CN104250241B - The preparation method of a kind of CB1 antagonist EGCG and the medicine of preparation - Google Patents

Abstract

The invention discloses the preparation method of a kind of CB1 antagonist EGCG and with treatment cerebrovascular disease, smoking cessation and the slimming medicine that it is prepared, mainly comprise the following steps: the structure of recombined human CB1 carrier for expression of eukaryon；CB1 high expressed HEK293 cell membrane and the preparation of membrane flexibility post；Affinity chromatography separates, preparation EGCG.Chinese herbal medicine monomer molecule cannabinoid receptors CB1 antagonist EGCG prepared by the present invention, action temperature and, untoward reaction is the most small, has good development prospect and use value.

Description

The preparation method of a kind of CB1 antagonist EGCG and the medicine of preparation

Technical field

The invention belongs to CB1 antagonist application, particularly relate to the system of a kind of CB1 antagonist EGCG Preparation Method and treatment cerebrovascular disease, smoking cessation and the slimming medicine prepared with it.

Background technology

Cannabinoid receptors is a kind of g protein coupled receptor, has two kinds of subtype acceptor CB1 and CB2.CB1 is subject to Body is expressed in the peripheral tissues such as central nervous system, lung, liver, kidney.Maincenter CB1 receptor main with Appetite-suppressing, minimizing are ingested relevant, and periphery CB1 receptor is main and regulation energy i (in vivo) metabolism, alleviates body Weight is relevant.CB1 receptor is the receptor that a class affects mankind's appetite and energy metabolism, and Rimonabant is first Selectively acting is in the antagonist of CB1 receptor, and CB1 antagonist is treatment obesity, giving up smoking and treating brain lacks The important target that blood disorders is sick, selectively acting is of great interest in the antagonist of CB1 receptor.

The method of screening CB1 antagonist is mainly pharmaceutical chemical method and sets up CB1 cell screening at present Model carries out high-flux medicaments sifting.Specific as follows:

One, Academy of Military Medicine, PLA/Military Medical Science Institute of PLA, pharmaceutical chemistry is special " design of CB1 receptor antagonist, synthesis are with biological for the Master's thesis such as industry Master degree candidate Tao Xiaoxuan, Zheng Zhibing Activity rating ", by the interaction of CB1 receptor antagonist Yu its receptor, establish " 4-methyl isophthalic acid H-bis- Arylpyrazoles " structure activity relationship of CB1 receptor antagonist.Based on the structure activity relationship having built up, and fully Consider the structural similarity of CB1 Yu CB2, antagonist cross under the premise such as film ability, with Rimonabant work For lead compound, its pyrazole ring 3 is carried out structural modification, devise and include quaternary ammonium salt, urea groups class With the compound that three classes such as guanidine base class have relatively high-hydrophilic, it is desirable to obtain there is high-affinity and be difficult to pass through The maincenter side effect selectively acting of minimizing to greatest extent of blood brain barrier is short of money in the CB1 receptor of peripheral tissues Anti-agent.

Two, East China University of Science's Pharmaceutical Engineering Speciality, the Master's thesis such as Hu Hui, Zhu Weihong " cannabinoid 1(CB1) The screening of receptor small molecular antagonists and model thereof are set up ".They set up CB1 receptor small molecular antagonists sieve Select platform, specific as follows:

1) by cotransfection CB1 receptor and Ga15 plasmid on CHO-K1 cell strain, stable expression is set up The cell strain (hCB1R-CHO) of CB1 receptor and Ga15；

2) utilize hCB1R-CHO cell strain, set up based on detection Ca2+ oscillations CB1 receptor antagonist screening mould Type, and to various experiment conditions (such as cell density, the concentration of Fluo-4AM and fluorescent dye incubation time etc.) It is optimized, is allowed to meet the requirement of high-flux medicaments sifting；

3) model that application is set up, explores screening novel C B1 that specificity is strong, affinity is high, toxic and side effects is low Receptor selective antagonists.Applying this model, paper has screened 364 micromolecular compounds, will screen To reactive compound do through GTP-Eu Competition binding assay, toxicity test, cross-over experiment etc. and to test further Card, successfully finds a novel C B1 receptor antagonist, and its IC50 value reaches 1.3 μMs.

At present, existing medicine Rimonabant untoward reaction is obvious, is suspended sale of in the world.EGCG passes through Antagonism CB1 receptor, can be used for cerebrovascular disease, loses weight and give up smoking, have no report both at home and abroad.How to integrate Go out the CB1 antagonist screening route of complete set, it is thus achieved that one can produce available by acting on CB1 In preventing and treating cerebrovascular disease, lose weight and the new drug given up smoking is the difficult problem that annoying people in the recent period.

Summary of the invention

The purpose of the embodiment of the present invention is to provide a kind of for verifying that CB1 antagonist EGCG treats cerebrovascular The method of disease, smoking cessation and fat-reducing effect, it is intended to solve to annoying how integrating out of people in the recent period a set of complete Whole CB1 antagonist screening route, it is thus achieved that one can be used for preventing and treating brain by acting on CB1 generation Angiopathy, the problem of new drug lost weight and give up smoking.

The embodiment of the present invention is achieved in that the preparation method of a kind of CB1 antagonist EGCG, and this is short of money Anti-agent EGCG preparation method includes step:

Step one, the structure of recombined human CB1 carrier for expression of eukaryon；

The extraction of total tissue RNA: take cerebral tissue and weigh 108mg, adds 1.0ml Trizol, puts into homogenate Device grinds on ice, proceeds to 1.5mlEP pipe, mixing, after standing 5min, add 200 μ l chloroforms, low Temperature centrifuge, 4 DEG C, 12000rpm, centrifugal 15min, take upper water and move into EP pipe mutually, add equal-volume Isopropanol, mixing, room temperature stand 1Omin, low-temperature centrifugation, add 1ml pre-cooling 0.1%DEPC water 75% washing with alcohol precipitation of preparation, super-clean bench air-dries 30min, adds 400 μ l DEPC and processes water dissolution Precipitation, agarose gel electrophoresis, observe under uviol lamp, it is judged that the integrity of RNA, uv-spectrophotometric Meter measures OD value and calculates RNA concentration, and-80 DEG C save backup；

Design of primers and synthesis: announce people's CB1 gene order design primer according to Genbank, draw in upstream 5 ' addition HindIII enzyme action points of thing, downstream primer 5 ' adds EcoR I enzyme action point, biological by Shanghai English fine horse Technology Co., Ltd. synthesizes；

The structure of pcDNA3.1-hCB1: Reverse Transcriptase kit reverse transcription obtains total cDNA, carries out PCR gram Grand CB1 gene, reaction condition: 94 DEG C of denaturations 4min；94 DEG C of degeneration 30s；56 DEG C of annealing 30s；74℃ Extend 45s；Last 74 DEG C extend 7min, it is contemplated that fragment length 1400bp, product 1% agarose gel Electrophoresis, cut glue and with DNA gel reclaim kits reclaim, purpose fragment HindIII of recovery and EcoR I double digestion 2h, reclaims purification, simultaneously with HindIII and EcoR I enzyme enzyme action pcDNA3.1, returns Receive purified linear plasmid vector, by obtained endonuclease bamhi with 3: 1 ratios with T4DNA Ligase at 20 DEG C Connect 24h, connection product is transformed in competence DH5 α, the coating LB solid training containing ampicillin Supporting base, the single positive colony of picking also expands recombiant plasmid, and plasmid kit extracts pcDNA3.1-hCB1, Double digestion PCR identifies, carries out order-checking and identifies；

CB1 eukaryotic expression vector transfection: recombinant vector pcDNA3.1-hCB1 is transiently transfected into HEK293 Cell, transfection uses Lipofectamine2000 reagent to carry out, and after transfection, 48h collects cell, and preparation is thin Cellular lysate liquid and total serum IgE carry out next step and analyze；

Step 2, CB1 high expressed HEK293 cell membrane and the preparation of membrane flexibility post:

Be taken at 37 DEG C, 5%CO2 and bag and appropriateness under the conditions of, be incubated at the DMEM containing 10% hyclone In culture fluid, routine passage, cell attachment growth monolayer covers bottom culture dish 90%, 0.25 trypsin Liquid digests, and collects cell, and blood counting chamber counts, and takes 8 × 106 cells, and 4 DEG C, 1000g is centrifuged 8min, Adding 10mL pre-cooling phosphate buffer piping and druming suspendible, condition is ibid centrifuged 10min, repeats aforesaid operations one Secondary, pour out supernatant and add 8mL hypotonic buffer liquid, ice cube is homogenized, low temperature ultrasonic rupture of membranes 30min, 4 DEG C Under the conditions of again 12000g be centrifuged 20min, upper liquid of inclining, add 5mLTris-HCl buffer resuspended, Standby, measure tissue sample and bovine serum albumin titer at spectrophotometer 680nm, measure optical density value, Dying method with coomassie brilliant blue measures Membrane protein's amount, takes chromatographic column post core, prepares CB1 cell chromatographic column；

Step 3, affinity chromatography separates, preparation EGCG

Self-control membrane flexibility post is received high performance liquid chromatograph, Folium Camelliae sinensis extract microporous filter membrane 0.45 μm Filtering, sample introduction 20 μ L, chromatographic condition is: column temperature 37 DEG C；Flow velocity 0.5mL min^-1, pH7.4, flow phase Phosphate buffer 50mmol min^-1, ultraviolet detection wavelength 280nm flowing balances each other sample introduction after 2h 20 μ L separate, and collect 3.5-4.0min eluent, i.e. obtain CB1 selective antagonist EGCG.

Further, CB1 forward primer CNR1-f:

5 '-CGGGATCCGCCACCATGAAGTCGATCCTAGATG-3 ' downstream primer CNR1 r:5 '-GGAATTCCTTTTTCTGTGCAGCCACAA-3 '.

Further, the authentication method of antagonist EGCG is:

Infrared spectrum analysis: results of IR EGCG is at 1690cm^-1There is the last one stretching vibration at place Absworption peak υ, 1190～1240cm^-1There is the asymmetric stretching vibration absworption peak υ of a group stronger at place, 1450～1600cm^-1There is phenyl ring framework characteristic vibration absorption peak at place, and spectrogram is consistent with EGCG standard diagram, Thus the obtained CB1 antagonist agent of preliminary confirmation is EGCG,

Uv absorption wavelength detecting: take separation sample, measures maximum absorption wavelength with ultraviolet-visible chromatograph, Find at 273.4nm, have absorption maximum；

High-efficient liquid phase chromatogram technique analysis: using C18 chromatographic column, flowing is methanol mutually: water: acetic acid (23: 75: 2, v/v), flow velocity 1.0mL.min^-1, column temperature 35 DEG C, detection wavelength is 272nm, efficient liquid phase Chromatography quantitative analysis affinity chromatography separated EGCG purity 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 aqueous solvent dissolves, and uses to rat, is administered way Footpath is lumbar injection/oral.

Another object of the present invention is to provide a kind of preparation method system with above-mentioned CB1 antagonist EGCG Standby cerebrovascular disease, smoking cessation and slimming medicine.

Chinese herbal medicine monomer molecule cannabinoid receptors CB1 antagonist EGCG prepared by the present invention, action temperature and, no Good reaction is the most small, has good development prospect and use value.

Accompanying drawing explanation

Fig. 1 be the embodiment of the present invention provide for verify CB1 antagonist EGCG treatment cerebrovascular disease, Smoking cessation and the method flow diagram of fat-reducing effect.

Detailed description of the invention

In order to make the purpose of the present invention, technical scheme and advantage clearer, 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 show that the present invention provides for verifying that CB1 antagonist EGCG treats cerebrovascular disease, ring The flow process of the method for cigarette and fat-reducing effect.For convenience of description, illustrate only part related to the present invention.

What embodiments of the invention provided is used for verifying that CB1 antagonist EGCG treats cerebrovascular disease, smoking cessation With the method for fat-reducing effect, the method comprises the following steps:

S101: build the eukaryotic expression HEK293 cell of CB1 receptor；

S102: with HEK293 cell by cellular membrane chromatography, screening and separating goes out CB1 part Galla Turcica (Galla Helepensis) Catechin gallate 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 EGCG and terminate the random of smoking effect Double blind experiment, verifies EGCG function of smoking cessation.

In step S104, the method for checking EGCG treating cerebral ischemia is:

Using bolt line to block SD rat, middle cerebral artery occlusion method MCAO method is set up rats with focal brain and is lacked Blood model, each treated animal drug administration by injection 2d in advance, and 0.5h before modeling, after modeling, 4h, 12h are again It is administered 3 times respectively；

Modeling 24h laggard row neurological deficit score, measures the cerebral tissue water yield and cerebral infarct size percentage is when carried out Pathological examination, and change with laser Doppler flowmetry detection MCAO rat brain blood flow.

In step S104, measure the EGCG method to the antiobesity action of high fat diet obesity-induced mice For:

Mice model of obesity is set up in the induction of high fat diet method, gives EGCG30mg/kg and treat 4 weeks, inspection Survey changes of body mass and surrounding genital fat content, adipose cell form and serum cholesterol, trigalloyl sweet The change of oil content.

In step S104, foundation and the group technology of obese model are as follows:

ICR mice is randomly divided into Normal group and high lipid food group, and normal group is from experiment start to finish All giving normal diet to feed, high lipid food group gives high lipid food and feeds, and modeling maintains 2 months；

After modeling terminates, taking the mice beyond normal group mice average body quality 20% is obese model；

Filter out qualified mice 40, be randomly divided into 4 groups, give respectively solvent, EGCGmg/kg group, 10mg/kg Rimonabant；10 mouse stomaches of normal group give solvent, are administered and maintain 4 weeks.

In step S104, the Indexs measure of 4 combination lattice mices is as follows:

Accumulative food consumption quantity, per hour record 1 time in monitoring 12h after first administration；

Within the whole cycle, every day, the food ration of mice and the weight of every mice often organized in record, and sees Examine the active situation of mice；

Fasting 12h after end, extracts eyeball and takes blood, measure serum triacylglycerol, T-CHOL, strips out complete Portion's Right Lower Abdomen butt crack fatty tissue, surrounding genital fatty tissue are also weighed；

Taking genitals week fat 2 fritters to fix with 4% formalin, paraffin section, HE dyes, the most identical Visual field fat cell hypertrophy form.

In step S104, EGCG is as follows to smoking cessation effect double-blind study method:

Use randomized, double-blind placebo-controlled study method, 43 examples are met into set condition and signs informed consent The smoker of book is divided into the EGCG i.e. treatment group of group, 200mg/ sheet and placebo group, EGCG group 25 example, Placebo group 18 example, 12 weeks courses for the treatment of；

During observing treatment, different time point successful quitting rate smoking capacity≤1/d, smoking reduce effective rates Period smoking capacity relatively treats the ratio of front minimizing >=50%, and evaluates visuality and crave for intensity test and appraisal table；

With urine cotinine and expiration carbonomonoxide 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 recombined human CB1 carrier for expression of eukaryon

The extraction of 1.1 total tissue RNA: take cerebral tissue and weigh 108mg, adds 1.0ml Trizol, puts into even Slurry device grinds on ice, proceeds to 1.5mlEP pipe, mixing, after standing 5min, add 200 μ l chloroforms, Refrigerated centrifuge (4 DEG C, 12000rpm) is centrifuged 15min, takes upper water and moves into EP pipe, addition etc. mutually The isopropanol of volume, mixing, room temperature stands 1Omin, low-temperature centrifugation, adds the 0.1%DEPC of 1ml pre-cooling 75% washing with alcohol precipitation of water preparation, super-clean bench air-dries 30min, adds 400 μ l DEPC and processes water-soluble Solve precipitation, agarose gel electrophoresis, observe under uviol lamp, it is judged that the integrity of RNA, ultraviolet spectrometry light Degree meter measures OD value and calculates RNA concentration, and-80 DEG C save backup；

1.2 design of primers and synthesis: announce people's CB1 gene order design primer according to Genbank, upper 5 ' addition HindIII enzyme action points of trip primer, downstream primer 5 ' adds EcoR I enzyme action point, by Shanghai English fine horse Bioisystech Co., Ltd synthesizes；

CB1 forward 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 Clone's CB1 gene, reaction condition: 94 DEG C of denaturations 4min；94 DEG C of degeneration 30s；56 DEG C of annealing 30s； 74 DEG C extend 45s；Last 74 DEG C extend 7min, it is contemplated that fragment length 1400bp, product 1% agarose Gel electrophoresis, cuts glue and reclaims kits recovery, purpose fragment HindIH of recovery with DNA gel With EcoR I double digestion 2h, reclaim purification, simultaneously with HindIII and EcoR I enzyme enzyme action pcDNA3.1, Reclaim purified linear plasmid vector, by above-mentioned two the obtained endonuclease bamhis of step with 3: 1 ratios T4DNA Ligase connects 24h at 20 DEG C, is transformed in competence DH5 α by connection product, is coated with the benzyl penicillium sp Han ammonia The LB solid medium of element, the single positive colony of picking also expands recombiant plasmid, and plasmid kit extracts PcDNA3.1-hCB1, double digestion PCR identify, send Bao Bio-Engineering Company to carry out order-checking and identify；

1.4CB1 eukaryotic expression vector transfection: recombinant vector pcDNA3.1-hCB1 is transiently transfected into HEK293 cell, transfection uses Lipofectamine2000 reagent to carry out, and concrete grammar sees Lipofectamine2000 operation instructions, after transfection, 48h collects cell, prepares cell pyrolysis liquid and total serum IgE Etc. carrying out next step experimental analysis；

2, CB1 high expressed HEK293 cell membrane and the preparation of membrane flexibility post:

Be taken at 37 DEG C, 5%CO2 and bag and appropriateness under the conditions of, be incubated at the DMEM containing 10% hyclone In culture fluid, routine passage, cell attachment growth monolayer covers bottom culture dish about 90%, 0.25 Trypsin Enzyme liquid digests, and collects cell, and blood counting chamber counts, and takes about 8 × 10⁶Cell, 4 DEG C, 1000g is centrifuged 8min, adds 10mL pre-cooling phosphate buffer piping and druming suspendible, and condition is ibid centrifuged 10min, repeats above-mentioned Operation once, pours out supernatant and adds 8mL hypotonic buffer liquid, be homogenized, low temperature ultrasonic rupture of membranes 30min in ice cube, Under the conditions of 4 DEG C, 12000g is centrifuged 20min again, upper liquid of inclining, and adds 5mLTris-HCl buffer weight Outstanding, standby, measure tissue sample and bovine serum albumin titer at spectrophotometer 680nm, measure light close Angle value, dying method with coomassie brilliant blue measures Membrane protein's amount, takes chromatographic column post core, prepares CBl cell chromatograph Post；

3, affinity chromatography separates, prepares EGCG

Self-control membrane flexibility post is received high performance liquid chromatograph, Wuxi, Chongqing City (2013 produce per year) then Folium Camelliae sinensis extract microporous filter membrane (0.45 μm) filters, and sample introduction 20 μ L, chromatographic condition is: column temperature 37 DEG C；Stream Speed 0.5mL min^-1, pH7.4, flow phase phosphate buffer 50mmo1 min^-1, ultraviolet detection wavelength The 280nm flowing sample introduction 20 μ L after 2h that balances each other separates, and collects 3.5-4.0min eluent, i.e. obtains CBl Selective antagonist EGCG.

The authentication method of antagonist EGCG is:

Infrared spectrum analysis: results of IR EGCG is at 1690cm^-1There is the last one stretching vibration at place Absworption peak υ, 1190～1240cm^-1There is the asymmetric stretching vibration absworption peak υ of a group stronger at place, 1450～1600cm^-1There is phenyl ring framework characteristic vibration absorption peak at place, and spectrogram is consistent with EGCG standard diagram, Thus the obtained CBl antagonist agent of preliminary confirmation is EGCG.

Uv absorption wavelength detecting: take separation sample, measures maximum absorption wavelength with ultraviolet-visible chromatograph, Find at 273.4nm, have absorption maximum, consistent with the Pharmacopoeia of the People's Republic of China.

High-efficient liquid phase chromatogram technique analysis: use C18 chromatographic column (4.6mm × 250mm, 5 μm), flowing It is methanol mutually: water: acetic acid (23: 75: 2, v/v), flow velocity 1.0mL.min^-1, column temperature 35 DEG C, detection Wavelength is 272nm.High performance liquid chromatography quantitative analysis affinity chromatography separated EGCG purity 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 document report.

The above analysis is identified, it is CB1 antagonist EGCG that used affinity chromatography separates obtained material.

The using method of antagonist:

After the EGCG used at present is mainly aqueous solvent dissolving, using to rat, route of administration is abdominal cavity Injection/oral (gavage).

Below in conjunction with the accompanying drawings and the application principle of the present invention is further described by specific embodiment.

1, cerebrovascular proves experiment

1.1 test methods: use bolt line blocking experiment model SD rat, middle cerebral artery occlusion method (MCAO Method) set up evaluating focal brain ischemia in rats, each treated animal drug administration by injection 2d in advance, and before modeling 0.5 H, after modeling, 4h, 12h are administered 3 times the most respectively.Modeling 24h laggard row neurological deficit score, measures brain group Knit the water yield and cerebral infarct size percentage when carries out pathological examination, and detect with laser Doppler flowmetry MCAO rat brain blood flow changes.

1.2 experimental results:

1.2.1 on neurological deficit score and the impact of brain water content

After MCAO rat anesthesia is clear-headed, visible hemiplegia sample symptom occurs, mainly shows as operation in various degree Receiving in offside forelimb, take on inward turning, muscular tension reduces, and pushes away right shoulder to side shifting, resistance decline, portion Transfer thing is turn-taked to offside.Rat model is carried out Neurological deficits and brain water content measures.

Result shows: model group Neurological deficits and brain water content are tieed up apparently higher than sham-operation, EGCG Injection group compares with model group, and the neuroethology obstacle of rat is substantially improved, and brain water content significantly drops Low.Prompting EGCG has obvious protective function to MCAO rat.

1.2.2 the impact on cerebral infarct size

After EGCG injection group is administered, contrasting rats in sham-operated group, cerebral infarct size percentage rate is remarkably decreased.

1.2.3 the impact on Pathologic histology of brain

Rats in sham-operated group neuron and brain parenchymal cell structure are normal, and kernel understands, even dyeing, Cell peripheral gap slightly expands.Under model control group rat encephaloscope, visible substantially large area softens stove, have in Mesh-like.Cell peripheral gap substantially expands, swelling, and Nissl body reduces or disappears, nuclear hyperchromatism, pyknosis Or dissolve.Cerebral tissue vasodilation, hence it is evident that fill big belly.EGCG group rat cerebral tissue softens stove and is obviously reduced, The most collections infarction.Cell peripheral gap is the most broadening, right neuron unit and brain parenchymal cell, capillary coil pipe week Enclosing increase, neuronal cell core mild swelling, kernel understands, it is seen that glial cells hyperplasia, in degeneration, and water Swollen change.

To sum up result, and other research worker existing, it was demonstrated that EGCG be can be used for by antagonism CB1 receptor The prevention of cerebrovascular disease and treatment.

2, measure EGCG the weight-reducing experiment of high fat diet obesity-induced mice is studied

Mice model of obesity is set up in the induction of high fat diet method, gives EGCG30mg/kg and treat 4 weeks, inspection Survey changes of body mass and surrounding genital fat content, adipose cell form and serum cholesterol, trigalloyl sweet The change of oil content.Result: EGCG plays antiobesity action by suppression CB1 receptor, can substantially reduce little The food intake dose of Mus and weight, reduce containing of surrounding genital fat, serum cholesterol and triacylglycerol Amount.Conclusion: EGCG can be reduced appetite and produce antiobesity action by suppression CB1 receptor, can be reduced simultaneously Blood lipid level.

The foundation of 2.1 obese models and packet

ICR mice is randomly divided into Normal group and high lipid food group.Normal group is from experiment start to finish All giving normal diet to feed, high lipid food group gives high lipid food and feeds, and modeling maintains 2 months.Modeling After end, taking the mice beyond normal group mice average body quality 20% is obese model.Filter out qualified little Mus 40, is randomly divided into 4 groups, gives solvent, EGCGmg/kg group, 10mg/kg Li Mona respectively Class.10 mouse stomaches of normal group give solvent, are administered and maintain 4 weeks.

2.2 Indexs measure

Accumulative food consumption quantity, per hour record 1 time in monitoring 12h after first administration.In whole experiment week In phase, every day, the food ration of mice and the weight of every mice often organized in record, and observes the activity of mice Situation.Experiment terminates rear fasting 12h, extracts eyeball and takes blood, measures serum triacylglycerol, T-CHOL. Strip out whole Right Lower Abdomen butt crack fatty tissue, surrounding genital fatty tissue and weigh.Take genitals week fat Fat 2 fritter is fixed with 4% formalin, paraffin section, and HE dyes, and compares same field of view fat cell hypertrophy shape State.

2.3 food ration impacts

EGCG30mg/kg mice food ration is below model group at each time point.With Rimonabant to 12 The effect that in h, suppression mice ingests is similar to.Do different dosing dosage group research to find, EGCG30mg/kg The effect of group suppression food ration is the strongest.Compared to model group, in 12h, the mice food ration of administration group substantially subtracts Few, the food ration of 30mg/kg and 10mg/kg Rimonabant group mice 12h have dropped 38.4% respectively, 43.0%.

In 4 weeks, the effect of 30mg/kgEGCG group suppression mice food ration is the strongest, and sustained drug inhibits little The food ration of Mus, and there is not inhibitory action in the food ration of normal group and model group mice.Same dose Under, the food inhibitory action of Rimonabant is stronger than EGCG.

The impact on Mice Body quality of 2.4 medicines

The weight of normal group and model group mice remains stable.EGCG group and Rimonabant all have reduction The effect of obesity mice weight.The decline about 8.0% to weight of the 30mg/kgEGCG group.10 Mg/kg Rimonabant effect is better than EGCG effect.

2.5 impacts on mice body fat content

Compared to normal group, model group mice body fat content dramatically increases.EGCG and Rimonabant are controlled After treatment, the fat weight of mouse propagation device periphery declines, and 30mg/kgEGCG significantly reduces fat content.

2.6 impacts on mouse adipocytes volume

Model group adipose cell is relatively large in diameter, and visual field inner cell number is less；After 30mg/kgEGCG treatment, little Adips Mus fat cell dia diminishes, and intracellular fat content is less the most obvious.

2.7 on mice serum triacylglycerol and the impact of cholesterol level

The obesity mice serum cholesterol of high fat diet induction and the content of triacylglycerol significantly raise.EGCG After the treatment of 30mg/kg dosage group, the content of mice serum cholesterol and triacylglycerol is remarkably decreased.

3, smoking cessation effect double blind experiment is studied by EGCG

Use randomized, double-blind placebo-controlled study method, 43 examples are met into set condition and signs informed consent The smoker of book is divided into EGCG group (treatment group；200mg/ sheet) and placebo group, EGCG group 25 example, Placebo group 18 example, 12 weeks courses for the treatment of；Different time point successful quitting rate (smoking capacity≤1 during observing treatment / d), smoking reduce effective percentage (during treatment, smoking capacity relatively treats the ratio of front minimizing >=50%), and evaluate can Intensity test and appraisal table is craved for depending on property；With urine cotinine and expiration carbonomonoxide concentration checking tobacco smoking status.

Result shows: after treatment, and treatment group and placebo group successful quitting rate are respectively 47.19% and 12. 71%, smoking reduces effective percentage and is respectively 37.41% and 11.82%, and two groups of differences have statistical significance (P<0.01)；Treat treatment at the 2nd～12 weekends group and crave for the score of intensity, CO exhalation amount respectively less than comfort Agent group (P < 0.01)；Treatment group urine cotinine content was less than placebo group (P < 0. at the 8th week and the 12nd week 01).Treatment period, two groups of volunteers all occurred without serious adverse events.

The foregoing is only presently preferred embodiments of the present invention, not in order to limit the present invention, all at this Any amendment, equivalent and the improvement etc. made within bright spirit and principle, should be included in the present invention Protection domain within.

Claims (3)

1. the preparation method of a CB1 antagonist EGCG, it is characterised in that this antagonist EGCG preparation method includes step:

Step one, the structure of recombined human CB1 carrier for expression of eukaryon；

nullThe extraction of total tissue RNA: take cerebral tissue and weigh 108mg，Add 1.0ml Trizol，Put in homogenizer and grind on ice，Proceed to 1.5mlEP pipe，Mixing，200 μ l chloroforms are added after standing 5min，Refrigerated centrifuge，4℃，12000rpm，Centrifugal 15min，Take upper water and move into EP pipe mutually，Add isopyknic isopropanol，Mixing，Room temperature stands 10min，Low-temperature centrifugation，Add 75% washing with alcohol precipitation of the 0.1%DEPC water preparation of 1ml pre-cooling，Super-clean bench air-dries 30min，Add 400 μ l DEPC and process water dissolution precipitation，Agarose gel electrophoresis，Observe under uviol 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's CB1 gene order design primer according to Genbank, at 5 ' addition HindIII enzyme action points of forward primer, downstream primer 5 ' adds EcoR I enzyme action point, Shanghai Ying Jun Bioisystech Co., Ltd synthesize；

The structure of pcDNA3.1-hCB1: Reverse Transcriptase kit reverse transcription obtains total cDNA, carries out PCR and clones CB1 gene, reaction condition: 94 DEG C of denaturations 4min；94 DEG C of degeneration 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 recovery with DNA gel, purpose fragment HindIII reclaimed and EcoR I double digestion 2h, reclaim purification, simultaneously with HindIII and EcoR I enzyme enzyme action pcDNA3.1, reclaim purified linear plasmid vector, obtained endonuclease bamhi is connected 24h with 3: 1 ratios T4DNA Ligase at 20 DEG C, connection product is transformed in competence DH5 α, the coating LB solid medium containing ampicillin, the single positive colony of picking also expands recombiant plasmid, plasmid kit extracts pcDNA3.1-hCB1, double digestion PCR identifies, carry out order-checking to identify；

CB1 eukaryotic expression vector transfection: recombinant vector pcDNA3.1-hCB1 is transiently transfected into HEK293 cell, transfection uses Lipofectamine2000 reagent to carry out, and after transfection, 48h collects cell, prepares cell pyrolysis liquid and total serum IgE carries out next step and analyzes；

Step 2, CB1 high expressed HEK293 cell membrane and the preparation of membrane flexibility post:

It is taken at 37 DEG C, 5% CO₂And under the conditions of saturated humidity, be incubated in the DMEM culture fluid containing 10% hyclone, routine passage, cell attachment growth monolayer covers bottom culture dish 90%, and cell is collected in 0.25 trypsin solution digestion, and blood counting chamber counts, and takes 8 × 10⁶Cell, 4 DEG C, 1000g is centrifuged 8min, add 10mL pre-cooling phosphate buffer piping and druming suspendible, condition is ibid centrifuged 10min, repeat aforesaid operations once, pour out supernatant and add 8mL hypotonic buffer liquid, ice cube is homogenized, low temperature ultrasonic rupture of membranes 30min, under the conditions of 4 DEG C, 12000g is centrifuged 20min again, incline upper liquid, add 5mLTris-HCl buffer resuspended, standby, tissue sample and bovine serum albumin titer is measured at spectrophotometer 680nm, measure optical density value, dying method with coomassie brilliant blue measures Membrane protein's amount, take chromatographic column post core, prepare CB1 cell chromatographic column；

Step 3, affinity chromatography separates, preparation EGCG

Self-control membrane flexibility post is received high performance liquid chromatograph, and Folium Camelliae sinensis extract microporous filter membrane 0.45 μm filters, and sample introduction 20 μ L, chromatographic condition is: column temperature 37 DEG C；Flow velocity 0.5mL min^-1, pH7.4, flow phase phosphate buffer 50mmol min^-1, the ultraviolet detection wavelength 280nm flowing sample introduction 20 μ L after 2h that balances each other separates, and collects 3.5-4.0min eluent, i.e. obtains CB1 selective antagonist EGCG.

2. antagonist EGCG preparation method as claimed in claim 1, it is characterised in that CB1 forward 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 characterised in that the authentication method of antagonist EGCG is:

Infrared spectrum analysis: results of IR EGCG is at 1690cm^-1There is the last one stretching vibration absworption peak υ at place, 1190～1240cm^-1There is the asymmetric stretching vibration absworption peak υ of a group stronger at place, 1450～1600cm^-1There is phenyl ring framework characteristic vibration absorption peak at place, and spectrogram is consistent with EGCG standard diagram, and thus the obtained CB1 antagonist agent of preliminary confirmation is EGCG,

Uv absorption wavelength detecting: take separation sample, measures maximum absorption wavelength with ultraviolet-visible chromatograph, finds there is absorption maximum at 273.4nm；

High-efficient liquid phase chromatogram technique analysis: use C18 chromatographic column, flowing is mutually for methanol: water: acetic acid is 23: 75: 2, v/v, flow velocity 1.0mL.min^-1, column temperature 35 DEG C, detection wavelength is 272nm, and high performance liquid chromatography quantitative analysis affinity chromatography separated EGCG purity 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).