CN104250241A - Preparation method for CB1 antagonist EGCG and prepared drug - Google Patents

Preparation method for CB1 antagonist EGCG and prepared drug Download PDF

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CN104250241A
CN104250241A CN201310566708.9A CN201310566708A CN104250241A CN 104250241 A CN104250241 A CN 104250241A CN 201310566708 A CN201310566708 A CN 201310566708A CN 104250241 A CN104250241 A CN 104250241A
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egcg
antagonist
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cell
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CN104250241B (en
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李晶
封玉玲
肖忠华
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Chongqing Three Gorges Medical College
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Chongqing Three Gorges Medical College
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/58—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4
    • C07D311/60—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with aryl radicals attached in position 2
    • C07D311/62—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with aryl radicals attached in position 2 with oxygen atoms directly attached in position 3, e.g. anthocyanidins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor

Abstract

The invention discloses a preparation method for a CB1 antagonist EGCG and the prepared drug for cerebrovascular disease treatment, smoking cessation and weight losing. The main steps include: construction of recombinant human CB1 eukaryotic expression vector; preparation of a CB1 high expression HEK293 cell membrane and a cell membrane chromatographic column; affinity chromatography separation and preparation of EGCG. The Chinese herbal monomer molecular cannabinoid receptor CB1 antagonist EGCG prepared by the method provided by the invention has mild effect, extremely small adverse reaction, and good development prospects and use value.

Description

A kind of preparation method of CB1 antagonist EGCG and the medicine of preparation
Technical field
The invention belongs to CB1 antagonist Application Areas, particularly relate to preparation method and the treatment cerebrovascular disease prepared with it, smoking cessation and the slimming medicine of a kind of CB1 antagonist EGCG.
Background technology
Cannabinoid receptors is a kind of g protein coupled receptor, has two kinds of subtype acceptor CB1 and CB2.CB1 acceptor is expressed in the peripheral tissues such as central nervous system, lung, liver, kidney.Maincenter CB1 acceptor main with depress appetite, reduce and ingest relevant, and periphery CB1 acceptor is mainly and the metabolism of control agent self-energy, loses weight relevant.CB1 acceptor is the acceptor that a class affects mankind's appetite and energy metabolism, Rimonabant is that first selectively acting is in the antagonist of CB1 acceptor, CB1 antagonist is the important target for the treatment of of obesity, smoking cessation and treatment Imaging in Patients with Cerebral Ischemia Disease, and selectively acting receives in the antagonist of CB1 acceptor and pays close attention to widely.
The method of current screening CB1 antagonist mainly pharmaceutical chemical method and set up CB1 cell screening model and carry out high-flux medicaments sifting.Specific as follows:
One, Academy of Military Medicine, PLA/Military Medical Science Institute of PLA, pharmaceutical chemistry Master postgraduate Tao Xiaoxuan, the Master's thesis such as Zheng Zhibing " design of CB1 receptor antagonist, synthesis and evaluated biological activity ", by the interaction of CB1 receptor antagonist and its acceptor, establish the structure activity relationship of " 4-methyl-1 H-diaryl pyrazoles " CB1 receptor antagonist.Based on the structure activity relationship set up, and taking into full account under the prerequisite such as the structural similarity of CB1 and CB2, the film ability excessively of antagonist, using Rimonabant as lead compound, structural modification is carried out to its pyrazole ring 3, devise three classes such as comprising quaternary ammonium salt, urea groups class and guanidine base class and there is compound compared with high-hydrophilic, expect to obtain there is high-affinity and reduce maincenter side effect selectively acting to greatest extent in the CB1 receptor antagonist of peripheral tissues not easily through hemato encephalic barrier.
Two, East China University of Science's Pharmaceutical Engineering Speciality, the Master's thesis such as Hu Hui, Zhu Weihong " cannaboid 1(CB1) screening of acceptor small molecular antagonists and model thereof set up ".They set up CB1 acceptor small molecular antagonists Screening Platform, specific as follows:
1) by cotransfection CB1 acceptor and Ga15 plasmid on CHO-K1 cell strain, the cell strain (hCB1R-CHO) of stably express CB1 acceptor and Ga15 is set up;
2) hCB1R-CHO cell strain is utilized, set up based on detection Ca2+ oscillations CB1 receptor antagonist screening model, and various experiment condition (concentration and fluorescence dye incubation time etc. as cell density, Fluo-4AM) is optimized, make it the requirement meeting high-flux medicaments sifting;
3) model of application foundation, explores the novel C B1 receptor selective antagonists that screening specificity is strong, avidity is high, toxic side effect is low.Apply this model, paper has screened 364 micromolecular compounds, do further checking by screening the active compound obtained through GTP-Eu Competition binding assay, toxicity test, cross-over experiment etc., successfully find a novel C B1 receptor antagonist, its IC50 value reaches 1.3 μMs.
At present, existing medicine Rimonabant untoward reaction is obvious, is suspended sale of in the world.EGCG, by antagonism CB1 acceptor, can be used for cerebrovascular disease, fat-reducing and smoking cessation, has no report both at home and abroad.How integrating out the CB1 antagonist screening route of complete set, obtaining that one can be used for preventing and treating cerebrovascular disease by acting on CB1 generation, the new drug of fat-reducing and smoking cessation is the difficult problem that annoying people in the recent period.
Summary of the invention
The object of the embodiment of the present invention is to provide a kind of for verifying that CB1 antagonist EGCG treats the method for cerebrovascular disease, smoking cessation and fat-reducing effect, being intended to solve the CB1 antagonist screening route how integrating out complete set that annoying people in the recent period, obtaining the problem of new drug that can be used for preventing and treating by acting on CB1 generation cerebrovascular disease, fat-reducing and smoking cessation.
The embodiment of the present invention is achieved in that the preparation method of a kind of CB1 antagonist EGCG, and this antagonist EGCG preparation method comprises step:
Step one, the structure of recombinant human CB1 carrier for expression of eukaryon;
The extraction of total tissue RNA: get cerebral tissue and to weigh 108mg, add 1.0ml Trizol, put into homogenizer in grinding on ice, proceed to 1.5mlEP pipe, mixing, 200 μ l chloroforms are added after leaving standstill 5min, refrigerated centrifuge, 4 DEG C, 12000rpm, centrifugal 15min, get upper water and move into EP pipe mutually, add isopyknic Virahol, mixing, room temperature leaves standstill 1Omin, low-temperature centrifugation, add 75% washing with alcohol precipitation of the 0.1%DEPC water preparation of 1ml precooling, the air-dry 30min of super clean bench, add 400 μ l DEPC process water dissolution precipitations, agarose gel electrophoresis, observe under ultraviolet lamp, judge the integrity of RNA, ultraviolet spectrophotometer measures OD value and calculates RNA concentration,-80 DEG C save backup,
Design of primers and synthesis: announce people CB1 gene order according to Genbank and design primer, add HindIII enzyme point of contact at 5 ' of upstream primer, downstream primer 5 ' adds EcoR I enzyme point of contact, synthesized by Shanghai Ying Jun Bioisystech Co., Ltd;
The structure of pcDNA3.1-hCB1: Reverse Transcriptase kit reverse transcription obtains total cDNA, carries out PCR and clones CB1 gene, reaction conditions: 94 DEG C of denaturation 4min, 94 DEG C of sex change 30s, 56 DEG C of annealing 30s, 74 DEG C extend 45s, last 74 DEG C extend 7min, expection fragment length 1400bp, product 1% agarose gel electrophoresis, cut glue and reclaim kits with DNA gel and reclaim, the object fragment HindIII reclaimed and EcoR I double digestion 2h, reclaim purifying, cut pcDNA3.1 with HindIII and EcoR I enzyme enzyme simultaneously, reclaim purified linear plasmid vector, obtained endonuclease bamhi is connected 24h with 3: 1 ratios T4DNA Ligase at 20 DEG C, product conversion will be connected in competence DH5 α, coating is containing the LB solid medium of penbritin, the single positive colony of picking the recombinant plasmid that increases, plasmid kit extracts pcDNA3.1-hCB1, double digestion PCR identifies, carry out order-checking qualification,
CB1 eukaryotic expression vector transfection: recombinant vectors pcDNA3.1-hCB1 is transiently transfected into HEK293 cell, transfection employing Lipofectamine2000 reagent carries out, and 48h collecting cell after transfection, prepares cell pyrolysis liquid and total serum IgE carries out next step analysis;
Step 2, the preparation of CB1 high expression level HEK293 cytolemma and membrane flexibility post:
Be taken at 37 DEG C, 5%CO2 and bag and appropriate condition under, be incubated in the DMEM nutrient solution containing 10% foetal calf serum, routine passage, cell attachment growth individual layer to cover bottom culture dish 90%, 0.25 trypsin solution digestion, collecting cell, blood counting chamber counts, get 8 × 106 cells, 4 DEG C, the centrifugal 8min of 1000g, add 10mL precooling phosphoric acid buffer piping and druming suspendible, the same centrifugal 10min of condition, repeat aforesaid operations once, incline and supernatant liquor and add 8mL hypotonic buffer liquid, homogenate in ice cube, low temperature ultrasonic rupture of membranes 30min, the centrifugal 20min of 12000g again under 4 DEG C of conditions, to incline upper liquid, add 5mLTris-HCl damping fluid resuspended, for subsequent use, spectrophotometer 680nm place measures tissue sample and bovine serum albumin reference liquid, measure optical density value, dying method with coomassie brilliant blue measures Membrane protein's amount, get chromatographic column post core, obtained CB1 cell chromatographic column,
Step 3, affinity chromatography is separated, preparation EGCG
Self-control membrane flexibility post is received high performance liquid chromatograph, tea leaf extract millipore filtration 0.45 μm filtration, sample introduction 20 μ L, chromatographic condition is: column temperature 37 DEG C; Flow velocity 0.5mLmin -1, pH7.4, moving phase phosphate buffered saline buffer 50mmolmin -1, ultraviolet detection wavelength 280nm sample introduction 20 μ L after moving phase balance 2h is separated, and collects 3.5-4.0min elutriant, namely obtains CB1 selective antagonist EGCG.
Further, CB1 upstream primer CNR1-f:
5 '-CGGGATCCGCCACCATGAAGTCGATCCTAGATG-3 ' downstream primer CNR1-r:5 '-GGAATTCCTTTTTCTGTGCAGCCACAA-3 '.
Further, the authentication method of antagonist EGCG is:
Infrared spectroscopy: results of IR EGCG is at 1690cm -1there is the last one stretching vibration absorption peak υ at place, at 1190 ~ 1240cm -1there is the asymmetric stretching vibration absorption peak υ of a group stronger at place, at 1450 ~ 1600cm -1there is phenyl ring framework characteristic vibration absorption peak at place, and spectrogram is consistent with EGCG standard diagram, and the preliminary confirmation CB1 antagonist agent that obtains is EGCG thus,
Uv-absorbing wavelength detecting: get sample separation, measures maximum absorption wavelength with ultraviolet-visible chromatographic instrument, finds there is maximum absorption at 273.4nm place;
High-efficient liquid phase chromatogram technique analysis: adopt C18 chromatographic column, moving phase is methyl alcohol: water: acetic acid (23: 75: 2, v/v), flow velocity 1.0mL.min -1, column temperature 35 DEG C, determined wavelength is 272nm, and high performance liquid chromatography quantitative analysis affinity chromatography is separated EGCG purity and reaches more than 98%;
Nuclear magnetic resonance map 3.00 (1H) .5.11 (1H) of nuclear magnetic resonance spectroscopy: EGCG, 5.53 (1H), 6.05 (2H), 6.65 (2H), 7.09 (2H).
Further, the using method of antagonist EGCG is: after water solvent dissolves, and uses to rat, and route of administration is abdominal injection/oral.
The cerebrovascular disease that another object of the present invention is to provide a kind of preparation method of above-mentioned CB1 antagonist EGCG to prepare, smoking cessation and slimming medicine.
Herbal medicine monomer molecule cannabinoid receptors CB1 antagonist EGCG prepared by the present invention, action temperature and, untoward reaction is extremely small, has good development prospect and use value.
Accompanying drawing explanation
Fig. 1 be the embodiment of the present invention provide for verifying that CB1 antagonist EGCG treats the method flow diagram of cerebrovascular disease, smoking cessation and fat-reducing effect.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with embodiment, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
Fig. 1 shows provided by the invention for verifying that CB1 antagonist EGCG treats the flow process of method of cerebrovascular disease, smoking cessation and fat-reducing effect.For convenience of explanation, illustrate only part related to the present invention.
Embodiments of the invention provide for verifying that CB1 antagonist EGCG treats the method for cerebrovascular disease, smoking cessation and fat-reducing effect, the method comprises the following steps:
S101: the eukaryotic expression HEK293 cell building CB1 acceptor;
S102: with HEK293 cell by cellular membrane chromatography, screening and separating goes out CB1 part nutgall catechin gallic acid ester EGCG;
S103: verify that this part EGCG is CB1 antagonist through disposable calcium current detection kit;
S104: checking EGCG anti-cerebral ischemia, antiobesity action; Carry out the randomized, double-blind experiment that EGCG stops smoking effect, checking EGCG function of smoking cessation.
In step S104, the method for checking EGCG treating cerebral ischemia is:
Adopt bolt line to block SD rat, middle cerebral artery occlusion method MCAO method sets up evaluating focal brain ischemia in rats, and each treated animal is drug administration by injection 2d in advance, and before modeling 0.5h, 4h, 12h administration 3 times respectively again after modeling;
The laggard row neurological deficit score of modeling 24h, measures the cerebral tissue water yield and cerebral infarct size percentage when carries out pathological examination, and detects the change of MCAO rat brain volume of blood flow with laser Doppler flowmetry.
In step S104, measuring EGCG to the method for the antiobesity action of high fat diet obesity-induced mice is:
Mice model of obesity is set up to the induction of high fat diet method, gives EGCG30mg/kg treatment 4 weeks, the change of detection bodies quality change and surrounding genital lipid content, adipocyte form and serum cholesterol, triacylglycerol content.
In step S104, foundation and the clustering method of obese model are as follows:
ICR mouse is divided at random Normal group and high lipid food group, normal group all gives normal diet to end and feeds from experiment, and high lipid food group gives high lipid food and feeds, and modeling maintains 2 months;
After modeling terminates, getting the mouse exceeding normal group mouse average body quality 20% is obese model;
Filter out qualified mouse 40, be divided into 4 groups at random, give solvent, EGCGmg/kg group, 10mg/kg Rimonabant respectively; Normal group 10 mouse stomaches give solvent, and administration maintains 4 weeks.
In step S104, the Indexs measure of 4 combination lattice mouse is as follows:
Accumulative food consumption quantity in 12h is monitored, record per hour 1 time after first administration;
Within the whole cycle, every day, the food ration of mouse and the weight of every mouse often organized in record, and observe the active situation of mouse;
Fasting 12h after terminating, extracts eyeball and gets blood, measure serum triacylglycerol, total cholesterol, strips out whole Right Lower Abdomen butt crack fatty tissue, surrounding genital fatty tissue weighing;
Get all fatty 2 fritters, 4% formalins of sexual organ to fix, paraffin section, HE dyes, and compares same field of view fat cell hypertrophy form.
In step S104, EGCG is as follows to smoking cessation effect double-blind study method:
Adopt randomized, double-blind placebo-controlled study method, to meet 43 examples into set condition and the smoker signing Informed Consent Form is divided into EGCG group and treatment group, 200mg/ sheet and placebo, EGCG group 25 is routine, placebo 18 is routine, 12 weeks courses for the treatment of;
Observe different time point successful quitting rate smoking capacity≤1 of treatments period/d, smoking reduce effective rates during smoking capacity comparatively treat the ratio of front minimizing >=50%, and evaluate visuality and crave for intensity test and appraisal table;
To urinate cotinine and expiration carbon monoxide concentration checking tobacco smoking status.
The step of the preparation method of the antagonist of the embodiment of the present invention is:
1, the structure of recombinant human CB1 carrier for expression of eukaryon
The extraction of 1.1 total tissue RNA: get cerebral tissue and to weigh 108mg, add 1.0ml Trizol, put into homogenizer in grinding on ice, proceed to 1.5mlEP pipe, mixing, 200 μ l chloroforms are added after leaving standstill 5min, refrigerated centrifuge (4 DEG C, 12000rpm) centrifugal 15min, get upper water and move into EP pipe mutually, add isopyknic Virahol, mixing, room temperature leaves standstill 1Omin, low-temperature centrifugation, add 75% washing with alcohol precipitation of the 0.1%DEPC water preparation of 1ml precooling, the air-dry 30min of super clean bench, add 400 μ l DEPC process water dissolution precipitations, agarose gel electrophoresis, observe under ultraviolet lamp, judge the integrity of RNA, ultraviolet spectrophotometer measures OD value and calculates RNA concentration,-80 DEG C save backup,
1.2 design of primers and synthesis: announce people CB1 gene order according to Genbank and design primer, add HindIII enzyme point of contact at 5 ' of upstream primer, downstream primer 5 ' adds EcoR I enzyme point of contact, synthesized by Shanghai Ying Jun Bioisystech Co., Ltd;
CB1 upstream primer CNR1-f:5 '-CGGGATCCGCCACCATGAAGTCGATCCTAGATG-3 ' downstream primer CNR1-r:5 '-GGAATTCCTTTTTCTGTGCAGCCACAA-3 ';
The structure of 13pcDNA3.1-hCB1: Reverse Transcriptase kit reverse transcription obtains total cDNA, carries out PCR and clones CB1 gene, reaction conditions: 94 DEG C of denaturation 4min, 94 DEG C of sex change 30s, 56 DEG C of annealing 30s, 74 DEG C extend 45s, last 74 DEG C extend 7min, expection fragment length 1400bp, product 1% agarose gel electrophoresis, cut glue and reclaim kits with DNA gel and reclaim, the object fragment HindIH reclaimed and EcoR I double digestion 2h, reclaim purifying, cut pcDNA3.1 with HindIII and EcoR I enzyme enzyme simultaneously, reclaim purified linear plasmid vector, above-mentioned two endonuclease bamhis that step obtains are connected 24h with 3: 1 ratios T4DNALigase at 20 DEG C, product conversion will be connected in competence DH5 α, coating is containing the LB solid medium of penbritin, the single positive colony of picking the recombinant plasmid that increases, plasmid kit extracts pcDNA3.1-hCB1, double digestion PCR identifies, Bao Bio-Engineering Company is sent to carry out order-checking qualification,
1.4CB1 eukaryotic expression vector transfection: recombinant vectors pcDNA3.1-hCB1 is transiently transfected into HEK293 cell, transfection adopts Lipofectamine2000 reagent to carry out, concrete grammar is see Lipofectamine2000 working instructions, 48h collecting cell after transfection, prepares cell pyrolysis liquid and total serum IgE etc. and carries out next step experimental analysis;
2, the preparation of CB1 high expression level HEK293 cytolemma and membrane flexibility post:
Be taken at 37 DEG C, 5%CO2 and bag and appropriate condition under, be incubated in the DMEM nutrient solution containing 10% foetal calf serum, routine passage, cell attachment growth individual layer covers about 90%, 0.25 trypsin solution digestion bottom culture dish, collecting cell, blood counting chamber counts, and gets about 8 × 10 6cell, 4 DEG C, the centrifugal 8min of 1000g, add 10mL precooling phosphoric acid buffer piping and druming suspendible, the same centrifugal 10min of condition, repeat aforesaid operations once, incline and supernatant liquor and add 8mL hypotonic buffer liquid, homogenate in ice cube, low temperature ultrasonic rupture of membranes 30min, the centrifugal 20min of 12000g again under 4 DEG C of conditions, to incline upper liquid, add 5mLTris-HCl damping fluid resuspended, for subsequent use, spectrophotometer 680nm place measures tissue sample and bovine serum albumin reference liquid, measure optical density value, dying method with coomassie brilliant blue measures Membrane protein's amount, get chromatographic column post core, obtained CBl cell chromatographic column,
3, affinity chromatography is separated, prepares EGCG
Self-control membrane flexibility post is received high performance liquid chromatograph, and Wuxi, Chongqing City then (2013 produce per year) tea leaf extract millipore filtration (0.45 μm) filters, sample introduction 20 μ L, and chromatographic condition is: column temperature 37 DEG C; Flow velocity 0.5mLmin -1, pH7.4, moving phase phosphate buffered saline buffer 50mmo1min -1, ultraviolet detection wavelength 280nm sample introduction 20 μ L after moving phase balance 2h is separated, and collects 3.5-4.0min elutriant, namely obtains CBl selective antagonist EGCG.
The authentication method of antagonist EGCG is:
Infrared spectroscopy: results of IR EGCG is at 1690cm -1there is the last one stretching vibration absorption peak υ at place, at 1190 ~ 1240cm -1there is the asymmetric stretching vibration absorption peak υ of a group stronger at place, at 1450 ~ 1600cm -1there is phenyl ring framework characteristic vibration absorption peak at place, and spectrogram is consistent with EGCG standard diagram, and the preliminary confirmation CBl antagonist agent that obtains is EGCG thus.
Uv-absorbing wavelength detecting: get sample separation, measures maximum absorption wavelength with ultraviolet-visible chromatographic instrument, finds there is maximum absorption at 273.4nm place, consistent with the Pharmacopoeia of the People's Republic of China.
High-efficient liquid phase chromatogram technique analysis: adopt C18 chromatographic column (4.6mm × 250mm, 5 μm), moving phase is methyl alcohol: water: acetic acid (23: 75: 2, v/v), flow velocity 1.0mL.min -1, column temperature 35 DEG C, determined wavelength is 272nm.High performance liquid chromatography quantitative analysis affinity chromatography is separated EGCG purity and reaches more than 98%.
Nuclear magnetic resonance map 3.00 (1H) .5.11 (1H) of nuclear magnetic resonance spectroscopy (1HNMR): EGCG.5.53(1H)，6.05(2H)，6.65(2H)，7.09(2H)。Measurement result is consistent with bibliographical information.
The above analysis identify, institute adopt affinity chromatography separation the material that obtains be CB1 antagonist EGCG.
The using method of antagonist:
The EGCG used at present is after mainly water solvent dissolves, and use to rat, route of administration is abdominal injection/oral (gavage).
Below in conjunction with drawings and the specific embodiments, application principle of the present invention is further described.
1, cerebro-vascular diseases proves experiment
1.1 test methods: adopt bolt line blocking experiment model SD rat, middle cerebral artery occlusion method (MCAO method) sets up evaluating focal brain ischemia in rats, each treated animal is drug administration by injection 2d in advance, and before modeling 0.5h, 4h, 12h respectively administration 3 times again after modeling.The laggard row neurological deficit score of modeling 24h, measures the cerebral tissue water yield and cerebral infarct size percentage when carries out pathological examination, and detects the change of MCAO rat brain volume of blood flow with laser Doppler flowmetry.
1.2 experimental results:
1.2.1 on the impact of neurological deficit score and brain water content
After MCAO rat anesthesia is clear-headed, visible hemiplegia sample symptom occurs, main manifestations is operation offside forelimb adduction in various degree, shoulder inward turning, and muscular tension reduces, and pushes away right shoulder and moves to offside, and resistance declines, and Some Animals is turn-taked to offside.Neurological deficits and brain water content mensuration are carried out to rat model.
Result shows: model group Neurological deficits and brain water content are tieed up apparently higher than sham-operation, and EGCG injection group compares with model group, and the neuroethology obstacle of rat obviously improves, and brain water content significantly reduces.Prompting EGCG has obvious provide protection to MCAO rat.
1.2.2 on the impact of cerebral infarct size
After the administration of EGCG injection group, contrast rats in sham-operated group, cerebral infarct size percentage significantly declines.
1.2.3 on the impact of Pathologic histology of brain
Rats in sham-operated group neurone and brain parenchymal cell structure normal, kernel is clear, even dyeing, and cell peripheral gap slightly expands.Under model control group rat encephaloscope, visible obviously big area softens stove, and what have is mesh-like.Cell peripheral gap obviously expands, swelling, and nissl bodies reduces or disappears, nuclear hyperchromatism, pyknosis or dissolving.Cerebral tissue vasodilation, obviously fills big belly.EGCG group rat cerebral tissue softens stove and obviously reduces, how collections infarct.Cell peripheral gap is slightly broadening, right neuron unit and brain parenchymal cell, increases around capillary coil pipe, neuronal cell core mild swelling, and kernel is clear, visible glial cells hyperplasia, and in sex change, oedema changes.
To sum up result, and other researchists existing, prove that EGCG can be used for the prevention and therapy of cerebrovascular disease by antagonism CB1 acceptor.
2, measure EGCG to study the weight-reducing experiment of high fat diet obesity-induced mice
Mice model of obesity is set up to the induction of high fat diet method, gives EGCG30mg/kg treatment 4 weeks, the change of detection bodies quality change and surrounding genital lipid content, adipocyte form and serum cholesterol, triacylglycerol content.Result: EGCG, by suppressing CB1 receptor exerts antiobesity action, significantly can reduce food intake and the weight of mouse, reduces the content of surrounding genital fat, serum cholesterol and triacylglycerol.Conclusion: EGCG, by suppressing CB1 acceptor, reduces appetite and produces antiobesity action, can reduce blood lipid level simultaneously.
The foundation of 2.1 obese models and grouping
ICR mouse is divided at random Normal group and high lipid food group.Normal group all gives normal diet to end and feeds from experiment, and high lipid food group gives high lipid food and feeds, and modeling maintains 2 months.After modeling terminates, getting the mouse exceeding normal group mouse average body quality 20% is obese model.Filter out qualified mouse 40, be divided into 4 groups at random, give solvent, EGCGmg/kg group, 10mg/kg Rimonabant respectively.Normal group 10 mouse stomaches give solvent, and administration maintains 4 weeks.
2.2 Indexs measure
Accumulative food consumption quantity in 12h is monitored, record per hour 1 time after first administration.Within whole experimental period, every day, the food ration of mouse and the weight of every mouse often organized in record, and observe the active situation of mouse.Experiment terminates rear fasting 12h, extracts eyeball and gets blood, measure serum triacylglycerol, total cholesterol.Strip out whole Right Lower Abdomen butt crack fatty tissue, surrounding genital fatty tissue weighing.Get all fatty 2 fritters, 4% formalins of sexual organ to fix, paraffin section, HE dyes, and compares same field of view fat cell hypertrophy form.
2.3 food ration impacts
EGCG30mg/kg mouse food ration at each time point lower than model group.Similar to the effect suppressing mouse to ingest in 12h with Rimonabant.Do the research of different dosing dosage group to find, EGCG30mg/kg group suppresses the effect of food ration the strongest.Compared to model group, in 12h, the mouse food ration of administration group obviously reduces, and the food ration of 30mg/kg and 10mg/kg Rimonabant group mouse 12h have dropped 38.4%, 43.0% respectively.
In 4 weeks, 30mg/kgEGCG group suppresses the effect of mouse food ration the strongest, and sustained drug inhibits the food ration of mouse, and the food ration of normal group and model group mouse does not occur restraining effect.Under same dose, the food restraining effect of Rimonabant is slightly stronger than EGCG.
2.4 medicines are on the impact of Mice Body quality
The weight of normal group and model group mouse remains stable.EGCG group and Rimonabant all have the effect reducing obesity mice weight.30mg/kgEGCG group is to the decline of weight about 8.0%.The effect of 10mg/kg Rimonabant is better than EGCG effect.
2.5 impacts on mouse body fat content
Compared to normal group, model group mouse body fat content significantly increases.The fat weight of EGCG and the rear mouse propagation device periphery of Rimonabant treatment declines, 30mg/kgEGCG significantly reduce fat content.
2.6 impacts on mouse adipocytes volume
Model group adipocyte diameter is comparatively large, and visual field inner cell number is less; After 30mg/kgEGCG treatment, mouse adipocytes diameter diminishes, and in cell, lipid content is less comparatively obvious.
2.7 impacts on mice serum triacylglycerol and cholesterol level
The obesity mice serum cholesterol of high fat diet induction and the content of triacylglycerol significantly raise.After the treatment of EGCG30mg/kg dosage group, the content of mice serum cholesterol and triacylglycerol significantly declines.
3, EGCG studies smoking cessation effect double blind experiment
Adopt randomized, double-blind placebo-controlled study method, 43 examples to be met into set condition and the smoker signing Informed Consent Form is divided into EGCG group (treatment group; 200mg/ sheet) and placebo, EGCG group 25 example, placebo 18 example, 12 weeks courses for the treatment of; Observe the different time point successful quitting rate of treatments period (smoking capacity≤1/d), smoking reduces efficient (treatments period smoking capacity comparatively treats the ratio of front minimizing >=50%), and evaluate visuality and crave for intensity test and appraisal table; To urinate cotinine and expiration carbon monoxide concentration checking tobacco smoking status.
Result shows: after treatment, treatment group and placebo successful quitting rate are respectively 47.19% and 12.71%, and smoking minimizing is efficient is respectively 37.41% and 11.82%, and two groups of differences have statistical significance (P<0.01); Treat 2nd ~ 12 weekend treatment group crave for the score of intensity, CO exhalation amount is all less than placebo (P<0.01); Treatment group urine cotinine content the 8th week and the 12nd week lower than placebo (P<0.01).Treatments period two groups of volunteers all occur without serious adverse events.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.

Claims (5)

1. a preparation method of CB1 antagonist EGCG, is characterized in that, this antagonist EGCG preparation method comprises step:
Step one, the structure of recombinant human CB1 carrier for expression of eukaryon;
The extraction of total tissue RNA: get cerebral tissue and to weigh 108mg, add 1.0ml Trizol, put into homogenizer in grinding on ice, proceed to 1.5mlEP pipe, mixing, 200 μ l chloroforms are added after leaving standstill 5min, refrigerated centrifuge, 4 DEG C, 12000rpm, centrifugal 15min, get upper water and move into EP pipe mutually, add isopyknic Virahol, mixing, room temperature leaves standstill 10min, low-temperature centrifugation, add 75% washing with alcohol precipitation of the 0.1%DEPC water preparation of 1ml precooling, the air-dry 30min of super clean bench, add 400 μ l DEPC process water dissolution precipitations, agarose gel electrophoresis, observe under ultraviolet lamp, judge the integrity of RNA, ultraviolet spectrophotometer measures OD value and calculates RNA concentration,-80 DEG C save backup,
Design of primers and synthesis: announce people CB1 gene order according to Genbank and design primer, add HindIII enzyme point of contact at 5 ' of upstream primer, downstream primer 5 ' adds EcoR I enzyme point of contact, synthesized by Shanghai Ying Jun Bioisystech Co., Ltd;
The structure of pcDNA3.1-hCB1: Reverse Transcriptase kit reverse transcription obtains total cDNA, carries out PCR and clones CB1 gene, reaction conditions: 94 DEG C of denaturation 4min, 94 DEG C of sex change 30s, 56 DEG C of annealing 30s, 74 DEG C extend 45s, last 74 DEG C extend 7min, expection fragment length 1400bp, product 1% agarose gel electrophoresis, cut glue and reclaim kits with DNA gel and reclaim, the object fragment HindIII reclaimed and EcoR I double digestion 2h, reclaim purifying, cut pcDNA3.1 with HindIII and EcoR I enzyme enzyme simultaneously, reclaim purified linear plasmid vector, obtained endonuclease bamhi is connected 24h with 3:1 ratio T4DNA Ligase at 20 DEG C, product conversion will be connected in competence DH5 α, coating is containing the LB solid medium of penbritin, the single positive colony of picking the recombinant plasmid that increases, plasmid kit extracts pcDNA3.1-hCB1, double digestion PCR identifies, carry out order-checking qualification,
CB1 eukaryotic expression vector transfection: recombinant vectors pcDNA3.1-hCB1 is transiently transfected into HEK293 cell, transfection employing Lipofectamine2000 reagent carries out, and 48h collecting cell after transfection, prepares cell pyrolysis liquid and total serum IgE carries out next step analysis;
Step 2, the preparation of CB1 high expression level HEK293 cytolemma and membrane flexibility post:
Be taken at 37 DEG C, 5%CO2 and bag and appropriate condition under, be incubated in the DMEM nutrient solution containing 10% foetal calf serum, routine passage, cell attachment growth individual layer covers 90%, 0.25 trypsin solution digestion bottom culture dish, collecting cell, blood counting chamber counts, and gets 8 × 10 6cell, 4 DEG C, the centrifugal 8min of 1000g, add 10mL precooling phosphoric acid buffer piping and druming suspendible, the same centrifugal 10min of condition, repeat aforesaid operations once, incline and supernatant liquor and add 8mL hypotonic buffer liquid, homogenate in ice cube, low temperature ultrasonic rupture of membranes 30min, the centrifugal 20min of 12000g again under 4 DEG C of conditions, to incline upper liquid, add 5mLTris-HCl damping fluid resuspended, for subsequent use, spectrophotometer 680nm place measures tissue sample and bovine serum albumin reference liquid, measure optical density value, dying method with coomassie brilliant blue measures Membrane protein's amount, get chromatographic column post core, obtained CB1 cell chromatographic column,
Step 3, affinity chromatography is separated, preparation EGCG
Self-control membrane flexibility post is received high performance liquid chromatograph, tea leaf extract millipore filtration 0.45 μm filtration, sample introduction 20 μ L, chromatographic condition is: column temperature 37 DEG C; Flow velocity 0.5mLmin -1, pH7.4, moving phase phosphate buffered saline buffer 50mmolmin -1, ultraviolet detection wavelength 280nm sample introduction 20 μ L after moving phase balance 2h is separated, and collects 3.5-4.0min elutriant, namely obtains CB1 selective antagonist EGCG.
2. antagonist EGCG preparation method as claimed in claim 1, is characterized in that, CB1 upstream primer CNR1-f:
5 '-CGGGATCCGCCACCATGAAGTCGATCCTAGATG-3 ' downstream primer CNR1-r:5 '-GGAATTCCTTTTTCTGTGCAGCCACAA-3 '.
3. antagonist EGCG preparation method as claimed in claim 1, it is characterized in that, the authentication method of antagonist EGCG is:
Infrared spectroscopy: results of IR EGCG is at 1690cm -1there is the last one stretching vibration absorption peak υ at place, at 1190 ~ 1240cm -1there is the asymmetric stretching vibration absorption peak υ of a group stronger at place, at 1450 ~ 1600cm -1there is phenyl ring framework characteristic vibration absorption peak at place, and spectrogram is consistent with EGCG standard diagram, and the preliminary confirmation CB1 antagonist agent that obtains is EGCG thus,
Uv-absorbing wavelength detecting: get sample separation, measures maximum absorption wavelength with ultraviolet-visible chromatographic instrument, finds there is maximum absorption at 273.4nm place;
High-efficient liquid phase chromatogram technique analysis: adopt C18 chromatographic column, moving phase is methyl alcohol: water: acetic acid (23:75:2, v/v), flow velocity 1.0mL.min -1, column temperature 35 DEG C, determined wavelength is 272nm, and high performance liquid chromatography quantitative analysis affinity chromatography is separated EGCG purity and reaches more than 98%;
Nuclear magnetic resonance map 3.00 (1H) .5.11 (1H) of nuclear magnetic resonance spectroscopy: EGCG, 5.53 (1H), 6.05 (2H), 6.65 (2H), 7.09 (2H).
4. antagonist EGCG preparation method as claimed in claim 1, it is characterized in that, the using method of antagonist EGCG is: after water solvent dissolves, and uses to rat, and route of administration is abdominal injection/oral.
5. the cerebrovascular disease prepared by the preparation method of the CB1 antagonist EGCG described in claim 1-4 any one, smoking cessation and slimming medicine.
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