CN105755097A - Method for comprehensively screening anti-leukemia active constituents from natural indigo and method for determining anti-leukemia active constituent targets - Google Patents
Method for comprehensively screening anti-leukemia active constituents from natural indigo and method for determining anti-leukemia active constituent targets Download PDFInfo
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Abstract
The invention provides a method for comprehensively screening anti-leukemia active constituents from natural indigo and a method for determining anti-leukemia active constituent targets.In the anti-leukemia active constituents, a comprehensive two-dimensional K562/CMC system is constructed, two-dimensional gas chromatography and time-of-flight mass spectrometry are combined, and a K562 cell membrane chromatographic column is constructed to serve as a first-dimensional chromatographic column so as to be beneficial to optimum separation of various constituents in the natural indigo; in a process of determining the anti-leukemia active constituent targets, Pharm Mapper Server software and Target Hunter software are used for subjecting the active constituents to reverse experiments to determine the active constituent targets, Venny analysis software is used for subjecting docking targets of the Pharm Mapper Server software and the Target Hunter software to common target analysis, and a forward docking experiment is combined with a cell membrane chromatography competition experiment, a kinase activity assay experiment or a surface plasmon resonance experiment to be used for target verification, so that the targets of the active constituents for leukemia cells can be determined rapidly and accurately.
Description
Technical field
The invention belongs to biological technical field, be specifically related in a kind of Indigo Naturalis of screening comprehensively anti-leukocythemia liveness composition and determine its target calibration method.
Background technology
Indigo Naturalis is the leaf of acanthaceous vegetable acanthaceous indigo, polygonaceae plant polygonum tinctorium ait. or cross China Tech plant Isatis indigotica Fort. or the processed prepared dried powder of stem and leaf or agglomerate.Research in recent years shows: containing plurality of active ingredients such as indigo, indirubin, couroupitine As in Indigo Naturalis, wherein, indirubin has leukemia composition.But, research confirms, indirubin can not substitute leukemia effect of Indigo Naturalis completely, speculate accordingly, not yet someone carried out correlational study at present to also have other leukemia compositions, but the concrete kind of these compositions, and the action target that these compositions are on leukaemia in Indigo Naturalis.
Summary of the invention
The present invention carries out for solving the problems referred to above.First purpose of the present invention is in that to provide a kind of method of anti-leukocythemia liveness composition in comprehensive screening Indigo Naturalis；Second purpose of the present invention is in that to provide one to determine these anti-leukocythemia liveness composition target calibration methods, in utilization, the 3rd purpose of the present invention determines that anti-leukocythemia liveness composition target calibration method carries out screening and the checking of isorhamnetin target.
In order to realize first purpose, present invention employs following technical scheme:
A kind of method of anti-leukocythemia liveness composition in comprehensive screening Indigo Naturalis, it is characterised in that comprise the following steps: step 1, prepares Indigo Naturalis extracting solution；Step 2, builds full two dimension K562/CMC system, and the first dimension chromatographic column in this full two dimension K562/CMC system is K562 membrane flexibility post, and the second dimension chromatographic column is C18 chromatographic column, and the second dimension Coupled columns has flight time mass spectrum；Step 3, Indigo Naturalis extracting solution in step one is squeezed in full two dimension K562/CMC system, the first dimension chromatographic column is adopted to carry out first time separation, adopt the second dimension chromatographic column that the first outflow component tieed up in chromatographic column is easily separated again, adopt flight time mass spectrum that the outflow component of Two way chromatograms is analyzed, obtain flowing out the structural information of component.By constantly switching ten direction changeover valves in full two dimension K562/CMC system, obtain multiple potential leukemia composition；Step 4, activity checking, the impact of propagation and apoptosis by investigating the leukemia composition K562 leukaemia on being in logarithmic (log) phase is verified, and obtains different anti-leukocythemia liveness compositions.
Wherein, in step, the step of preparation Indigo Naturalis extracting solution comprises the following steps: step 1-1, dry Indigo Naturalis powder is mixed with the ratio that mass ratio is 1:10 with chloroformic solution (chloral hydrate containing 2%), in a heated condition, adopt ultrasonic vibration, obtain the extracting solution containing active component；Step 1-2, by the extracting solution micro-pore-film filtration through 0.22 μm, obtains described Indigo Naturalis extracting solution.
In step 2, building full two dimension K562/CMC system and include the preparation of K562 membrane flexibility post, method is as follows: step 2-1, takes 3.7 × 107Individual K562 cell prepares K562 membrane pellet, and is resuspended in 5mL normal saline by K562 membrane pellet, forms suspension；Step 2-2, reacts suspension and a certain amount of silica gel to 5min when 4 DEG C and vacuum oscillation, forms the fixing phase of cell membrane；Step 2-3, adopts liquid phase pump and loads fixing for described cell membrane mutually in chromatographic column for mobile phase with the phosphate buffer of 10mmol/L, and dress column flow rate gradient optimizing is as follows: 0-5min, flow velocity rises to 1.0mL min-1 from 0.2mL min-1；5-5.5min, flow velocity is maintained at 1.0mL min-1,30s, balances chromatographic column subsequently under the flow velocity of 0.2mL min-1.
Including isorhamnetin and couroupitine A by anti-leukocythemia liveness composition in the Indigo Naturalis that said method obtains, it can be used for preparation prevention or treatment leukemia medicament.
In order to realize second purpose of the present invention, one provided by the invention determines anti-leukocythemia liveness composition target calibration method, comprise the following steps: step one, adopt software PharmMapperServer and TargetHunter that active component is reversely docked, respectively obtain multiple docking target；Step 2, application Venny analyzes software and the docking target of above two software carries out total target analysis, then according to matching degree score, normalization fit score and z ' mark are ranked up, obtains the target that probability is maximum；Step 3, the maximum target of the probability that step 2 obtained carries out forward docking experiment and is verified, it is determined that target.
Meanwhile, target is verified by the basis of forward docking experiment in conjunction with membrane flexibility competitive assay, kinase activity test experience, surface plasma resonance experiment.
In order to realize the 3rd purpose of the present invention, the invention provides one and determine isorhamnetin target calibration method, comprise the following steps: step 1, adopt software PharmMapperServer and TargetHunter that active component is reversely docked, respectively obtain multiple docking target；Step 2, application Venny analyzes software and the docking target of above two software carries out total target analysis, obtain eight total targets, then according to matching degree score, normalization fit score and z ' mark are ranked up, it is determined that the maximum target of probability is nonreceptor tyrosine kinase；Step 3, nonreceptor tyrosine kinase step 2 obtained carries out forward docking experiment and is verified, it is determined that target.
Said method also includes the step that the effect molecular mechanism of isorhamnetin is explored, the method adopting cell cycle detection and Western-Blotting is explored, and isorhamnetin blocks K562 cell in the G2/M phase by Src/ATR/Wee1/CDK1/CyclinB signal path.
Invention effect and effect
According to the method for anti-leukocythemia liveness composition in comprehensive screening Indigo Naturalis provided by the invention, owing to constructing full two dimension K562/CMC system, by two-dimensional gas chromatography and flight time mass spectrum coupling, and build K562 membrane flexibility post as the first dimension chromatographic column, be conducive to reaching components various in Indigo Naturalis the separating effect of optimum.
Additionally, these anti-leukocythemia liveness composition target calibration methods are determined according to provided by the invention, determine the target of active component owing to adopting PharmMapperServer and TargetHunter software that active component carries out reversely experiment, adopt Venny analysis software that the docking target of both the above software carries out total target analysis, forward docking experiment is adopted to carry out target checking in conjunction with the experiment of membrane flexibility competitive assay, kinase activity test experience or surface plasma resonance, it is possible to determine the active component target to leukaemia fast, accurately.
Accompanying drawing explanation
Fig. 1 is the full two dimension K562/CMC binding mode figure of the present invention.Wherein, (a) is two ten-way valves binding mode figure of two dimension K562/CMC system full when being in position 1；B () is two ten-way valves binding mode figure of two dimension K562/CMC full when being in position 2.
Fig. 2 is the 3D figure that Plays product mixed solution of the present invention obtains in the full two-dimentional system analysis of K562/CMC；
Fig. 3 (a) is the 3D figure that the Indigo Naturalis extract in the present invention obtains in the full two-dimentional system analysis of K562/CMC；Fig. 3 (b) is the structural formula of the potential anti-leukocythemia liveness compound identified through time-of-flight mass spectrometry (TOFMS).
Fig. 4 (a) is the isorhamnetin measured of the CCK-8 in the present invention result figure to the inhibited proliferation of K562 cell；Fig. 4 (b) and Fig. 4 (c) is the isorhamnetin of the Annexin V-FITC detection in the present invention result figure to K562 apoptosis facilitation.
Fig. 5 is the schematic diagram analyzing the total target spot obtained in the present invention through PharmMapperServer and TargetHunter.
Fig. 6 is the result schematic diagram that action target spot is Src kinases receptors verifying isorhamnetin in the present invention.Wherein, the numeral 1～5 in figure represents the isorhamnetin of variable concentrations respectively, and (a) is isorhamnetin and Src kinases receptors joint mode figure；B () is the result of membrane flexibility competitive assay；C () is kinase activity testing result；D () is the result figure of surface plasma resonance experiment；E () is the sensing figure after the isorhamnetin removal blank of the variable concentrations in (d).
Fig. 7 is the result figure that isorhamnetin passes through the Src receptors modulate cell cycle.Wherein, (a) and (b) is the result of the G2/M phase that is arrested in by K562 leukemic cells of isorhamnetin dose dependent；C () is that isorhamnetin directly acts on Src receptor, regulated the result figure of cell cycle by ATR/Wee1/CDK1/CyclinB.
Detailed description of the invention
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described.
One, antileukemie active component potential in screening Indigo Naturalis
1, preparation Indigo Naturalis extracting solution, comprises the following steps:
Step 1, mixes dry Indigo Naturalis powder with the ratio that mass ratio is 1:10 with chloroformic solution (chloral hydrate containing 2%), heating-up temperature less than 70 DEG C when, adopt ultrasonic vibration, obtain the extracting solution containing active component；Step 2, by the extracting solution micro-pore-film filtration through 0.22 μm, obtains Indigo Naturalis extracting solution.
2, full two dimension K562/CMC system is built
Full two dimension K562/CMC system adopts Agilent1200 series of high efficiency chromatograph of liquid (Agilent, the U.S.).As it is shown in figure 1, full two dimension K562/CMC system include the first high-pressure liquid phase delivery pump 1, automatic sampler 2, as first 3, two ten direction changeover valve 4 (model: RheodyneMXP9960 of K562 membrane flexibility post tieing up chromatographic column；Supplier: IDEXCorporation, U.S.), first pre-column the 5, second high-pressure liquid phase delivery pump the 6, the 3rd high-pressure liquid phase delivery pump the 7, second pre-column 8, as the second C tieing up chromatographic column18Reversed phase chromatographic column 9 and flight time mass spectrum 10.
As shown in Fig. 1 (a), first high-pressure liquid phase delivery pump 1 is modular pump, connect with automatic sampler 2 and as the first K562 membrane flexibility post 3 tieing up chromatographic column, K562 membrane flexibility post 3 connects with the 1. position in two ten direction changeover valves 4,2. position in two ten direction changeover valves 4 connects the entrance point of the first pre-column 5,5. position connects the port of export of the first pre-column 5, and 5. position connects with 6. position, and 6. position is used for emptying the first pre-column 5.
Second high-pressure liquid phase delivery pump 6 and the 3rd high-pressure liquid phase delivery pump 7 collectively constitute a binary pump, connect with the 9. position in two ten direction changeover valves 4,9. position connects with 10. position, 10. position connects the entrance point of the second pre-column, the port of export connects with 10. position, 10. position is connected with 3. position by 10. position, and 3. position is by 4. position with as the second C tieing up chromatographic column18Reversed phase chromatographic column 9 connects, C18Being partly in flight time mass spectrum 10 of reversed phase chromatographic column 9 is analyzed.
Build the parts overwhelming majority in full two dimension K562/CMC system and belong to instrument from tape member, need to change according to the difference of actual separation composition only for the chromatographic column separated.In the present invention, the groundwork building full two dimension K562/CMC system is in that K562 membrane flexibility Column preparation, and preparation method comprises the steps:
Step 1, takes 3.5 × 107Individual K562 cell, use 10mmol/L phosphate buffer (PBS) eccentric cleaning three times under 1000 × g rotating speed, each 5min, take precipitation 10mmol/LPBS suspendible, use JY92-IIN Ultrasonic Cell Disruptor that cell suspension is crushed, ultrasonic power is 400W, carries out altogether 5 times, and total time is 90s.
Step 2, by broken homogenate centrifugal 10min under 1000 × g, takes supernatant, more centrifugal 20min obtains membrane pellet under 12000 × g.Take precipitation and be resuspended in 5mL normal saline, then suspension and 0.04g silica gel are reacted when vacuum concussion 5min, form the fixing phase of cell membrane, after stirring 30min, in 4 DEG C of condition left overnight.
Step 3, fills post, carries out under 4 DEG C of conditions, adopts liquid phase pump and loads fixing for cell membrane mutually in chromatographic column for mobile phase with the phosphate buffer of 10mmol/L, and dress column flow rate gradient optimizing is as follows: 0-5min, and flow velocity is from 0.2mL min-1Rise to 1.0mL min-1；5-5.5min, flow velocity is maintained at 1.0mL min-1, 30s, subsequently at 0.2mL min-1Flow velocity under balance chromatographic column.
The membrane flexibility post prepared should be stored under 4 DEG C of conditions.
3, the investigation of full two dimension K562/CMC system stability
Configuring certain density imatinib and dexamethasone mixed solution as titer, then sieved by full two dimension K562/CMC system by this titer, investigate the stability of system, result is as shown in Figure 2.As shown in Figure 2, two kinds of medicines can be completely separable, illustrates that the stability of system is better.
4, the screening of potential anti-leukocythemia liveness composition in Indigo Naturalis
Step 1, as shown in Fig. 1 (a), Indigo Naturalis extracting solution is by after automatic sampler 2 sample introduction, K562 membrane flexibility post 3 is easily separated, after phosphate buffer eluting via the first high-pressure liquid phase delivery pump 1 conveying, first component flows out and is enriched with in the first pre-column 5, and meanwhile, the second high-pressure liquid phase delivery pump 6 and the 3rd high-pressure liquid phase delivery pump 7 are used for balancing C18Reversed phase chromatographic column 9 and flight time mass spectrum 10；
Step 2, after 2.5min, switches two ten direction changeover valves 4, and as shown in Fig. 1 (b), now, the 2. position in two ten direction changeover valves 4 connects with 3. position.Binary pump by 9. position, 8. position, 3. position, 2. position, 5. position and 4. position the first component in the first pre-column 5 is directed into C18Reversed phase chromatographic column 9 further separates, and carries out data analysis by flight time mass spectrum 10.Now, from K562 membrane flexibility post 3 flow out second component by 1. position and 10. position be enriched in the second pre-column 8；
Step 3, again two ten direction changeover valves 4 of switching so that present the annexation shown in Fig. 1 (a) in system, carry out the analysis of second component and the enrichment of the 3rd component.So switching 12 times so that the whole components in the first dimension chromatographic column can enter into C18-mass spectrum further separates analysis.
In the present invention, adopt mass analyzer to carry software (AgilentMassHunterWorkstation) data of mass spectrum collection are analyzed, initial data is derived, import to again in MATLAB7.10.0, carry out baseline calibration and peak alignment, then draw 3D collection of illustrative plates, result is compared, screening active component, selects result to see Fig. 3.As shown in Fig. 3 (a) and Fig. 3 (b), adopt this system to carry out the screening of leukemia composition in Indigo Naturalis extracting solution, successfully filtered out the active component that indirubin, couroupitine A and isorhamnetin are three kinds potential.
Two, the activity checking of potential antileukemie active component
Lateral reactivity composition is carried out cell proliferation and apoptosis activity detection.
1, the impact of on cell proliferation:
Take the logarithm the K562 leukaemia of trophophase, 1000g is centrifuged, it is laid in 96 orifice plates after resuspended counting, density is 5000 cells/well, is separately added into the isorhamnetin (0,12.5,25,50,100,200 μMs) of different final concentration, after 48h after overnight incubation, every hole adds 10 μ LCCK-8 reagent, 37 DEG C of incubators hatch 2h, adopt Synergy4 multi-wavelength microplate reader (BioTek, the U.S.) to measure the absorbance in every hole, 450nm place.
2, on apoptotic impact:
First, take the logarithm the K562 leukaemia of trophophase, 1000g is centrifuged, it is laid in 6 orifice plates after resuspended counting, density is 10000 cells/well, is separately added into the isorhamnetin (0,12.5,25,50 μMs) of different final concentration, after 48h after overnight incubation, 1000g centrifugal collecting cell, then wash once with 1 × PBS；Then, take the resuspended cell of 5-10 ten thousand, centrifugal 5 minutes of 1000g, abandons supernatant, adds 195 μ LAnnexinV-FITC in conjunction with liquid re-suspended cell gently；Then, add 5 μ LAnnexinV-FITC, mix gently；Finally, adding 10 μ L propidium iodide stain liquid, mix gently, room temperature (20-25 DEG C) lucifuge is hatched 10-20 minute, is subsequently placed in ice bath.
Two above experiment repeats 6 times respectively, data acquisition GraphpadPrism5 carries out statistical disposition, and data description meansigma methods ± standard deviation represents, compares employing administration group and compare with blank group between group, adopting t check analysis, the difference between P < 0.05 expression two groups is statistically significant.
Interpretation of result
Fig. 4 is isorhamnetin on the cell proliferation of K562 leukaemia and apoptotic affects result schematic diagram.
Fig. 4 (a) is the CCK-8 isorhamnetin the measured result figure to the inhibited proliferation of K562 cell.It follows that along with the increase of isorhamnetin concentration, the suppression ratio of K562 cell proliferation is increased by isorhamnetin, when the concentration of isorhamnetin is 200 μm of ol/L, to the suppression ratio of K562 cell proliferation close to 80%.
Fig. 4 (b) is the isorhamnetin the adopting Annexin V-FITC dyeing detection result figure to K562 apoptosis facilitation.It follows that along with the increase of isorhamnetin concentration, the apoptotic promotion rate of K562 is increased by isorhamnetin, when the concentration of isorhamnetin is 50 μm of ol/L, to the apoptotic suppression ratio of K562 higher than 80%.
Fig. 4 (c) is the isorhamnetin adopting the double; two dyeing detection of the Annexin V-FITC/PI result figure to K562 apoptosis facilitation.It can thus be appreciated that, comparing with matched group, the apoptosis of K562 cell is had facilitation by isorhamnetin, along with the increase of isorhamnetin concentration, the K562 cell being colored gets more and more, and illustrates that the apoptotic promotion rate of K562 is increased by isorhamnetin along with the increase of isorhamnetin concentration.
Inventor it is experimentally confirmed that couroupitine A can suppress the propagation of K562 cell too by same, promoted the apoptosis of K562 cell, has good anti-leukocythemia liveness.
Three, the screening of anti-leukocythemia liveness composition target and checking
Below with the screening of isorhamnetin target be verified as example, the screening of anti-leukocythemia liveness composition target in Indigo Naturalis and proof procedure are illustrated.The screening of other anti-leukocythemia liveness composition target is identical with isorhamnetin with the method for checking.
1, target sieving
As it is shown in figure 5, the process of target sieving comprises the steps:
Step one, adopts software PharmMapperServer and the TargetHunter that opens for free that active component is reversely docked, and respectively obtains 198 and 208 docking targets；
Step 2, application Venny analyzes software and the docking target of above two software carries out total target analysis, obtain eight total targets, then according to eight total targets are ranked up by matching degree score, normalization fit score and z ' mark, result is as shown in table 1, obtains the target that probability is maximum.
The score sequence of 1 eight total targets of table
Further according to target spot and cell membrane and leukemic dependency, result according to table 1, finds that nonreceptor tyrosine kinase (Src) is the maximum action target spot of isorhamnetin probability.
2, target checking
Adopt forward docking software LeDock that isorhamnetin and Src are docked, as shown in Fig. 6 (a), and be verified in conjunction with membrane flexibility competitive assay, kinase activity test experience, surface plasma resonance experiment, Src is verified as target.
(1), membrane flexibility competitive assay is adopted to be verified
With Dasatinib (Src inhibitor) solution for titer, investigating and add before and after the isorhamnetin of variable concentrations in mobile phase, whether the retention time of Dasatinib there occurs change.Method particularly includes: first steady to baseline with Spirit of Mindererus. balance K562 leukaemia's film post of 10mmol/L, sample introduction Dasatinib, record corresponding retention time.Then measure in the mobile phase of 10mmol/LPBS, be separately added into the variation tendency of Dasatinib retention time after the isorhamnetin active Chinese drug component monomer that concentration is 0,0.5,1,2 and 5 μm of ol/L.
(2) kinase activity detection, is adopted to be verified:
Adopt mobility to change method (Mobilityshiftassay) to detect, comprise the steps:
Step 1, prepares 1 × kinase buffer liquid (50mmol/LHEPES, pH7.5；
0.0015%Brij-35；10mmol/LMgCl2；2mmol/LDTT) with stop buffer (100mmol/LHEPES, pH7.5；0.015%Brij-35；0.2%CoatingReagent#3；50mmol/LEDTA)；
Step 2, compound prepares: first, configures the isorhamnetin mother solution of 10mmol/L with DMSO, then in 96 orifice plates, the isorhamnetin of 10 variable concentrations of configuration, and take respectively in every hole in 10 μ L one 96 new orifice plate of addition；Then, every hole adds 90 μ L1 × kinase buffer liquid, and after concussion mixing 10min, every hole takes 5 μ L and adds in 384 new orifice plates, and arranges secondary orifices (n=3)；
Step 3, kinase reaction: first, prepares 2.5 × enzymatic solution and 2.5 × peptide solution, enzyme will be added and obtain 2.5 × enzymatic solution in 1 × phosphate buffer, the peptide of FAM labelling and ATP are joined in 1 × kinase buffer liquid, prepares 2.5 × peptide solution；Secondly, 2.5 × enzymatic solution is added to the 384 orifice plate detection holes containing the 5 μ L isorhamnetin solution containing 10%DMSO, incubated at room 10min；Then, in detection hole, add 10 μ L2.5 × peptide solution, hatch the appointment time for 28 DEG C, carry out kinase reaction；Finally, add 25 μ L stop buffers and terminate reaction.
Step 4, carries out Caliper reading, and carries out data process.
(3) surface plasma resonance experiment is adopted to be verified:
Adopt CM5 chip, then adopt 1:1 combination model, by variable concentrations (0,1.95,3.91,7.81,15.62,31.25,62.50 μm of ol/L) isorhamnetin sample introduction.
Interpretation of result
Membrane flexibility competitive assay shows that the retention time of Dasatinib (Src inhibitor) is gradually shortened (Fig. 6 b) along with in mobile phase, the concentration of isorhamnetin raises；Vitro kinase activity test result indicate that the IC50 of Src kinase activity is 6.9 μm of ol/L (Fig. 6 c) by isorhamnetin；The K of the adhesion between surface plasma resonance experimental result display Src and isorhamnetindValue is 3.81 × 10-6Mol (Fig. 6 d and Fig. 6 e).Data above result proves that Src is an action target spot of isorhamnetin.
Four, the effect molecular mechanisms of isorhamnetin
1, cell cycle detection:
Take the logarithm the K562 leukaemia of trophophase, 1000g is centrifuged, it is laid in 6 orifice plates after resuspended counting, density is 10000 cells/well, the isorhamnetin (0,12.5,25,50 μm of ol/L) of different final concentration it is separately added into after overnight incubation, after 24h, 1000g centrifugal collecting cell, then wash once with 1 × PBS.
Cell is fixed: adds in 1 milliliter of ice bath pre-cooling 70% ethanol, blows and beats mixing gently, fixes 24 hours for 4 DEG C, then centrifugal 5 minutes of 1000g, sedimentation cell.Carefully absorb supernatant, add the PBS of about 1 milliliter of ice bath pre-cooling, re-suspended cell.Recentrifuge sedimentation cell, carefully absorbs supernatant.Gently with suitable cell dispersion at the bottom of attack centrifuge tube, it is to avoid cell is agglomerating.Adding 0.3 milliliter of propidium iodide stain liquid in every solencyte sample, slow and abundant re-suspended cell precipitates, and after 37 DEG C of lucifuge temperature are bathed 30 minutes, upper machine detects.
2, Western-Blotting experiment carries out according to the step in documents below:
Chen,X.etal.Comparativenormal/failingratmyocardiumcellmembranechromatographicanalysissystemforscreeningspecificcomponentsthatcounteractdoxorubicin-inducedheartfailurefromAcontiumcarmichaeli.Analyticalchemistry86,4748-4757,doi:10.1021/ac500287e(2014).Can also carry out according to ordinary skill in the art means.
Interpretation of result
Fig. 7 is cell cycle and Western-Blotting result figure.By Fig. 7's it is shown that isorhamnetin can pass through Src/ATR/Wee1/CDK1/CyclinB signal path retardance K562 cell in the G2/M phase, and then inducing cell apoptosis, play leukemia effect.
Embodiment effect and effect
The present embodiment discloses a kind of new anti-leukocythemia liveness composition in Indigo Naturalis: isorhamnetin.And the novel targets to isorhamnetin leukemia resisting action: nonreceptor tyrosine kinase (Src) has screened and has verified.Screening and the result of confirmatory experiment are further characterized by the isorhamnetin that the present embodiment screens and can effectively suppress the increment of K562 cell and promote apoptosis.This compound can directly act on Src, significantly inhibits the phosphorylation of Src, regulates cell cycle by cell block in the G2/M phase.The adhesion of surface plasma resonance data display isorhamnetin and Src is 3.81 μm of ol/L, and kinase activity detection finds that the kinase whose IC50 of Src is 6.9 μm of ol/L by isorhamnetin, is a good lead compound.
Claims (10)
1. the method for anti-leukocythemia liveness composition in a comprehensive screening Indigo Naturalis, it is characterised in that comprise the following steps:
Step 1, prepares Indigo Naturalis extracting solution；
Step 2, builds full two dimension K562/CMC system, and the first dimension chromatographic column in described full two dimension K562/CMC system is K562 membrane flexibility post, and the second dimension chromatographic column is C18Chromatographic column, described second dimension Coupled columns has flight time mass spectrum；
Step 3, described Indigo Naturalis extracting solution in step one is squeezed in described full two dimension K562/CMC system, described first dimension chromatographic column is adopted to carry out first time separation, adopt described second dimension chromatographic column that the described first outflow component tieed up in chromatographic column is easily separated again, adopt described flight time mass spectrum that the outflow component of described Two way chromatograms is analyzed, obtain flowing out the structural information of component
By constantly switching ten direction changeover valves in described full two dimension K562/CMC system, obtain multiple potential leukemia composition；
Step 4, activity checking, it is verified by investigating the described leukemia composition propagation of K562 leukaemia on being in logarithmic (log) phase and the impact of apoptosis, obtains different anti-leukocythemia liveness compositions.
2. the method for anti-leukocythemia liveness composition in comprehensive screening Indigo Naturalis according to claim 1, it is characterised in that:
Wherein, in step 1, the step preparing described Indigo Naturalis extracting solution comprises the following steps:
Step 1-1, mixes dry Indigo Naturalis powder with the ratio that mass ratio is 1:10 with the chloroformic solution containing the chloral hydrate that volume fraction is 2%, in a heated condition, adopts ultrasonic vibration, obtains the extracting solution containing active component；
Step 1-2, by the described extracting solution micro-pore-film filtration through 0.22 μm, obtains described Indigo Naturalis extracting solution.
3. the method for anti-leukocythemia liveness composition in comprehensive screening Indigo Naturalis according to claim 1, it is characterised in that:
Wherein, in described step 2, building described full two dimension K562/CMC system and include the preparation of K562 membrane flexibility post, the preparation method of described K562 membrane flexibility post comprises the following steps:
Step 2-1, takes 3.7 × 107Individual K562 cell prepares K562 membrane pellet, and is resuspended in 5mL normal saline by described K562 membrane pellet, forms suspension；
Step 2-2, reacts described suspension and a certain amount of silica gel to 5min when 4 DEG C and vacuum oscillation, forms the fixing phase of cell membrane；
Step 2-3, adopts liquid phase pump and loads fixing for described cell membrane mutually in chromatographic column for mobile phase with the phosphate buffer of 10mmol/L, and dress column flow rate gradient optimizing is as follows:
0-5min, flow velocity is from 0.2mL min-1Rise to 1.0mL min-1；
5-5.5min, flow velocity is maintained at 1.0mL min-1, 30s, subsequently at 0.2mL min-1Flow velocity under balance chromatographic column.
4. the method for anti-leukocythemia liveness composition in comprehensive screening Indigo Naturalis according to claim 1, it is characterised in that:
Wherein, in described Indigo Naturalis, anti-leukocythemia liveness composition includes isorhamnetin and couroupitine A.
5. isorhamnetin or couroupitine A purposes in preparation prevention or treatment leukemia medicament.
6. determining an anti-leukocythemia liveness composition target calibration method, the method screening according to any one of claims 1 to 3 of this anti-leukocythemia liveness composition obtains, it is characterised in that comprise the following steps:
Step 1, adopts software PharmMapperServer and TargetHunter that described active component is reversely docked, respectively obtains multiple docking target；
Step 2, application Venny analyzes software and the docking target of above two software carries out total target analysis, then according to matching degree score, normalization fit score and z ' mark are ranked up, obtains the target that probability is maximum；
Step 3, the maximum target of the described probability that described step 2 obtained carries out forward docking experiment and is verified, it is determined that target.
7. according to claim 6 determine anti-leukocythemia liveness composition target calibration method, it is characterised in that:
Wherein, in described step 3, it is also possible to carry out the checking of active component target in conjunction with one or more experiments in membrane flexibility competitive assay, kinase activity test experience, surface plasma resonance experiment.
8. the purposes in determining isorhamnetin target of the determination anti-leukocythemia liveness composition target calibration method described in any one of claim 6～7.
9. determining an isorhamnetin target calibration method, the method screening according to any one of claims 1 to 3 of this isorhamnetin obtains, it is characterised in that comprise the following steps:
Step 1, adopts software PharmMapperServer and TargetHunter that described active component is reversely docked, respectively obtains multiple docking target；
Step 2, application Venny analyzes software and the docking target of above two software carries out total target analysis, obtain eight total targets, then according to matching degree score, normalization fit score and z ' mark are ranked up, it is determined that the maximum target of probability is nonreceptor tyrosine kinase；
Step 3, the described nonreceptor tyrosine kinase described step 2 obtained carries out forward docking experiment and is verified, it is determined that target.
10. according to claim 9 determine isorhamnetin target calibration method, it is characterised in that also include:
The step that the effect molecular mechanism of isorhamnetin is explored, described isorhamnetin blocks K562 cell in the G2/M phase by Src/ATR/Wee1/CDK1/CyclinB signal path.
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| CN115337916A (en) * | 2022-07-29 | 2022-11-15 | 福建卫生职业技术学院 | Hydrogel type cell membrane semi-preparative chromatographic column and preparation method and application thereof |
| CN115337916B (en) * | 2022-07-29 | 2023-10-24 | 福建卫生职业技术学院 | Hydrogel type cell membrane semi-preparation chromatographic column and preparation method and application thereof |
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