CN106309423A - Application of chloro-substituted tetrahydrophenethyl chromone derivatives and pharmaceutical composition thereof in agilawood - Google Patents
Application of chloro-substituted tetrahydrophenethyl chromone derivatives and pharmaceutical composition thereof in agilawood Download PDFInfo
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Abstract
The invention discloses application of chloro-substituted tetrahydrophenethyl chromone derivatives and a pharmaceutical composition thereof which are extracted and separated from agilawood in prevention or treatment of inflammation-related diseases, autoimmune diseases and organ transplantation rejection, especially prevention or treatment of systemic inflammatory response, pneumonia, bronchitis, hepatitis, enteritis, rheumatoid arthritis, systemic lupus erythematosus, kidney transplantation rejection, liver transplantation rejection and hematopoietic stem cell transplantation rejection.
Description
Technical field
The present invention relates to four chloro tetrahydrochysene phenethyl chromone derivatives in prevention or treatment inflammation related disease, autoimmune disease and organ transplantation
Application in repulsion, belongs to pharmaceutical technology field.
Background technology
Human immune system includes the natural immunity and adaptive immunity, plays an important role in the body injury defending various interior extrinsic factors to cause.But
It is that immunologic function excessive activation also results in serious tissue injury.Relevant to immunologic function excessive activation disease mainly have inflammation related disease,
Autoimmune disease and organ-graft refection.Natural immunity excessive activation mainly results in inflammation related disease or increases the weight of inflammation related disease body damage
Wound.Adaptive immunity excessive activation causes autoimmune disease and organ-graft refection.
Prevention at present or the drug main glucocorticoid to be had for the treatment of inflammation related disease and cox-2 inhibitors are the NSAID (non-steroidal anti-inflammatory drug) of representative.Though
So glucocorticoid and NSAID (non-steroidal anti-inflammatory drug) have preferable antiinflammatory action, but adverse effect seriously limits their application.Sugar cortical hormone
Element prolonged application can cause metabolic dysfunction, NSAID (non-steroidal anti-inflammatory drug) prolonged application can cause gastric ulcer.Prevent or treat autoimmune disease and device
The drug main glucocorticoid to be had of official's transplant rejection and the adaptive immunity inhibitor such as ciclosporin, tacrolimus.Glucocorticoid life-time service can be led
Cause metabolic dysfunction, and the adaptive immunity inhibitor such as ciclosporin and tacrolimus has higher nephrotoxicity.Therefore, develop new structure and
The natural immunity of mechanism of action and adaptive immunity inhibitor are for prevention or treatment inflammation related disease, autoimmune disease and organ-graft refection
Significant.
Lignum Aquilariae Resinatum is the timber that Isolated From Thymelaeaceae Species Lignum Aquilariae Resinatum contains resin.Chinese Pharmacopoeia is recorded Lignum Aquilariae Resinatum and is had promoting the circulation of QI to relieve pain, warming middle-JIAO to arrest vomiting, helping inspiration to relieve asthma
Effect, is used for treating chest and abdomen feeling of distension and oppression pain, gastrofrigid vomiting singultus, QI rising in reverse order of suffering from a deficiency of the kidney dyspnea with rapid respiration.The antiinflammatory of these effects and therapeutical effect and modern medicine is made
With quite similar.The present inventor the phenethyl chromone derivative being widely present in Lignum Aquilariae Resinatum has been carried out extract separate and Structural Identification, and to they
The natural immunity and adaptive immunity inhibitory action are screened, to find that the natural immunity with new mechanism of action and adaptive immunity inhibitor are used
In prevention or treatment inflammation related disease, autoimmune disease and organ-graft refection.
Summary of the invention
The present inventor, through long-term extraction separation and Structural Identification, obtains the most known and unknown phenethyl chromone derivative from Lignum Aquilariae Resinatum, passes through
Pharmacological screening and deep pharmacodynamic study, find 3 known chloros tetrahydrochysene phenethyl chromone derivative (G1, G3 and G4) of following form first
(Chem.Pharm.Bull, 2003,51 (5): 560-564；Helvetica Chimica Acta, 2012,95:1657-1665) and 1 new chloro tetrahydrochysene
Phenethyl chromone derivative (G2) has the good natural immunity and specific immunity inhibitory action.
It is an object of the invention to provide 1 new chloro tetrahydrochysene phenethyl chromone derivative (G2) selected from above table.
Another object of the present invention is to provide the preparation method of the new chloro tetrahydrochysene phenethyl chromone derivative G2 selected from above table.
Four chloro tetrahydrochysene phenethyl chromone derivatives that a further object of the present invention is to provide in the above table are by the natural immunity and special
Property immunosuppressant application in prevention or treatment inflammation related disease, autoimmune disease and organ-graft refection.
It is still another object of the present invention to provide containing the pharmaceutical composition of one or more compounds in effective dose above table by the natural immunity and
Specific immunity suppression application in prevention or treatment inflammation related disease, autoimmune disease and organ-graft refection.
The present invention finds a kind of new chloro tetrahydrochysene acetophenone derivs first from Lignum Aquilariae Resinatum, and concrete structure the most above-mentioned tabular compound G2, its feature exists
In chlorine atom and 3 hydroxyls in chromone ring both sides, it is stereoisomer with compound G4.
It is as follows that described compound G2 extracts separating step:
Taking Lignum Aquilariae Resinatum medicinal powder, 95% alcohol heating reflux extracts, is evaporated to extractum shape, and add water suspendible, respectively with petroleum ether, ethyl acetate
Extraction.Take Ethyl acetate extract extractum, add silica gel mixed sample, be splined on silicagel column (200-300 mesh), successively with petroleum ether-ethyl acetate (8: 1-1: 1),
Chloroform-methanol (20: 1-1: 3) gradient elution, obtains 7 stream parts such as GYF1～GYF7.Take stream part GYF7, further through silica gel (200-300
Mesh) pillar layer separation, methanol dichloromethane (1: 12) gradient elution, TLC detects, and merges 6 stream parts such as obtaining GYF7-1～GYF7-6.
Take stream part GYF7-3, through silica gel (200-300 mesh) pillar layer separation, methanol dichloromethane (1: 12) eluting, obtain GYF7-3-1～GYF7-3-5
Deng 5 stream parts.Taking stream part GYF7-3-3 to separate through ODS reversed-phase column, methanol-water (50: 50) eluting, eluent separates through half preparation liquid phase post,
Acetonitrile-water (12: 88) eluting, obtains compound G2 (tR=90min).
Compound G1, G3 and G4 of the present invention can be prepared according to method well known in the art.
The present invention passes through macrophage strain RAW264.7 cell and BV-2 cell screening, it was demonstrated that compound G1, G2, G3 and G4 have suppression antibacterial
Lipopolysaccharide stimulates RAW264.7 cell and the nitric oxide production effect of BV-2 emiocytosis.
The present invention passes through enzyme-linked immunoassay, it was demonstrated that it is many that the compound G4 that suppression macrophages secrete Effect of Nitric Oxide is the strongest has suppression antibacterial fat
The RAW264.7 emiocytosis inflammatory factor TNF-α of sugar stimulation and the effect of IL-6.
The present invention passes through Flow cytometry, it was demonstrated that G4 has the effect of suppression Murine Neutrophil activation.
The present invention passes through Flow cytometry, it was demonstrated that G4 has suppression mouse spleen CD4+The effect of T cell activation.
The present invention passes through mouse system inflammatory response model, it was demonstrated that intravenous injection bacteria lipopolysaccharide can be suppressed to lead by gastric infusion compound G4
The systemic inflammatory responses caused.
The present invention is tested by Western-Blot, it was demonstrated that compound G4 can be exempted from by the suppression JNK signal path suppression natural immunity and specificity
Epidemic disease.
The present invention demonstrates compound G1, G2, G3 and G4 and pharmaceutical composition thereof by above pharmacological experiment can be by the natural immunity and special
Property immunosuppressant application in prevention or treatment inflammation related disease, autoimmune disease and organ-graft refection.
The pharmaceutical composition containing compound G1, G2, G3 and G4 of the present invention can be prepared according to method well known in the art.
The compound of the present invention or the pharmaceutical composition containing them can be administered in a unit, route of administration can be administered orally, intravenous injection, flesh
Meat injection, nasal cavity, oral mucosa, skin or rectum etc..
The compound of the present invention or the form of administration containing their pharmaceutical composition can be liquid dosage form, solid dosage forms.As liquid dosage form can be
True solution class, colloidal type, particulate formulations, emulsion dosage form, mixed suspension form.Other dosage form such as tablet, capsule, drop pill, aerosol, pill, powder
Agent, solution, suspensoid, Emulsion, granule, suppository, lyophilized injectable powder etc..
The dosage of the pharmaceutical composition of the compound of the present invention depends on many factors, such as, to prevent or to treat the character of disease and serious journey
Degree, patient or the sex of animal, age, body weight, personality and individual reaction, route of administration, administration number of times, therapeutic purposes, the therefore present invention
Therapeutic dose can have change on a large scale.In general, the Suitable dosage ranges of the every day of the compounds of this invention: the compound that the present invention relates to
Consumption is 0.1～10mg/Kg body weight, can once take or divide and take for 2～4 times.Compound G1, G2, G3, G4 of the present invention or containing them
Pharmaceutical composition can individually take, or merge use with other treatment medicine or symptomatic drugs.
The present invention compound prevention or treatment inflammation related disease can be systemic inflammatory responses, pneumonia, bronchitis, hepatitis, nephritis,
Gastritis, enteritis, neuroinflamation, scytitis etc..
The compound prevention of the present invention or the autoimmune disease for the treatment of can be rheumatoid arthritis, systemic lupus erythematosus (sle), systemic vascular
Inflammation, scleroderma, autoimmune hemolytic anemia, autoimmunity aplastic anemia, chronic lymphocytic thyroiditis etc..
The compound prevention of the present invention or the organ-graft refection for the treatment of can be renal transplant rejection, liver transplantation repulsion, skin graft rejection, heart shifting
Plant repulsion, hematopoietic stem cell transplantation is repelled.
Accompanying drawing explanation
Accompanying drawing is that compound G4 is to NF-κ B path and the Western-Blot detection figure of MAPK path regulation effect.
Detailed description of the invention
Below with reference to embodiment, invention is described further, but does not limit the scope of the invention.
Embodiment 1: the preparation of compound G2
(1) experiment material
Medical material: Lignum Aquilariae Resinatum.It is accredited as Isolated From Thymelaeaceae Species Lignum Aquilariae Resinatum Aquilaria through Beijing University of Chinese Medicine's Chinese medicine modern study center professor Tu Pengfei
Sinensis (Lour.) Gilg contains the timber of resin.Specimen (CX2012029) deposits in Beijing University of Chinese Medicine's Chinese medicine modern study center mark
This storehouse.
Reagent: the chemical reagent such as ethanol, petroleum ether, ethyl acetate, chloroform, methanol, dichloromethane, acetonitrile are analytical pure, purchased from Beijing
Factory.
Instrument and material: U.S.'s Rudolph Autopol IV automatic polarimeter；Shimadzu Corporation of Japan UV-2450 spectrophotometer；U.S. Thermo
Nicolet Nexus 470FT-IR Fourier transformation infrared spectrometer；U.S.'s Varian Inova-500 type nuclear magnetic resonance analyser；Japan Shimadzu from
Sub-trap time of-flight mass spectrometer (IT-ToF-MS)；U.S. Waters2535 semipreparative high performance liquid chromatography instrument；JA50002 precision electronic balance；
DHG-9070B air dry oven；DZF-6090 vacuum drying oven；Thin layer chromatography GF254Silica gel precoated plate and column chromatography silica gel (200-300
Mesh), purchased from Haiyang Chemical Plant, Qingdao；Merck company of Germany ODS C18 chromatographic column；Amersham Biosciences company of Sweden Sephadex
LH-20 chromatographic column；Analytical type HPLC chromatogram post: Diamonsil C18Chromatographic column (4.6 × 250mm, 5 μm)；Partly prepare reversed phase chromatographic column: Water
XTerra Prep C18Chromatographic column (10 × 250mm, 5 μm) and Shiseido Capcell PAK MG Prep C18Chromatographic column (10 × 250mm,
5μm)。
(2) experimental technique
Taking Lignum Aquilariae Resinatum medical material 6.9kg, 95% alcohol heating reflux extracts 3 times, and each 2.5h merges alcohol extract, is evaporated to extractum shape (3.39kg),
Add water suspendible, respectively with petroleum ether and ethyl acetate extraction.Obtain petroleum ether part 150g, Ethyl acetate extract 600g.Take Ethyl acetate extract leaching
Cream (400g), adds silica gel mixed sample, and row silica gel column chromatography (200-300 mesh), successively with petroleum ether-ethyl acetate (8: 1 → I: 1), chloroform-methanol
(20: 1 → 1: 3) gradient elution, obtains 7 stream parts such as GYF1～GYF7.Take stream part GYF7 (67g), further through silica gel (200-300 mesh)
Pillar layer separation, methanol dichloromethane (1: 12) gradient elution, TLC inspects, and merges similar stream part, obtains GYF7-1～GYF7-6 etc. 6
Stream part.Take stream part GYF7-3, row silica gel (200-300 mesh) pillar layer separation, methanol dichloromethane (1: 12) eluting, obtain GYF7-3-1～
GYF7-3-5 etc. 5 flow part.Taking stream part GYF7-3-3 to separate through ODS reversed-phase column, methanol-water (50: 50) eluting, target eluent is through half system
Standby liquid phase post separates, and acetonitrile-water (12: 88) eluting obtains yellow oil G2 (13mg, tR=90min).G2 is carried out spectral data mensuration
And structure elucidation.
(3) experimental result
Yellow oil G2,HRESIMS provides quasi-molecular ion peak m/z 401.0545 [M+Cl]-(meter
Calculation value 401.0564), molecular formula is C18H19O6Cl。IR(KBr)vmax3424,1659,1612,
1513,1435,1246,1180,1097,1037,827,796cm-1；1H and13C NMR data is shown in Table 1.
Table 1: compound G2's1H (500MHz) and13C (125MHz) NMR data
Embodiment 2: compound G1～G4 vitro inhibition mouse macrophage and neural microglia nitric oxide (NO) secretory action measure
(1) experiment material
Cell strain: RAW264.7 cell is purchased from Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences's cell centre；BV-2 cell is by the Chinese Academy of Medical Sciences
Chen Naihong researcher presents.
Reagent: compound G1～G4 is extracted separation by Beijing University of Chinese Medicine's Chinese medicine modern study center from Lignum Aquilariae Resinatum；DMEM high glucose medium and
DMEM/F12 culture medium is purchased from Corning Incorporated；Bacteria lipopolysaccharide is purchased from Beijing Baeyer enlightening biotechnology Co., Ltd (article No.: L2880)；One
Nitrogen oxide measures test kit (Griess reagent) purchased from Puli's lema gene Technology Co., Ltd. (article No.: E1030)；Thiazolyl blue is public purchased from Amresco
Department's (article No.: 0793)；Analytical pure dimethyl sulfoxide, purchased from Beijing Chemical Plant.
Instrument: Enspire Multimode Plate Reader is purchased from platinum Ai Ermo company of Germany.
(2) experimental technique
The RAW264.7 cell (DMEM high glucose medium) and BV-2 cell (DMEM/F12 culture medium) that are in exponential phase are inoculated into 96
In orifice plate, inoculum density is 20000 cells/well, volume 100 μ l, 37 DEG C and 5%CO2After hatching 24 hours, add compound G1～G4,
Each compound arranges 7 concentration (acellular toxic action), and each concentration sets 3 multiple holes, after 1 hour, adds bacteria lipopolysaccharide (LPS) to end
Concentration 0.5 μ g/ml, continues to hatch 24 hours.Take 50 μ l cell conditioned mediums, add Griess reagent 100 μ l, after reacting 10 minutes, measure 540nm
The absorbance of wavelength, and calculate four compounds half-inhibition concentration (IC to two kinds of emiocytosis NO50).Separately will Tissue Culture Plate be cultivated
Base discards, and adds 100 μ l MTT reagent (the 0.5mg/ml Thiazolyl blue solution of PBS preparation), and cell culture incubator continues to hatch 4h, then discards
MTT reagent, each hole adds the dmso solution first a ceremonial jade-ladle, used in libation of 150 μ l, measures the absorbance of 570nm wavelength, and calculates each concentration compound pair
RAW264.7 cell and the growth inhibition ratio of BV-2 cell.
(3) experimental result
By secretion nitric oxide model discrimination, find that compound G1～G4 all can significantly inhibit mouse macrophage RAW264.7 and microglia
BV-2 is at the post-stimulatory Secretion of Nitric Oxide of lipopolysaccharide, its half-inhibition concentration (IC50) as shown in table 2；Compound G1～G4 is at maximum concentration
When reaching 100 μMs still to RAW264.7 and microglia without growth inhibited effect.
Table 2: compound G1～G4 vitro inhibition NO secretory action measure
Embodiment 3: compound G4 vitro inhibition mouse macrophage inflammatory factor secretory action measures
(1) experiment material
Cell strain: RAW264.7 cell is purchased from Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences's cell centre.
Reagent: compound G4 is extracted separation by Beijing University of Chinese Medicine's Chinese medicine modern study center from Lignum Aquilariae Resinatum；DMEM high glucose medium and DMEM/F12
Culture medium is purchased from Corning Incorporated；Bacteria lipopolysaccharide is purchased from Beijing Baeyer enlightening biotechnology Co., Ltd (article No.: L2880)；Measure TNF-α
It is purchased from Wuhan doctor's moral Bioisystech Co., Ltd (article No.: EK0527 and EK0411) with the ELISA kit of IL-6.
Instrument: Enspire Multimode Plate Reader is purchased from platinum Ai Ermo company of Germany.
(2) experimental technique
Being inoculated in 96 orifice plates by the RAW264.7 cell being in exponential phase, inoculum density is 50000 cells/well, volume 100 μ l, 37 DEG C
And 5%CO2After hatching 24 hours, adding compound G4 and positive drug dexamethasone (Dexamethasone, DEX), dexamethasone is the denseest
Spending 10 μMs (acellular toxic actions), compound G4 final concentration is respectively 5,10,20 μMs, each concentration of each compound sets 3 multiple holes, 1 hour
After, addition bacteria lipopolysaccharide (LPS), to final concentration 0.5 μ g/ml, continues to hatch 24 hours.Take cell conditioned medium, utilize ELISA kit to survey
Determine TNF-α and IL-6 concentration in cell conditioned medium.
(3) experimental result
Measured by secretion inflammatory factor, find that compound G4 process group cell conditioned medium TNF-α and IL-6 concentration are substantially less than LPS process group,
As shown in table 3 (* P ＜ 0.05vs LPS).Show that compound G4 can suppress mouse macrophage RAW264.7 post-stimulatory at lipopolysaccharide
TNF-α and IL-6 secretion.
Table 3: compound G4 vitro inhibition inflammatory factor secretory action measures
Embodiment 4: compound G4 vitro inhibition neutrophilic granulocyte activation effect measures
(1) experiment material
Animal: Balb/c mice is purchased from Beijing laboratory animal company limited of dimension tonneau China.
Reagent: compound G4 is extracted separation by Beijing University of Chinese Medicine's Chinese medicine modern study center from Lignum Aquilariae Resinatum；PE labelling rat anti-mouse CD11b
Antibody, purchased from BD Biosciences company of the U.S. (article No.: 557397).
Instrument: FACSCantoTMII flow cytometer is purchased from BD Biosciences company of the U.S..
(2) experimental technique
Cervical dislocation puts to death 1 Balb/c mice, 75% alcohol-pickled 5 minutes, then aseptic in superclean bench removes mice both sides femur, cuts off
Femoral knee end, utilizes 1ml syringe to draw IMDM culture medium (containing 2% hyclone) by during medullary cell is flushed to centrifuge tube in femur.
Through 40 μm aperture aseptic strainer filtering cell and numeration of leukocyte, then seed cells in 24 orifice plates, keep white blood cell concentration 1 × 106Individual/
Hole, addition IMDM culture medium (containing 2% hyclone) to every hole culture medium cumulative volume 1ml.Add compound G4 and positive drug dexamethasone,
Dexamethasone final concentration 10 μMs (acellular toxic action), compound G4 final concentration is respectively 5,10,20 μMs, each concentration of each compound sets 3
Individual multiple hole, 37 DEG C and 5%CO2After hatching 1 hour, add 250 μ l conditioned mediums and (utilize the RAW264.7 that DMEM high glucose medium is cultivated
Cell, stimulates 24 hours through the LPS of 0.5 μ g/ml, TNF-α concentration 30ng/ml), 37 DEG C and 5%CO2Continue to hatch 1.5h.Then will be thin
Born of the same parents collect in centrifuge tube, 4 DEG C, and 200g is centrifuged 5 minutes, removes supernatant, and the 250 μ l ice-cold PBS containing 1% bovine serum albumin (BSA) is resuspended
Cell, adds PE labelling rat anti-mouse CD11b antibody, hatches 1 hour on ice.It is subsequently adding 1ml containing the PBS of 1%BSA, 4 DEG C, 200g
Centrifugal 5 minutes, removing supernatant, add the 1ml PBS containing 1%BSA, 4 DEG C, 200g is centrifuged 5 minutes, removes supernatant.It is eventually adding 300 μ l
PBS re-suspended cell containing 1%BSA, utilizes BD FACSCantoTMNeutrophilic granulocyte CD11b labelling in II flow cytomery medullary cell
Average fluorescent strength.
(3) experimental result
By Flow cytometry, find that compound G4 process group CD11b average fluorescent strength is substantially less than LPS process group, as shown in table 4 (*
P ＜ 0.05vs LPS).Show that compound G4 can suppress Murine Neutrophil in the post-stimulatory activation of inflammatory factor.
Table 4: compound G4 vitro inhibition neutrophilic granulocyte activation effect measures
Embodiment 5: in compound G4 body, antiinflammatory action measures
(1) experiment material
Animal: Balb/c mice is purchased from Beijing laboratory animal company limited of dimension tonneau China.
Reagent: compound G4 is extracted separation by Beijing University of Chinese Medicine's Chinese medicine modern study center from Lignum Aquilariae Resinatum；Bacteria lipopolysaccharide is purchased from Beijing Baeyer
Enlightening biotechnology Co., Ltd (article No.: L2880)；Measure the ELISA kit of TNF-α and IL-6 purchased from Wuhan doctor's moral biotechnology
Company limited (article No.: EK0527 and EK0411).
Instrument: Enspire Multimode Plate Reader is purchased from platinum Ai Ermo company of Germany.
(2) experimental technique
By 36 8 week old Balb/c mices, adaptability is raised 3 days, then animal stochastic averagina is divided into 6 groups, Vehicle controls group (Vehicle),
Lipopolysaccharide group (LPS), Dexamethasone group (DEX), compound G4 basic, normal, high dosage group.By gavage give compound G4 each dosage group and
Dexamethasone group corresponding dosage compound G4 and dexamethasone, Vehicle and LPS group gives equal-volume solvent (containing 1% sodium carboxymethyl cellulose
5% glucose solution).It is administered latter 1 hour, gives 5 treated animal 10mg/kg lipopolysaccharide, Vehicle beyond Vehicle group by intravenous injection
Group gives respective volume normal saline.After 1.5h, gathering each treated animal venous blood by winning eyeball, after 4 DEG C of solidification 1h, 4 DEG C, 800g is centrifuged
Take serum.ELISA kit (Wuhan doctor's moral Bioisystech Co., Ltd) is utilized to measure TNF-α and IL-6 concentration in serum.
(3) experimental result
Measured by ELISA, find that compound G4 is administered TNF-α and IL-6 concentration in treated animal serum and is substantially less than LPS process group, such as table 5
Shown (* P ＜ 0.05vs LPS).Show that compound G4 has good internal antiinflammatory action.
Table 5: in compound G4 body, antiinflammatory action measures
Embodiment 6: compound G4 vitro inhibition CD4+T cell activation effect measures
(1) experiment material
Animal: Balb/c mice is purchased from Beijing laboratory animal company limited of dimension tonneau China.
Reagent: compound G4 is extracted separation by Beijing University of Chinese Medicine's Chinese medicine modern study center from Lignum Aquilariae Resinatum；Suslik anti-mouse CD3e antibody (goods
Number: 557306), suslik anti-mouse CD28 antibody (article No.: 557393), FITC labelling rat anti-mouse CD4 antibody (article No.: 553046),
PE labelling suslik anti-mouse CD69 antibody (article No.: 553237), purchased from BD Biosciences company of the U.S..
Instrument: FACSCantoTMII flow cytometer is purchased from BD Biosciences company of the U.S..
(2) experimental technique
Take aseptic 24 well culture plates, Vehicle controls group (Vehicle), model group (Model), Dexamethasone group (DEX), compound G4 are set
5, the dosage groups such as 10,20 μMs, each group sets 3 multiple holes, and each hole of Vehicle controls group adds the PBS of 1ml, and other is respectively organized every hole and adds 1ml containing 2 μ g/ml
The PBS of suslik anti-mouse CD3e antibody, 4 DEG C of overnight incubation.Cervical dislocation puts to death 1 Balb/c mice, 75% alcohol-pickled 5 minutes, then
Aseptic in superclean bench take mouse spleen, on 50ml centrifuge tube, set up 40 μm aperture nylon leaching nets, spleen is put on filter screen, slowly adds
Enter the PBS of 20ml, and grind spleen with 5ml syringe core rod flush end.The spleen cell 200g of filtration is centrifuged 5 minutes, removes supernatant, add
Enter the 5ml 1640RPMI culture medium re-suspended cell containing 10 hyclones, and leukocyte is counted.Then suck the PBS in 24 orifice plates, use
1640RPMI culture medium is cleaned 2 times, then is inoculated into by splenocyte in 24 orifice plates, keeps white blood cell concentration 1 × 106Individual/hole, adds containing 10 tire cattle
The 1640RPMI culture medium of serum is to every hole culture medium cumulative volume 1ml.Positive drug ground is added again in dexamethasone and compound G4 each dosage group hole
Sai meter Song and compound G4, dexamethasone final concentration 10 μMs (acellular toxic action), compound G4 each dosage group final concentration is respectively 5,10,
20 μMs, 37 DEG C and 5%CO2Under the conditions of continue to hatch.After 1 hour, CD3e is coated in hole and adds suslik anti-mouse CD28 antibody, concentration 2 μ g/ml,
Continue to hatch 16 hours.Then collecting in centrifuge tube by cell, 4 DEG C, 200g is centrifuged 5 minutes, removes supernatant, and 250 μ l are pure containing 1% Sanguis Bovis seu Bubali
The ice-cold PBS re-suspended cell of albumen (BSA), adds FITC labelling rat anti-mouse CD4 antibody and PE labelling suslik anti-mouse CD69 resists
Body, hatches 1 hour on ice.Being subsequently adding the 1ml PBS containing 1%BSA, 4 DEG C, 200g is centrifuged 5 minutes, removes supernatant, adds 1ml and contains
The PBS of 1%BSA, 4 DEG C, 200g is centrifuged 5 minutes, removes supernatant.It is eventually adding the 300 μ l PBS re-suspended cell containing 1%BSA, utilizes BD
FACSCantoTMII flow cytomery CD4+T cell activation mark CD69 average fluorescent strength.
(3) experimental result
By Flow cytometry, find compound G4 each dosage group CD4+Cell CD69 average fluorescent strength is substantially less than Model group, such as table
Shown in 6 (* P ＜ 0.05vs Model).Show that compound G4 can suppress mice CD4+The activation of T cell.
Table 6: compound G4 vitro inhibition CD4+T cell activation effect measures
Embodiment 7: compound G4 mechanisms of anti-inflammatory is studied
(1) experiment material
Cell strain: RAW264.7 cell is purchased from Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences's cell centre.
Reagent and material: compound G4 is extracted separation by Beijing University of Chinese Medicine's Chinese medicine modern study center from Lignum Aquilariae Resinatum；DMEM high glucose medium and
DMEM/F12 culture medium is purchased from Corning Incorporated；Bacteria lipopolysaccharide is purchased from Beijing Baeyer enlightening biotechnology Co., Ltd (article No.: L2880)；RIPA
Lysate (article No.: P0013B), BCA protein quantification test kit (article No.: P0010), PAGE gel reagent preparation box (article No.: P0012A),
SDS-PAGE electrophoresis liquid (article No.: P0014B), Westernblot transferring film liquid (article No.: P0021B), super quick ECL chemical luminescence reagent kit (goods
Number: P0018), pvdf membrane (article No.: FFP39) is purchased from green skies biotechnology research institute；NF-κ B Pathway Sampler Kit (article No.: 9936),
MAPK Family Antibody Sampler Kit (article No.: 9926) and Phospho-MAPK Family Antibody Sampler Kit (goods
Number: 9910) purchased from Cell Signalling Technology company.
Instrument: ImageQuantLAS4000mini ultra sensitive chemical luminescence imaging instrument, purchased from General Electric company.
(2) experimental technique
Inoculating cell: be inoculated in 6 orifice plates by the RAW264.7 cell being in exponential phase, inoculum density is 200000 cells/well, volume
2ml, 37 DEG C and 5%CO2Hatch 24 hours；Drug treating cell: 6 orifice plate cells are set Vehicle controls group (Vehicle), model group (Model),
Compound G4 each dosage group (5,10,20 μMs), compound G4 each dosage group adds compound G4 to final concentration of 5,10,20 μMs, and 1 is little
Shi Hou, addition bacteria lipopolysaccharide (LPS), to model group and G4 each dosage group to final concentration 0.5 μ g/ml, continues to hatch 6 hours；Collection cell:
Abandoning cell culture medium, rinse cell 2 times with ice-cold PBS, the most each hole adds the ice-cold PBS of 2ml, scrapes collection cell with cell and is centrifuged to 2ml
Guan Zhong, 4 DEG C, 300g be centrifuged 5 minutes, abandon supernatant；Cell lysis: add the RIPA lysate of 400 μ l, ice bath cracking 30 in each solencyte
Minute, 4 DEG C, 10000g be centrifuged 10 minutes, collect supernatant；Protein concentration is quantitative: utilize BCA protein quantification kit measurement each sample albumen
Concentration, and with RIPA lysate, each sample concentration is transferred to same concentration；Prepare protein electrophoresis sample: draw each 200 μ l of above-mentioned protein sample, add
Enter 50 μ l protein electrophoresis sample-loading buffer (5 ×), mix rear 99 DEG C of metal baths 10 minutes, be then cooled to room temperature；Protein electrophoresis: utilize SDS-PAGE
10% polyacrylamide gel prepared by gel reagent preparation box, then by each for above-mentioned protein sample loading 10 μ l, and 90V constant voltage electrophoresis 2 hours: albumen electricity
Turn: pvdf membrane that foam-rubber cushion, filter paper, methanol were soaked and complete the polyacrylamide gel of electrophoretic procedures, filter paper, foam-rubber cushion are put in order
Turning in folder to electricity, then be fixed in electricity turn trough, 120V constant voltage ice bath electricity turns 1.5 hours；Close: the film after being turned by electricity takes out, and puts into TBS molten
5% skim milk of liquid preparation is closed 1 hour；One anti-hatches: the pvdf membrane after closing is cut into little bar by purpose band position, so
After be separately added into 5% skim milk preparation rabbit anti-mouse Actin antibody, p65 antibody, phospho-p65 antibody, p38 antibody, phospho-p38
Antibody, Erk antibody, phospho-Erk antibody, Jnk antibody, phospho-Jnk antibody (being 1: 1000 dilution), hatched in 4 DEG C of plastic packaging bags
Night；Washing: by hatch one anti-after pvdf membrane TBST wash 3 times, each 5 minutes；Two anti-hatch: again pvdf membrane is loaded plastic packaging
In Dai, add the goat anti-rabbit igg of the horseradish peroxidase-labeled of TBST preparation, incubated at room 2h；Washing: by hatch two anti-after PVDF
Film TBST washing 3 times, each 5 minutes；Chemiluminescence imaging: pvdf membrane is put into one by one ImageQuantLAS4000mini hypersensitive
In chemiluminescence imaging instrument, add the excess of imports quick ECL chemical luminescence for liquid, shoot chemiluminescence picture.
Concentration.
(3) experimental result
Detected by above-mentioned Westernblot, find that compound G4 processes RAW264.7 cell, the p65 of LPS activation NF-κ B path is not had
Inhibitory action, does not has inhibitory action to p38 and the Erk signal pathway activated of MAPK path yet, but in compound G4, high dose is to Jnk signal pathway activated
There is significant inhibitory action (see photo), show to suppress Jnk signal pathway activated to be probably compound G4 performance antiinflammatory action and other natural immunity presses down
System and the key mechanism of adaptive immunity suppression.
Claims (6)
1. the compound selected from following form in prevention or treats the application in inflammation related disease, autoimmune disease and organ-graft refection.
2. contain the pharmaceutical composition of arbitrary compound in claim 1 and in prevention or treat inflammation related disease, autoimmune disease and organ
Application in transplant rejection.
3. according to the application of claim 1 and 2, described inflammation related disease include systemic inflammatory responses, pneumonia, bronchitis, hepatitis,
Nephritis, gastritis, enteritis, neuroinflamation, scytitis.
4. according to the application of claim 1 and 2, it is characterised in that described autoimmune disease includes rheumatoid arthritis, systematicness
Lupus erythematosus.
5. according to the application of claim 1 and 2, it is characterised in that described organ transplantation includes that renal transplantation, liver transplantation, hematopoietic stem cell move
Plant, skin transplantation.
6. according to the application of claim 1 and 2, it is characterised in that described prevention or treatment inflammation related disease, autoimmune disease and
The mechanism of organ-graft refection is the suppression natural immunity and adaptive immunity function.
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| CN201510396970.2A CN106309423B (en) | 2015-07-09 | 2015-07-09 | The purposes of chloro tetrahydro phenethyl chromone derivative and its pharmaceutical composition in agalloch eaglewood |
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| CN201510396970.2A CN106309423B (en) | 2015-07-09 | 2015-07-09 | The purposes of chloro tetrahydro phenethyl chromone derivative and its pharmaceutical composition in agalloch eaglewood |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106924444A (en) * | 2017-04-27 | 2017-07-07 | 中国医学科学院药用植物研究所海南分所 | A kind of entire body perfume extract medicinal with suppression enteritis and its preparation method and application |
| CN108727322A (en) * | 2017-04-25 | 2018-11-02 | 北京中医药大学 | Phenethyl chromone dimer and its preparation method and pharmaceutical composition and purposes |
| CN110818669A (en) * | 2019-11-25 | 2020-02-21 | 中国医学科学院药用植物研究所海南分所 | Aquilaria sinensis tetrahydro 2- (2-phenethyl) chromone compound and separation method and application thereof |
| CN112898261A (en) * | 2021-01-27 | 2021-06-04 | 中国热带农业科学院热带生物技术研究所 | Compound for preventing and treating inflammation and preparation method and application thereof |
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2015
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| DONG CHEN ET AL.: "Nine 2-(2-Phenylethyl)chromone Derivatives from the Resinous Wood of Aquilaria sinensis and Their Inhibition of LPS-Induced NO Production in RAW 264.7 Cells", 《EUR. J. ORG. CHEM.》 * |
| WEI LI ET AL.: "2-(2-Phenylethyl)chromone derivatives from Chinese agarwood induced by artificial holing", 《FITOTERAPIA》 * |
| WU BO ET AL: "Eight New 2-(2-Phenylethyl) chromone (2-(2-Phenylethyl)-4H-1-benzopyran-4-one) Derivatives from Aquilaria malaccensis Agarwood", 《HELVETICA CHIMICA ACTA》 * |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108727322A (en) * | 2017-04-25 | 2018-11-02 | 北京中医药大学 | Phenethyl chromone dimer and its preparation method and pharmaceutical composition and purposes |
| CN108727322B (en) * | 2017-04-25 | 2020-10-20 | 北京中医药大学 | Phenethyl chromone dimer, preparation method thereof, pharmaceutical composition and application |
| CN106924444A (en) * | 2017-04-27 | 2017-07-07 | 中国医学科学院药用植物研究所海南分所 | A kind of entire body perfume extract medicinal with suppression enteritis and its preparation method and application |
| CN110818669A (en) * | 2019-11-25 | 2020-02-21 | 中国医学科学院药用植物研究所海南分所 | Aquilaria sinensis tetrahydro 2- (2-phenethyl) chromone compound and separation method and application thereof |
| CN110818669B (en) * | 2019-11-25 | 2022-11-29 | 中国医学科学院药用植物研究所海南分所 | Aquilaria sinensis tetrahydro 2- (2-phenethyl) chromone compound and separation method and application thereof |
| CN112898261A (en) * | 2021-01-27 | 2021-06-04 | 中国热带农业科学院热带生物技术研究所 | Compound for preventing and treating inflammation and preparation method and application thereof |
| WO2022160455A1 (en) * | 2021-01-27 | 2022-08-04 | 中国热带农业科学院热带生物技术研究所 | Compound for preventing and treating inflammation, and preparation method therefor and use thereof |
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| Publication number | Publication date |
|---|---|
| CN106309423B (en) | 2019-06-07 |
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