promote appetite, help gastro-intestinal digestion.Modern age pharmaceutical research shows with cancer-resisting, antibacterial, resisting ultraviolet radiation, resists
A variety of pharmacological functions such as oxidation, antiatherosclerosis, anti-caries shield tooth.
The report of alkaloids chemical composition about Keemun black tea bioactivity is fewer, if can be successfully from Qimen
Biologically active alkaloids derivative ingredient is researched and developed in black tea, it will weight is made to agricultural and medicine and other fields
It contributes.
Invention content
Technical problem to be solved by the invention is to provide a kind of imidazoles dimer alkaloids that can protect nerve.
In order to solve the above-mentioned technical problem, the present invention adopts the following technical scheme that：Imidazoles dimer alkaloid, by formula I or
Representation shown in formula II or formula III：
The present invention also provides application of the above-mentioned imidazoles dimer alkaloid in preparing nerve protection medicine.
A kind of neuroprotective medicine can be specifically provided, be made of above-mentioned imidazoles dimer alkaloid and pharmaceutic adjuvant.
The pharmaceutical dosage form of the drug includes oral type and injection-type.The oral type includes tablet, capsule, particle
Agent, pill etc.；The injection-type includes injection, mixed rotary liquid etc..Conventional method of the specific preparation method with reference to pharmaceutical field
It prepares, the auxiliary material that pharmaceutic adjuvant used selects pharmaceutical field general according to dosage form difference.
The present invention also provides the preparation methods of above-mentioned imidazoles dimer alkaloid, include the following steps：
(1) raw material crushes
Keemun black tea is taken, processing is crushed to it, obtains Keemun black tea powder；
(2) it extracts
Keemun black tea powder is extracted with ethanol water, obtains extracting solution, it is red that Qimen is made by being dried in extracting solution
Tea extraction；
(3) it isolates and purifies
Keemun black tea extract is dissolved with methanol aqueous solution, then the most of methanol of concentration removal, uses petroleum ether later
Extraction removes small polar substances and pigment, is finally extracted with dichloromethane, obtains dichloromethane position extractum A；
By the extractum A water dissolution of dichloromethane position, aqueous tartaric acid solution is added, removes insoluble matter, uses dichloro again later
Methane is extracted, and isometric sodium hydrate aqueous solution, which is added, after removal water layer is extracted, and dichloromethane layer and buck are obtained
Layer, buck layer are extracted with dichloromethane again after ammonium chloride is added, and obtain dichloromethane position medicinal extract B；
Separation and purification treatment is carried out to dichloromethane position medicinal extract B, obtains the imidazoles dimer alkaloid.
Further, in step (2), a concentration of the 95% of ethanol water used.
Further, in step (3), a concentration of the 2% of aqueous tartaric acid solution used, sodium hydrate aqueous solution it is a concentration of
1-2%.
Further, in step (3), separation and purification treatment includes silica gel column chromatography, Sephadex LH-20 gel column layers
Analysis, Toyopearl column chromatographies.
Further, separation and purification treatment the specific steps are：It is carried out for the first time after dichloromethane position medicinal extract B is dissolved
Silica gel column chromatography, first time silica gel column chromatography is with methylene chloride-methanol volume ratio 500:1 to 1:1 is used as gradient elution, collects it
Middle methylene chloride-methanol volume ratio 20:1 to 10:1 elution fraction carries out second of silica gel column chromatography, second of silica gel column chromatography
With methylene chloride-methanol volume ratio 50:1 to 1:1 is used as gradient elution, collects wherein methylene chloride-methanol volume ratio 10:1 washes
De- component carries out third time silica gel column chromatography, and third time silica gel column chromatography is with methylene chloride-methanol volume ratio 10:1 to 1:1 conduct
Gradient elution collects elution fraction and carries out Toyopearl column chromatography purifying, obtains imidazoles dimer alkaloid shown in formula I；
The methylene chloride-methanol volume ratio 30 that first time silica gel column chromatography is obtained:1 to 20:1 elution fraction carries out the 4th
Secondary silica gel column chromatography, the 4th silica gel column chromatography is with methylene chloride-methanol volume ratio 70:1 to 1:1 is used as gradient elution, collects
Wherein methylene chloride-methanol volume ratio 10:1 elution fraction carries out Sephadex LH-20 gel filtration chromatographies, Sephadex LH-
20 gel filtration chromatographies rush column with methanol, collect a component being eluted out at first and carry out Toyopearl column chromatography purifying, obtain
Imidazoles dimer alkaloid shown in formula II；
The methylene chloride-methanol volume ratio 20 that 4th silica gel column chromatography is obtained:1 elution fraction carries out the 5th silica gel
Column chromatography, the 5th silica gel column chromatography is with methylene chloride-methanol volume ratio 40:1 to 1:1 is used as gradient elution, collects elution group
Divide and carry out Toyopearl column chromatography purifying, obtains imidazoles dimer alkaloid shown in formula III.
Here " the ... it is secondary " it is not used to limit sequencing, it is only used for distinguishing, as long as respective components are deviate from,
It can implement the chromatography of next step.
Beneficial effects of the present invention are embodied in：
Imidazoles dimer alkaloid provided by the invention with medicinal actives is in the nerve cell by hydrogen peroxide-induced
There is certain protective role in damage model, can be used for preparing nerve protection medicine, has to agricultural and field of medicaments important
Meaning；And more wide foreground is provided using Keemun black tea for effective exploitation.
The preparation method of imidazoles dimer alkaloid of the present invention is simple for process, is easy to implement, and cost is relatively low, and it is very good to have
Application prospect.
Description of the drawings
Fig. 1 is the x-ray crystal structure figure of imidazoles dimer alkaloid shown in formula I.
Fig. 2 is the x-ray crystal structure figure of imidazoles dimer alkaloid shown in formula II.
Fig. 3 is that formula I, formula II and imidazoles dimer alkaloid shown in formula III damage hydrogen peroxide-induced human nerve cell
Inhibition test schematic diagram.
Specific implementation mode
With reference to embodiment, the invention will be further described：
This part carries out general description to the material and experimental method that are arrived used in present invention experiment.Although being
Realize that many materials and operating method used in the object of the invention are it is known in the art that still the present invention makees as far as possible herein
Detailed description.It will be apparent to those skilled in the art that hereinafter, if not specified, material therefor, equipment and operation of the present invention
Method is well known in the art.
Embodiment 1
The preparation of imidazoles dimer alkaloid
The explanation of 1.1 imidazoles dimer alkaloids
The structure of imidazoles dimer alkaloid is as shown in formula I or formula II or formula III：
1.2 preparation methods and result
(1) raw material crushes
200 kilograms of Keemun black teas are taken, it is 10.5mm sieves to be crushed to aperture, obtains Keemun black tea powder；
(2) it extracts
Keemun black tea powder is added in 1000 kilogram of 95% ethanol water, stirring extraction 10 hours is obtained by filtration
Extracting solution is concentrated into 400L and crosses 200 mesh screens, continued concentrated 200 mesh screen, finally spray to concentrate by extracting solution
Dry, 190 DEG C of inlet air temperature, 90 DEG C of leaving air temp collects dried powder and crosses 40 mesh screens, obtains black tea extract；
(3) it isolates and purifies
70% methanol aqueous solution of Keemun black tea extract is dissolved, then the most of methanol of concentration removal (is gone as far as possible
Remove, being subject to does not influence follow-up extracting and demixing), small polar substances and pigment are removed with petroleum ether extraction later, finally with two
Chloromethanes is extracted, and dichloromethane position extractum A is obtained；
By the extractum A water dissolution of dichloromethane position, 2% aqueous tartaric acid solution is added, insoluble matter is removed, later again with two
Chloromethanes is extracted, and isometric 1%-2% sodium hydrate aqueous solutions, which are added, after removal water layer is extracted, and dichloromethane is obtained
Layer and buck layer, buck layer are extracted with dichloromethane again after ammonium chloride is added, and obtain dichloromethane position medicinal extract B；
500 grams of dichloromethane position medicinal extract B are taken, is mixed with chloroform dissolving and carries out first time after sample through silica gel column chromatography, first
Secondary silica gel column chromatography is with methylene chloride-methanol volume ratio 500:1 to 1:1 is used as gradient elution, collects wherein methylene chloride-methanol
Volume ratio 20:1 to 10:1 elution fraction carries out second of silica gel column chromatography, and second of silica gel column chromatography is with methylene chloride-methanol
Volume ratio 50:1 to 1:1 is used as gradient elution, collects wherein methylene chloride-methanol volume ratio 10:1 elution fraction carries out third time
Silica gel column chromatography, third time silica gel column chromatography is with methylene chloride-methanol volume ratio 10:1 to 1:1 is used as gradient elution, collection to wash
De- component (column chromatography mainly removes a small amount of impurity herein, is not grouped point, it is main only there are one ingredient, but purity is inadequate,
Subsequent Toyopeal purifying for the inadequate compound of this purity i.e. for continuing to purify) carry out Toyopearl column chromatographies
Purifying, obtains imidazoles dimer alkaloid 12.0mg shown in formula I；
The methylene chloride-methanol volume ratio 30 that first time silica gel column chromatography is obtained:1 to 20:1 elution fraction carries out the 4th
Secondary silica gel column chromatography, the 4th silica gel column chromatography is with methylene chloride-methanol volume ratio 70:1 to 1:1 is used as gradient elution, collects
Wherein methylene chloride-methanol volume ratio 10:1 elution fraction carries out Sephadex LH-20 gel filtration chromatographies, Sephadex LH-
20 gel filtration chromatographies rush column with methanol, collect a component being eluted out at first and carry out Toyopearl column chromatography purifying, obtain
Imidazoles dimer alkaloid 11.5mg shown in formula II；
The methylene chloride-methanol volume ratio 20 that 4th silica gel column chromatography is obtained:1 elution fraction carries out the 5th silica gel
Column chromatography, the 5th silica gel column chromatography is with methylene chloride-methanol volume ratio 40:1 to 1:1 is used as gradient elution, collects elution group
Divide (being equally regardless of component here) to carry out Toyopearl column chromatography purifying, obtains imidazoles dimer alkaloid shown in formula III
8.0mg。
The character of 1.3 imidazoles dimer alkaloids is verified
1.3.1 the characteristic of imidazoles dimer alkaloid shown in formula I is as follows：
1), white powder dissolves in first alcohol and water, does not dissolve in DMSO；
2), 257-258 DEG C of fusing point；HR-ESI-MS:m/z 349.20895([M+H]+,calcd for C15H25N8O2 +,
349.21005),371.19019([M+Na]+,calcd for C15H24N8O2Na+,371.19199).Spectroscopic data of the nuclear magnetic resonance
It is shown in Table 1；Crystal data and structure refinement are shown in Table 2；X-ray crystal structure is shown in attached drawing 1.
(1H NMR are in 600MHz, 13C for the spectroscopic data of the nuclear magnetic resonance of imidazoles dimer alkaloid shown in 1. formula I of table
NMR is tested under the conditions of 125MHz, and δ units are ppm, and coupling constant J units are Hz, and solvent is deuterated methanol).
| Position | δH(J, Hz) | δC |
| 2 | 7.54s | 137.4 |
| 2' | 7.54s | 137.4 |
| 4 | 138.6 | |
| 4' | 138.6 | |
| 5 | 124.1 | |
| 5' | 124.1 | |
| 6 | 3.56s | 32.9 |
| 6' | 3.56s | 32.9 |
| 8 | 2.95s | 36.6 |
| 8' | 2.95s | 36.6 |
| 9 | 160.8 | |
| 9' | 160.8 | |
| 10 | 5.98br s | |
| 10' | 5.98br s | |
| 11 | 2.63d(4.2) | 28.0 |
| 11' | 2.63d(4.2) | 28.0 |
| 12 | 3.82s | 17.6 |
The crystal data of imidazoles dimer alkaloid shown in 2. formula I of table
All spectral datas pass through1H-1The ID NMR speetnas such as H COSY, HMQC and HMBC belong to, it was demonstrated that gained
The structure of compound.
1.3.2 the characteristic of imidazoles dimer alkaloid shown in formula II is as follows：
1), white powder dissolves in methanol, chloroform and DMSO；
2), 244-245 DEG C of fusing point；HR-ESI-MS:m/z 392.25102([M+H]+,calcd for C17H30N9O2 +,
392.25225),414.23422([M+Na]+,calcd for C17H29N9O2Na+,414.23419),805.47712([2M+
Na]+,calcd for C34H58N18O4Na+,805.47861).Spectroscopic data of the nuclear magnetic resonance is shown in Table 3；Crystal data and structure essence
It repaiies and is shown in Table 4；X-ray crystal structure is shown in attached drawing 2.
Imidazoles dimer alkaloid shown in 3. formula II of table spectroscopic data of the nuclear magnetic resonance (1H NMR in 600MHz,13C
NMR is tested under the conditions of 125MHz, and δ units are ppm, and coupling constant J units are Hz, and solvent is deuterated chloroform).
The crystal data of imidazoles dimer alkaloid shown in 4. formula II of table
All spectral datas pass through1H-1The ID NMR speetnas such as H COSY, HMQC and HMBC belong to and X-ray
Crystal data parses, it was demonstrated that the structure of gained compound.
1.3.3 the characteristic of imidazoles dimer alkaloid shown in formula III is as follows：
1), faint yellow oily dissolves in methanol, chloroform and DMSO；
2)、HR-ESI-MS:m/z 371.19084([M+Na]+,calcd for C15H24N8O2Na+,371.19199).Core
Resonance spectroscopic data is shown in Table 5.
Imidazoles dimer alkaloid shown in 5. formula III of table spectroscopic data of the nuclear magnetic resonance (1H NMR in 600MHz,13C
NMR is tested under the conditions of 125MHz, and δ units are ppm, and coupling constant J units are Hz, and solvent is deuterated DMSO).
| Position | δH(J, Hz) | δC |
| 2 | 7.49s | 136.37 |
| 4 | 139.90 | |
| 5 | 121.49 | |
| 7 | 158.08 | |
| 9 | 3.90s | 47.91 |
| 11 | 151.92 | |
| 13 | 7.56s | 138.34 |
| 15 | 115.41 | |
| 16 | 160.76 | |
| N8-H | 5.66q(4.8) | |
| N17-H | 8.04q(4.8) | |
| N1-CH3 | 3.53s | 32.05 |
| N6-CH3 | 2.81s | 36.03 |
| N8-CH3 | 2.47d(4.8) | 27.59 |
| N10-CH3 | 2.51s | 43.44 |
| N14-CH3 | 3.72s | 34.74 |
| N17-CH3 | 2.72d(4.8) | 25.57 |
All spectral datas pass through1H-1The ID NMR speetnas such as H COSY, HMQC and HMBC belong to, it was demonstrated that gained
The structure of compound.
Embodiment 2
The neurotrosis Protection of the external hydrogen peroxide-induced of imidazoles dimer alkaloid of the present invention
Cell culture：Human neuroblastoma (SH-SY5Y) is taken, with dual anti-containing 10% fetal calf serum and 1%
Dulbecco ＇ s modified Eagle (DMEM) culture medium, with certain condition, (37 DEG C of temperature, gas concentration lwevel 5% are wet
95%) degree is incubated in carbon dioxide constant incubator.It is passed in 96 orifice plates when cell density is to 80%-90%, it is thin per hole
Born of the same parents' number about 2 × 104It is a.It is being separately added into 200 μM of H2O220 μ L3- (4,5- are added after 24 hours with 1 μM of compound culture
Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT, 5mg/mL), 37 degree of lower cultures 4
Hour, it removes culture medium and 150 μ L dimethyl sulfoxide (DMSO)s is added.Absorbance value is surveyed at 490nm.
As shown in figure 3, relative to control group (Basal), can succeed inducing nerve cell damage after addition hydrogen peroxide, damage
Wound model is successfully established.Huperzine (huperzine A), which is added, and organizes has apparent neurocyte protection effect；It is added such as formula
I, formula II, imidazoles dimer alkaloid group shown in formula III are also shown with preferable Neuroprotective effect, to illustrate this
Invention imidazoles dimer alkaloid is to being there is certain protective effect by the neurotrosis of hydrogen peroxide-induced.
Above-mentioned experiment proves that the product that the present invention is prepared imidazoles dimer as shown in formula I, formula II, formula III is biological
Alkali has stronger protective effect to neurotrosis, they can be applied to the preparation in terms of inhibition neurotrosis drug.
It, can be by the imidazoles dimer alkaloid as shown in formula I, formula II, formula III by medically acceptable in specific implementation
A kind of nerve protection medicine is made in dosage and pharmaceutically general auxiliary material.The pharmaceutical dosage form includes oral type and injection-type etc..
The oral type includes tablet, capsule, granule, pill etc.；The injection-type includes injection, mixed rotary liquid etc..Specifically
Preparation method is prepared with reference to the conventional method of pharmaceutical field.
It should be understood that example as described herein and embodiment are not intended to restrict the invention, this field only for explanation
Technical staff can make various modifications or variation according to it, all within the spirits and principles of the present invention, made by it is any modification,
Equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
Claims (9)
1. imidazoles dimer alkaloid, which is characterized in that the representation shown in formula I or formula II or formula III：
2. application of the imidazoles dimer alkaloid as described in claim 1 in preparing nerve protection medicine.
3. neuroprotective medicine, which is characterized in that by imidazoles dimer alkaloid as described in claim 1 and medicinal auxiliary
Material is made.
4. neuroprotective medicine as claimed in claim 3, which is characterized in that the pharmaceutical dosage form of the drug includes oral
Type and injection-type.
5. the preparation method of imidazoles dimer alkaloid as described in claim 1, which is characterized in that include the following steps：
(1) raw material crushes
Keemun black tea is taken, processing is crushed to it, obtains Keemun black tea powder；
(2) it extracts
Keemun black tea powder is extracted with ethanol water, obtains extracting solution, by being dried, obtained Keemun black tea carries extracting solution
Take object；
(3) it isolates and purifies
Keemun black tea extract is dissolved with methanol aqueous solution, then the most of methanol of concentration removal, uses petroleum ether extraction later
Small polar substances and pigment are removed, is finally extracted with dichloromethane, dichloromethane position extractum A is obtained；
By the extractum A water dissolution of dichloromethane position, aqueous tartaric acid solution is added, removes insoluble matter, uses dichloromethane again later
It is extracted, isometric sodium hydrate aqueous solution, which is added, after removal water layer is extracted, and dichloromethane layer and buck layer, alkali are obtained
Water layer is extracted with dichloromethane again after ammonium chloride is added, and obtains dichloromethane position medicinal extract B；
Separation and purification treatment is carried out to dichloromethane position medicinal extract B, obtains the imidazoles dimer alkaloid.
6. the preparation method of imidazoles dimer alkaloid as claimed in claim 5, which is characterized in that in step (2), second used
A concentration of the 95% of alcohol solution.
7. such as the preparation method of imidazoles dimer alkaloid described in claim 5 or 6, which is characterized in that in step (3), institute
With a concentration of the 2% of aqueous tartaric acid solution, a concentration of 1-2% of sodium hydrate aqueous solution.
8. such as the preparation method of imidazoles dimer alkaloid described in claim 5 or 6, which is characterized in that in step (3), point
Include silica gel column chromatography, Sephadex LH-20 gel filtration chromatographies, Toyopearl column chromatographies from purification process.
9. the preparation method of imidazoles dimer alkaloid as claimed in claim 8, which is characterized in that the tool of separation and purification treatment
Body step is：First time silica gel column chromatography is carried out after dichloromethane position medicinal extract B is dissolved, first time, silica gel column chromatography was with dichloro
Methane-methanol volume ratio 500:1 to 1:1 is used as gradient elution, collects wherein methylene chloride-methanol volume ratio 20:1 to 10:1 washes
De- component carries out second of silica gel column chromatography, and second of silica gel column chromatography is with methylene chloride-methanol volume ratio 50:1 to 1:1 conduct
Gradient elution collects wherein methylene chloride-methanol volume ratio 10:1 elution fraction carries out third time silica gel column chromatography, third time silicon
Plastic column chromatography is with methylene chloride-methanol volume ratio 10:1 to 1:1 is used as gradient elution, collects elution fraction and carries out Toyopearl
Column chromatography purifies, and obtains imidazoles dimer alkaloid shown in formula I；
The methylene chloride-methanol volume ratio 30 that first time silica gel column chromatography is obtained:1 to 20:1 elution fraction carries out the 4th silicon
Plastic column chromatography, the 4th silica gel column chromatography is with methylene chloride-methanol volume ratio 70:1 to 1:1 is used as gradient elution, collects wherein
Methylene chloride-methanol volume ratio 10:1 elution fraction carries out Sephadex LH-20 gel filtration chromatographies, and Sephadex LH-20 are solidifying
Plastic column chromatography rushes column with methanol, collects a component being eluted out at first and carries out Toyopearl column chromatography purifying, obtains formula II
Shown in imidazoles dimer alkaloid；
The methylene chloride-methanol volume ratio 20 that 4th silica gel column chromatography is obtained:1 elution fraction carries out the 5th silica gel column layer
Analysis, the 5th silica gel column chromatography is with methylene chloride-methanol volume ratio 40:1 to 1:1 be used as gradient elution, collect elution fraction into
Row Toyopearl column chromatographies purify, and obtain imidazoles dimer alkaloid shown in formula III.
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| WO2012148140A2 (en) * | 2011-04-29 | 2012-11-01 | 주식회사 레고켐 바이오사이언스 | Imidazole-based alkaloid derivatives which have angiogenesis inhibition and antioxidant effects and production method thereof |
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