CN105755097B - Anti-leukocythemia liveness ingredient and its determining target calibration method in screening indigo naturalis comprehensively - Google Patents
Anti-leukocythemia liveness ingredient and its determining target calibration method in screening indigo naturalis comprehensively Download PDFInfo
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Abstract
The present invention provides one kind and screens anti-leukocythemia liveness ingredient in indigo naturalis comprehensively and determine its target calibration method, in anti-leukocythemia liveness ingredient, construct full two dimension K562/CMC system, two-dimensional gas chromatography and flight time mass spectrum are combined, and K562 membrane flexibility column is constructed as the first dimension chromatographic column, be conducive to the separating effect for being optimal components various in indigo naturalis；During determining its target, the reversed target for testing determining active constituent is carried out to active constituent using PharmMapper Server and TargetHunter software, shared target analysis is carried out to the docking target of both the above software using Venny analysis software, experiment combination cell membrane chromatography competitive assay, kinase activity test experience or surface plasma resonance experiment are docked using forward direction and carry out target checking, can fast, accurately determine active constituent to the target of leukaemia cell.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to anti-leukocythemia liveness ingredient and really in a kind of indigo naturalis of screening comprehensively
Its fixed target calibration method.
Background technique
Indigo naturalis is the leaf or the processed system of cauline leaf of acanthaceous vegetable acanthaceous indigo, polygonaceae plant indigo plant or cross China Tech plant woaded blue
The dried powder or agglomerate obtained.Research in recent years shows：Contain the plurality of active ingredients such as indigo, indigo red, couroupitine A in indigo naturalis,
In, indigo red has anti-leukocythemia ingredient.However, research confirms, indigo red cannot substitute anti-leukocythemia effect of indigo naturalis completely,
Speculate accordingly, there are also other anti-leukocythemia ingredients in indigo naturalis, but the specific type of these ingredients and these ingredients are in leukaemia
Not yet someone carried out correlative study to action target on cell at present.
Summary of the invention
The present invention is to carry out in order to solve the above problem.The first purpose of this invention is to provide a kind of screening comprehensively
The method of anti-leukocythemia liveness ingredient in indigo naturalis；Second object of the present invention is to provide a kind of these anti-leukocythemias of determination work
Property ingredient target calibration method, third object of the present invention using determine anti-leukocythemia liveness ingredient target calibration method carry out it is different
The screening and verifying of rhamnetin target.
In order to realize first purpose, present invention employs following technical solutions：
A kind of comprehensive method for screening anti-leukocythemia liveness ingredient in indigo naturalis, it is characterised in that include the following steps：Step
1, prepare indigo naturalis extracting solution；Step 2, full two dimension K562/CMC system, the first dimension in the full two dimension K562/CMC system are constructed
Chromatographic column is K562 membrane flexibility column, and the second dimension chromatographic column is C18 chromatographic column, and the second dimension Coupled columns have flight time matter
Spectrum；Step 3, the indigo naturalis extracting solution in step 1 is squeezed into full two dimension K562/CMC system, is carried out using the first dimension chromatographic column
It separates, the outflow component in the first dimension chromatographic column is separated again using the second dimension chromatographic column, when using flight for the first time
Between mass spectrum the outflow component of Two way chromatograms is analyzed, obtain outflow component structural information.By constantly switching complete two
Ten direction changeover valves in K562/CMC system are tieed up, a variety of potential anti-leukocythemia ingredients are obtained；Step 4, activity verifying, by examining
It examines anti-leukocythemia ingredient to verify the proliferation of K562 leukaemia cell and the influence of apoptosis that are in logarithmic phase, obtains difference
Anti-leukocythemia liveness ingredient.
Wherein, in step 1, prepare indigo naturalis extracting solution the step of include the following steps：Step 1-1, by dry indigo naturalis powder
Last and chloroformic solution (chloraldurate containing 2%) is with mass ratio for 1:10 ratio mixing, in a heated condition, using ultrasound into
Row oscillation, obtains the extracting solution containing active constituent；Step 1-2 is obtained described by extracting solution through 0.22 μm of micro-pore-film filtration
Indigo naturalis extracting solution.
In step 2, the preparation that full two dimension K562/CMC system includes K562 membrane flexibility column is constructed, method is as follows：Step
Rapid 2-1, takes 3.7 × 107A K562 cell prepares K562 membrane pellet, and K562 membrane pellet is resuspended in 5mL physiology
In salt water, suspension is formed；Step 2-2 reacts suspension with a certain amount of silica gel under the conditions of 4 DEG C and vacuum oscillation
5min forms cell membrane stationary phase；Step 2-3 uses liquid phase pump and incites somebody to action by mobile phase of the phosphate buffer of 10mmol/L
The cell membrane stationary phase is fitted into chromatographic column, and dress column flow rate gradient optimizing is as follows：0-5min, flow velocity is from 0.2mLmin-1 liter
To 1.0mLmin-1；5-5.5min, flow velocity are maintained at 1.0mLmin-1,30s, then under the flow velocity of 0.2mLmin-1
Balance chromatographic column.
Include Isorhamnetin and couroupitine A by anti-leukocythemia liveness ingredient in indigo naturalis obtained by the above method, can be used
In preparation prevention or treatment leukemia medicament.
In order to realize second object of the present invention, a kind of determining anti-leukocythemia liveness ingredient target provided by the invention
Method includes the following steps：Step 1, using software Pharm Mapper Server and Target Hunter to active constituent
It is reversely docked, respectively obtains multiple docking targets；Step 2, using Venny analysis software to pair of above two software
It connects target and carries out shared target analysis, be then ranked up according to matching degree score, normalization fit score and z ' score,
Obtain the maximum target of possibility；Step 3, a possibility that step 2 is obtained maximum target carry out positive docking test into
Row verifying, determines target.
Meanwhile combination cell membrane chromatography competitive assay, kinase activity test experience, table on the basis of forward direction docking experiment
Target is verified in plasma resonance experiment in face.
In order to realize third object of the present invention, the present invention provides a kind of determining Isorhamnetin target calibration method, packets
Include following steps：Step 1, active constituent is carried out using software Pharm Mapper Server and Target Hunter reversed
Docking, respectively obtains multiple docking targets；Step 2, it is carried out using docking target of the Venny analysis software to above two software
Shared target analysis, obtains eight shared targets, then according to matching degree score, normalization fit score and z ' score into
Row sequence determines that the maximum target of possibility is nonreceptor tyrosine kinase；Step 3, non-receptor junket ammonia step 2 obtained
Acid kinase carries out positive docking and is verified, and determines target.
Further include the steps that the effect molecular mechanism to Isorhamnetin is explored in the above method, is examined using the cell cycle
It surveys and the method for Western-Blotting is explored, Isorhamnetin is believed by Src/ATR/Wee1/CDK1/Cyclin B
Number access retardance K562 cell is in the G2/M phase.
Invention action and effect
The method of anti-leukocythemia liveness ingredient in the comprehensive screening indigo naturalis provided according to the present invention, due to constructing full two dimension
Two-dimensional gas chromatography and flight time mass spectrum are combined, and construct K562 membrane flexibility column as first by K562/CMC system
Chromatographic column is tieed up, the separating effect for being optimal components various in indigo naturalis is conducive to.
In addition, these anti-leukocythemia liveness ingredient target calibration methods of the determination provided according to the present invention, due to using
PharmMapper Server and TargetHunter the software target for testing determining active constituent reversed to active constituent progress,
Shared target analysis is carried out to the docking target of both the above software using Venny analysis software, experiment knot is docked using forward direction
It closes membrane flexibility competitive assay, kinase activity test experience or surface plasma resonance experiment and carries out target checking, it can be fast
Target of fast, the accurate determining active constituent to leukaemia cell.
Detailed description of the invention
Fig. 1 is full two dimension K562/CMC binding mode figure of the invention.Wherein, when (a) is that two ten-way valves are in position 1
The binding mode figure of full two dimension K562/CMC system；It (b) is the effect of two dimension K562/CMC full when two ten-way valves are in position 2
Ideograph.
Fig. 2 is the 3D figure that Plays product mixed solution of the present invention is obtained 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 is obtained in the full two-dimentional system analysis of K562/CMC；Fig. 3 (b)
It is the structural formula of the potential anti-leukocythemia liveness compound through time-of-flight mass spectrometry (TOFMS) identification.
Fig. 4 (a) is result of the Isorhamnetin to the inhibited proliferation of K562 cell of the CCK-8 measurement in the present invention
Figure；Fig. 4 (b) and Fig. 4 (c) is that the Isorhamnetin of V-FITC of the Annexin detection in the present invention promotes to make to K562 Apoptosis
Result figure.
Fig. 5 is showing for the shared target spot analyzed in the present invention through PharmMapper Server and TargetHunter
It is intended to.
Fig. 6 is that the action target spot of verifying Isorhamnetin in the present invention is the result schematic diagram of Src kinases receptors.Wherein, scheme
In number 1~5 respectively indicate the Isorhamnetin of various concentration, (a) is Isorhamnetin and Src kinases receptors joint mode figure；
(b) be membrane flexibility competitive assay verification result；It (c) is kinase activity testing result；It (d) is surface plasma resonance reality
The result figure tested；(e) be various concentration in (d) Isorhamnetin removal blank after sensing figure.
Fig. 7 is the result figure that Isorhamnetin passes through the Src receptors modulate cell period.Wherein, (a) and (b) is Isorhamnetin
The dose-dependent result that K562 leukemic cells are arrested in the G2/M phase；(c) be Isorhamnetin directly act on Src by
Body adjusts the result figure of cell cycle by ATR/Wee1/CDK1/Cyclin B
Specific embodiment
Illustrate a specific embodiment of the invention below in conjunction with attached drawing.
One, potential antileukemie active constituent in indigo naturalis is screened
1, indigo naturalis extracting solution is prepared, is included the following steps：
Step 1, by dry indigo naturalis powder and chloroformic solution (chloraldurate containing 2%) with mass ratio for 1:10 ratio
Mixing is vibrated using ultrasound under conditions of heating temperature is no more than 70 DEG C, obtains the extracting solution containing active constituent；
Step 2, extracting solution is obtained into indigo naturalis extracting solution through 0.22 μm of micro-pore-film filtration.
2, full two dimension K562/CMC system is constructed
Full two dimension K562/CMC system uses 1200 series of high efficiency liquid chromatograph of Agilent (Agilent, the U.S.).Such as
Shown in Fig. 1, full two dimension K562/CMC system includes the first high pressure liquid phase delivery pump 1, autosampler 2, as the first dimension chromatography
3, two ten direction changeover valves, 4 (models of K562 membrane flexibility column of column：Rheodyne MXP9960；Supplier：IDEX
Corporation, the U.S.), the first pre-column 5, the second high pressure liquid phase delivery pump 6, third high pressure liquid phase delivery pump 7, the second pre-column
8, the C as the second dimension chromatographic column18Reverse-phase chromatographic column 9 and flight time mass spectrum 10.
As shown in Fig. 1 (a), the first high pressure liquid phase delivery pump 1 is modular pump, with autosampler 2 and as the first dimension
The K562 membrane flexibility column 3 of chromatographic column is connected to, and K562 membrane flexibility column 3 is connected to the 1. position in two ten direction changeover valves 4,
2. position in two ten direction changeover valves 4 is connected to the input end of the first pre-column 5, and 5. position is connected to the outlet end of the first pre-column 5,5. position and
6. position is connected to, 6. position is for emptying the first pre-column 5.
Second high pressure liquid phase delivery pump 6 and third high pressure liquid phase delivery pump 7 collectively constitute a binary pump and two ten logical
9. position connection in switching valve 4,9. position is connected to 10. position, and 10. position is connected to the input end of the second pre-column, and outlet end is connected to 10. position,
10. position is connected to by 10. position with 3. position, 3. position is by 4. position and as the C of the second dimension chromatographic column18Reverse-phase chromatographic column 9 is connected to, C18
A part of reverse-phase chromatographic column 9, which enters in flight time mass spectrum 10, to be analyzed.
The component overwhelming majority constructed in full two dimension K562/CMC system belongs to instrument from tape member, only for separation
Chromatographic column needs are replaced according to the difference of actual separation ingredient.In the present invention, the main of full two dimension K562/CMC system is constructed
Work is prepared by K562 membrane flexibility column, and preparation method includes the following steps：
Step 1,3.5 × 10 are taken7A K562 cell is turned using 10mmol/L phosphate buffer (PBS) in 1000 × g
Three times, each 5min takes precipitating to be suspended with 10mmol/L PBS, using JY92-IIN Ultrasonic Cell Disruptor to thin to the lower eccentric cleaning of speed
Born of the same parents' suspension is crushed, ultrasonic power 400W, is carried out 5 times altogether, total time 90s.
Step 2, broken homogenate is centrifuged 10min at 1000 × g, takes supernatant, then be centrifuged 20min at 12000 × g
Obtain membrane pellet.It takes precipitating to be resuspended in 5mL physiological saline, suspension and 0.04g silica gel is then shaken into condition in vacuum
Lower reaction 5min forms cell membrane stationary phase, after stirring 30min, stands overnight under the conditions of 4 DEG C.
Step 3, column is filled, is carried out under the conditions of 4 DEG C, uses liquid phase pump and with the phosphate buffer of 10mmol/L for flowing
Mutually cell membrane stationary phase is fitted into chromatographic column, dress column flow rate gradient optimizing is as follows：0-5min, flow velocity is from 0.2mLmin-1It rises
To 1.0mLmin-1；5-5.5min, flow velocity are maintained at 1.0mLmin-1, 30s, then in 0.2mLmin-1Flow velocity under put down
Weigh chromatographic column.
The membrane flexibility column prepared should be stored under the conditions of 4 DEG C.
3, the investigation of full two dimension K562/CMC system stability
Certain density Imatinib and dexamethasone mixed solution are configured as titer, then passes through the titer
Full two dimension K562/CMC system is sieved, and investigates the stability of system, as a result as shown in Figure 2.As shown in Figure 2, two kinds of drugs
Can be completely separable, illustrate that the stability of system is preferable.
4, in indigo naturalis potential anti-leukocythemia liveness ingredient screening
Step 1, as shown in Fig. 1 (a), after indigo naturalis extracting solution passes through 2 sample introduction of autosampler, in K562 membrane flexibility column
Separated in 3, via the first high pressure liquid phase delivery pump 1 conveying phosphate buffer elution after, first group distribute and
It is enriched in first pre-column 5, meanwhile, the second high pressure liquid phase delivery pump 6 and third high pressure liquid phase delivery pump 7 are for balancing C18Reverse phase color
Compose column 9 and flight time mass spectrum 10；
Step 2, after 2.5min, switch two ten direction changeover valves 4, as shown in Fig. 1 (b), at this point, two ten direction changeover valves 4
In 2. position be connected to 3. position.Binary pump passes through 9. position, 8. position, 3. position, 2. position, 5. position and 4. position will be in the first pre-column 5
First component is directed into C18It is further separated in reverse-phase chromatographic column 9, and data is carried out by flight time mass spectrum 10
Analysis.At this point, the second component flowed out from K562 membrane flexibility column 3 is enriched in the second pre-column 8 by 1. position and 10. position
In；
Step 3, switch two ten direction changeover valves 4 again, so that connection relationship shown in Fig. 1 (a) is presented in system, into
The analysis of the second component of row and the enrichment of third component.So switching 12 times, so that whole components in the first dimension chromatographic column
It is able to enter C18Further separation analysis is carried out in mass spectrum.
In the present invention, software (Agilent MassHunter Workstation) is carried to mass spectrum using mass spectrometer
The data of acquisition are analyzed, and initial data is exported, then are imported into MATLAB7.10.0, and baseline calibration and peak pair are carried out
Together, 3D map is then drawn, result is compared, active constituent is screened, result is selected to see Fig. 3.Such as Fig. 3 (a) and Fig. 3
(b) shown in, the screening of anti-leukocythemia ingredient in indigo naturalis extracting solution is carried out using the system, has successfully filtered out indigo red, tryptamines
Three kinds of potential active constituents of ketone and Isorhamnetin.
Two, the activity verifying of potential antileukemie active constituent
Cell Proliferation and apoptosis activity detection are carried out to potential activity ingredient.
1, the influence of cell proliferation：
The K562 leukaemia cell of logarithmic growth phase, 1000g centrifugation are resuspended after counting and are laid in 96 orifice plates, and density is
5000 cells/wells are separately added into the Isorhamnetin (0,12.5,25,50,100,200 μM) of different final concentrations after overnight incubation,
After 48h, 10 μ L CCK-8 reagents are added in every hole, and 37 DEG C of incubators are incubated for 2h, using Synergy4 multi-wavelength microplate reader
(BioTek, the U.S.) measures the absorbance value in every hole at 450nm.
2, to the influence of Apoptosis：
Firstly, the K562 leukaemia cell of logarithmic growth phase, 1000g centrifugation are resuspended after counting and are laid in 6 orifice plates, it is close
Degree is 10000 cells/wells, and the Isorhamnetin (0,12.5,25,50 μM) of different final concentrations, 48h are separately added into after overnight incubation
Afterwards, cell is collected by centrifugation in 1000g, then washed once with 1 × PBS；Then, the cell for taking 5-10 ten thousand to be resuspended, 1000g are centrifuged 5 points
Clock abandons supernatant, 195 μ L Annexin V-FITC combination liquid is added, cell is gently resuspended；Then, 5 μ L Annexin V- are added
FITC is mixed gently；Finally, 10 μ L propidium iodide stain liquid are added, mix gently, room temperature (20-25 DEG C), which is protected from light, is incubated for 10-20
Minute, it is subsequently placed in ice bath.
Two above experiment is repeated 6 times respectively, and data carry out statistical disposition, data description using Graphpad Prism5
It is indicated with average value ± standard deviation, comparison among groups use administration group compared with blank group, using t check analysis, P<0.05 table
Difference between showing two groups is statistically significant.
Interpretation of result
Fig. 4 is Isorhamnetin to the cell Proliferation of K562 leukaemia cell and the influence result schematic diagram of Apoptosis.
Fig. 4 (a) is result figure of the Isorhamnetin to the inhibited proliferation of K562 cell of CCK-8 measurement.It follows that
With the increase of Isorhamnetin concentration, Isorhamnetin increases the inhibiting rate of K562 cell Proliferation, when the concentration of Isorhamnetin is
When 200 μm of ol/L, to the inhibiting rate of K562 cell Proliferation close to 80%.
Fig. 4 (b) is the Isorhamnetin using V-FITC of Annexin dyeing detection to K562 Apoptosis facilitation
Result figure.It follows that Isorhamnetin increases the promotion rate of K562 Apoptosis with the increase of Isorhamnetin concentration, when
When the concentration of Isorhamnetin is 50 μm of ol/L, 80% is higher than to the inhibiting rate of K562 Apoptosis.
Fig. 4 (c) is to promote to make to K562 Apoptosis using the Isorhamnetin of the double dyeing detections of V-FITC/PI of Annexin
Result figure.It follows that comparing with control group, Isorhamnetin has facilitation to the apoptosis of K562 cell, with different
The increase of rhamnetin concentration, the K562 cell being colored is more and more, illustrates Isorhamnetin to the promotion rate of K562 Apoptosis
Increase with the increase of Isorhamnetin concentration.
Inventor is by same it is experimentally confirmed that couroupitine A is similarly able to suppress the proliferation of K562 cell, promotion K562
The apoptosis of cell has good anti-leukocythemia liveness.
Three, the screening and verifying of anti-leukocythemia liveness ingredient target
Screening below by taking the screening of Isorhamnetin target and verifying as an example, to anti-leukocythemia liveness ingredient target in indigo naturalis
It is illustrated with verification process.The method and Isorhamnetin phase of screening and the verifying of other anti-leukocythemia liveness ingredient targets
Together.
1, target sieving
As shown in figure 5, the process of target sieving includes the following steps：
Step 1, using open for free software Pharm Mapper Server and Target Hunter to active constituent into
The reversed docking of row, respectively obtains 198 and 208 docking targets；
Step 2 carries out shared target analysis to the docking target of above two software using Venny analysis software, obtains
Then eight shared targets are arranged eight shared targets according to matching degree score, normalization fit score and z ' score
Sequence, the results are shown in Table 1, obtains the maximum target of possibility.
The score of 1 eight shared targets of table sorts
According to the result of table 1, nonreceptor tyrosine kinase is found further according to target spot and the correlation of cell membrane and leukaemia
It (Src) is the maximum action target spot of Isorhamnetin possibility.
2, target checking
Isorhamnetin and Src are docked using forward direction docking software LeDock, as shown in Fig. 6 (a), and combination cell
Membrane chromatography competitive assay, kinase activity test experience, surface plasma resonance experiment are verified, and are carried out to Src as target
Verifying.
(1), it is verified using membrane flexibility competitive assay
Using Dasatinib (Src inhibitor) solution as titer, the different sandlwood that various concentration is added in mobile phase is investigated
Whether plain front and back, the retention time of Dasatinib are changed.Specific method is：It is first flat with the ammonium acetate solution of 10mmol/L
Weighing, K562 leukaemia cell's film column is steady to baseline, and sample introduction Dasatinib records corresponding retention time.Then it measures
The Isorhamnetin active Chinese drug component monomer that concentration is 0,0.5,1,2 and 5 μm of ol/L is separately added into the mobile phase of 10mmol/L PBS
The variation tendency of Dasatinib retention time afterwards.
(2), it is verified using kinase activity detection：
Change method (Mobility shift assay) using mobility to be detected, include the following steps：
Step 1, prepare 1 × kinase buffer liquid (50mmol/L HEPES, pH7.5；
0.0015%Brij-35；10mmol/L MgCl2；2mmol/L DTT) and terminate liquid (100mmol/L HEPES,
pH 7.5；0.015%Brij-35；0.2%Coating Reagent#3；50mmol/L EDTA)；
Step 2, compound prepares：Firstly, with the Isorhamnetin mother liquor of DMSO configuration 10mmol/L, then in 96 orifice plates
In, the Isorhamnetin of 10 various concentrations is configured, and take 10 μ L to be added in a 96 new orifice plates respectively in every hole；Then,
90 μ L1 × kinase buffer liquid is added in every hole, and after concussion mixes 10min, every hole takes 5 μ L to be added in 384 new orifice plates, and pair is arranged
Hole (n=3)；
Step 3, kinase reaction：Firstly, preparing 2.5 × enzyme solutions and 2.5 × peptide solution, 1 × phosphorus will be added in enzyme
2.5 × enzyme solutions are obtained in phthalate buffer, and the peptide and ATP of FAM label are added in 1 × kinase buffer liquid, prepared
Obtain 2.5 × peptide solution；Secondly, 2.5 × enzyme solutions are added to the Isorhamnetin solution for containing 10%DMSO containing 5 μ L
384 orifice plate detection holes in, be incubated at room temperature 10min；Then, 10 μ L2.5 × peptide solution is added into detection hole, 28 DEG C incubate
It educates specified time, carries out kinase reaction；Finally, 25 μ L stop buffers, which are added, terminates reaction.
Step 4, Caliper reading is carried out, and carries out data processing.
(3) it is verified using surface plasma resonance experiment：
Using CM5 chip, 1 is then used:1 binding model, by various concentration (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 to increase with the concentration of Isorhamnetin in mobile phase, and (Src inhibits Dasatinib
Agent) retention time (Fig. 6 b) is gradually shortened；Vitro kinase activity the experimental results showed that Isorhamnetin to Src kinase activity
IC50 is 6.9 μm of ol/L (Fig. 6 c)；The K of the surface plasma resonance experiment binding force between Src and Isorhamnetin as the result is shownd
Value is 3.81 × 10-6Mol (Fig. 6 d and Fig. 6 e).Above data result proves that Src is an action target spot of Isorhamnetin.
Four, the effect molecular mechanisms of Isorhamnetin
1, the cell cycle is detected：
The K562 leukaemia cell of logarithmic growth phase, 1000g centrifugation are resuspended after counting and are laid in 6 orifice plates, and density is
10000 cells/wells are separately added into the Isorhamnetin (0,12.5,25,50 μm of ol/L) of different final concentrations after overnight incubation, for 24 hours
Afterwards, cell is collected by centrifugation in 1000g, then washed once with 1 × PBS.
Cell is fixed：1 milliliter of ice bath is added to be pre-chilled in 70% ethyl alcohol, gently piping and druming mixes, and 4 DEG C are fixed 24 hours, then
1000g is centrifuged 5 minutes, sedimentation cell.It is careful to absorb supernatant, the PBS of about 1 milliliter of ice bath pre-cooling is added, cell is resuspended.Again from
Heart sedimentation cell, carefully absorbs supernatant.Gently attack centrifuge tube bottom avoids cell agglomerating with appropriate cell dispersion.Every solencyte
0.3 milliliter of propidium iodide stain liquid is added in sample, cell precipitation slowly and is sufficiently resuspended, after 37 DEG C are protected from light warm bath 30 minutes,
Upper machine is detected.
2, Western-Blotting experiment is carried out according to the step in following documents：
Chen,X.et al.Comparative normal/failing rat myocardium cell membrane
chromatographic analysis system for screening specific components that
counteract doxorubicin-induced heart failure from Acontium
carmichaeli.Analytical chemistry 86,4748-4757,doi:10.1021/ac500287e(2014).?
It can be carried 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, the result shows that, Isorhamnetin can pass through
Src/ATR/Wee1/CDK1/Cyclin B signal access blocks K562 cell in the G2/M phase, and then induces cell apoptosis, and plays
Anti-leukocythemia effect.
Embodiment action and effect
The present embodiment discloses a kind of new anti-leukocythemia liveness ingredient in indigo naturalis：Isorhamnetin.And it is anti-to Isorhamnetin
The novel targets of leukaemia effect：Nonreceptor tyrosine kinase (Src) is screened and has been verified.The result of screening and confirmatory experiment
The Isorhamnetin for further confirming that the present embodiment is screened can effectively inhibit the increment of K562 cell and promote apoptosis.This compound
Src can be directly acted on, the phosphorylation of Src is significantly inhibited, adjusts the cell cycle for cell block in the G2/M phase.Surface etc. from
Sub-resonance data show that the binding force of Isorhamnetin and Src are 3.81 μm of ol/L, and kinase activity detection discovery Isorhamnetin is to Src
The IC50 of kinases is 6.9 μm of ol/L, is a good lead compound.
Claims (9)
1. a kind of method of anti-leukocythemia liveness ingredient in indigo naturalis of screening comprehensively, it is characterised in that include the following steps：
Step 1, indigo naturalis extracting solution is prepared；
Step 2, full two dimension K562/CMC system is constructed, the first dimension chromatographic column in the full two dimension K562/CMC system is K562
Membrane flexibility column, the second dimension chromatographic column is C18Chromatographic column, the second dimension Coupled columns have flight time mass spectrum；
Step 3, the indigo naturalis extracting solution in step 1 is squeezed into the full two dimension K562/CMC system, using described first
Tie up chromatographic column carry out first time separation, using it is described second dimension chromatographic column to it is described first dimension chromatographic column in outflow component again
It is separated, is analyzed using outflow component of the flight time mass spectrum to the Two way chromatograms, obtain outflow component
Structural information,
By constantly switching ten direction changeover valves in the full two dimension K562/CMC system, obtain a variety of potential anti-leukocythemias at
Point；
Step 4, activity verifying, by investigating the anti-leukocythemia ingredient to the proliferation of the K562 leukaemia cell in logarithmic phase
Influence with apoptosis is verified, and different anti-leukocythemia liveness ingredients is obtained.
2. the method for anti-leukocythemia liveness ingredient in comprehensive screening indigo naturalis according to claim 1, it is characterised in that：
Wherein, in step 1, the step of preparing the indigo naturalis extracting solution includes the following steps：
Step 1-1, the chloroformic solution by dry indigo naturalis powder and the chloraldurate for being 2% containing volume fraction are with mass ratio
1:10 ratio mixing, in a heated condition, is vibrated using ultrasound, obtains the extracting solution containing active constituent；
Step 1-2 obtains the indigo naturalis extracting solution by the extracting solution through 0.22 μm of micro-pore-film filtration.
3. the method for anti-leukocythemia liveness ingredient in comprehensive screening indigo naturalis according to claim 1, it is characterised in that：
Wherein, in the step 2, the preparation that the full two dimension K562/CMC system includes K562 membrane flexibility column is constructed,
The preparation method of the K562 membrane flexibility column includes the following steps：
Step 2-1, takes 3.7 × 107A K562 cell prepares K562 membrane pellet, and the K562 membrane pellet is resuspended
In 5mL physiological saline, suspension is formed；
The suspension is reacted under the conditions of 4 DEG C and vacuum oscillation 5min with a certain amount of silica gel, is formed thin by step 2-2
After birth stationary phase；
Step 2-3 is used liquid phase pump and is filled the cell membrane stationary phase using the phosphate buffer of 10mmol/L as mobile phase
Enter in chromatographic column, dress column flow rate gradient optimizing is as follows：
0-5min, flow velocity is from 0.2mLmin-1Rise to 1.0mLmin-1；
5-5.5min, flow velocity are maintained at 1.0mLmin-1, 30s, then in 0.2mLmin-1Flow velocity under balance chromatographic column.
4. the method for anti-leukocythemia liveness ingredient in comprehensive screening indigo naturalis according to claim 1, it is characterised in that：
Wherein, anti-leukocythemia liveness ingredient includes Isorhamnetin and couroupitine A in the indigo naturalis.
5. a kind of determining anti-leukocythemia liveness ingredient target calibration method, the anti-leukocythemia liveness ingredient according to claim 1~3 in
Described in any item methods screen to obtain, which is characterized in that include the following steps：
Step 1, it is reversed right to be carried out using software Pharm Mapper Server and Target Hunter to the active constituent
It connects, respectively obtains multiple docking targets；
Step 2, shared target analysis is carried out to the docking target of above two software using Venny analysis software, then according to
Matching degree score, normalization fit score and z ' score are ranked up, and obtain the maximum target of possibility；
Step 3, the maximum target of the possibility that the step 2 obtains is carried out positive docking to verify, is determined
Target.
6. determining anti-leukocythemia liveness ingredient target calibration method according to claim 5, it is characterised in that：
Wherein, in the step 3, can be combined with membrane flexibility competitive assay, kinase activity test experience, surface etc. from
One or more of sub-resonance experiment experiment carries out the verifying of active constituent target.
7. the described in any item determining anti-leukocythemia liveness ingredient target calibration methods of claim 5~6 are determining Isorhamnetin target
Purposes in mark.
8. a kind of determining Isorhamnetin target calibration method, Isorhamnetin method described in any one of claim 1 to 3
Screening obtains, which is characterized in that includes the following steps：
Step 1, it is reversed right to be carried out using software Pharm Mapper Server and Target Hunter to the active constituent
It connects, respectively obtains multiple docking targets；
Step 2, shared target analysis is carried out to the docking target of above two software using Venny analysis software, obtains eight
Then shared target is ranked up according to matching degree score, normalization fit score and z ' score, determine possibility maximum
Target be nonreceptor tyrosine kinase；
Step 3, the nonreceptor tyrosine kinase that the step 2 obtains is carried out positive docking to verify, is determined
Target.
9. determining Isorhamnetin target calibration method according to claim 8, which is characterized in that further include：
The step of exploring to the effect molecular mechanism of Isorhamnetin, the Isorhamnetin passes through Src/ATR/Wee1/CDK1/
CyclinB signal path blocks K562 cell in the G2/M phase.
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| Characterization of anti-leukemia components from Indigo naturalis using comprehensive twodimensional K562/cell membrane chromatography and in silico target identification;Xunxun Wu等;《Scientific Reports》;20160506;1-13 * |
| 异鼠李素体内外抗肿瘤作用及其机制的研究;朱玲;《中国博士学位论文全文数据库（医药卫生辑）》;20060615;全文 * |
| 色胺酮对人白血病细胞株K562细胞增殖抑制、凋亡诱导的影响;缪珊等;《中国药理学通报》;20090228;第25卷(第2期);256-259 * |
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