CN105717250B - Method for fast screening topoisomerase I inhibitor from natural product - Google Patents
Method for fast screening topoisomerase I inhibitor from natural product Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/884—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample organic compounds
Abstract
The invention discloses a method for fast screening a topoisomerase I inhibitor from a natural product, and relates to the analysis field of natural products. The method comprises the following steps of: (1) preparation of an enzyme binding reaction buffer solution; (2) preparation of a to be detected sample; (3) preparation of a standard solution; (4) binding reaction of the to-be-detected sample and normal and inactive topoisomerase I; (5) chromatography-mass spectrometry detection analysis of reaction sample; and (6) enrichment ratio of the topoisomerase I to the inhibitor. The ultrafiltration membrane technology and the chromatography-mass spectrometry united technology united method are applied to the screening of the topoisomerase I inhibitor, the sample analysis speed is fast and the specificity is strong so as to conveniently recognize a medicine ligand combined with a biological target molecule; meanwhile, the sample analysis dosage is less, and the spectrogram information quantity is large so as to realize the fast screening of the lead compound, the method has great advantage on the aspect of researching mutual effect of medicine small molecule ligand and biological macromolecule receptor; the method is suitable for analyzing in-vitro enrichment ratio of the natural product extract or monomer compound to the topoisomerase I inhibitor.
Description
Invention field
The present invention relates to topoisomerase I in Analysis of Natural Products field, more particularly to a kind of rapid screening natural product
The method of inhibitor；Specifically, the present invention is from natural product extraction using ultrafiltration membrane technique with chromatograph, mass spectrometric hyphenated technique
The method of rapid screening topoisomerase I inhibitor in thing or monomer compound.
Background technology
DNA topoisomerase Is, are a kind of indispensable enzymes being widely present in protokaryon and eukaryote body, its catalytic site
Tyrosinyl residues to attack DNA single-stranded so that enzyme and DNA 3'- phosphodiester bonds are formed and are covalently attached, and cause single-stranded disconnected of DNA
Split, so as to pass through to adjust superhelix, it is chain, go the unhitching effect of chain and nucleic acid, impact DNA topological structure.Targeted drug is
Be applied to the state-of-the-art medicine for the treatment of of cancer at present, it by with tumor occur, grow necessary specific target spot effect come
Prevent the growth of tumor cell.Research finds that topoisomerase shows the height not being affected by other factors in tumor cell
The topoisomerase I content and activity of horizontal expression, particularly colon cancer, cervical cancer and ovarian cancer etc. is thin far above regular
Born of the same parents, especially in S phase tumor cells, activity is greatly improved.Therefore, topoisomerase I inhibitor optionally Inhibit proliferaton phase
DNA of tumor cell is replicated, and is prevented tumor cell fast breeding, and then is killed tumor cell.As topoisomerase I inhibitor has
There are numerous superioritys, National Cancer Institute drug Mechanism analysis computer network system is by topoisomerase I inhibitor
One of six big series antineoplastic medicaments of primary study are classified as, the enzyme becomes the important novel targets of design new anticancer drug, right
The research of its inhibitor can provide new clinical medicine and technological means for the treatment of tumor.
The conventional screening method of topoisomerase I inhibitor generally uses spectrographic method, Surface Plasmon Resonance, X-ray and dissipates
(Chenxi Yao, Na Na, Lingyun Huang, Dacheng He, the Jin such as color method, calorimetry and nuclear magnetic resonance method
Ouyang.Analytica Chimica Acta, 2013,79,60-66；Vanisree Mulabagal, Angela
I.Calderon, Analytical Chemistry, 2010,82,3616-3621), said method is convenient and swift, can be certain
Screening requirements are met in degree.But spectrographic method is also easy to produce false negative and false positive results because there are ambient interferences；Nuclear-magnetism is altogether
Detection process of shaking can not provide detailed features of protein-ligand interaction in quick exchange reaction etc..Ultrafiltration mass spectrum is used in conjunction
Technology combines the characteristics such as the excellent separation function of ultrafiltration apparatus and mass-spectrometric technique high speed, high sensitivity, high accuracy, with dividing
Fast speed, high specificity and high-throughout feature are analysed, the Drug Ligand combined with bio-target molecule can be easily recognized, while
Sample analysis consumption is few, spectrogram information amount big, in terms of Medicine small molecule part and biomacromolecule acceptor interaction research
With very big advantage (Shun Xiao, Runru Yu, Ni Ai, Xiaohui Fan.Journal of Pharmaceutical
And Biomedical Analysis, 2015,104,67-74；Xingxin Yang, Dan Wang, Zhiwei Yang, et
Al.Journal of Chromatography A, 2015,1413,33-46).
The content of the invention
It is an object of the invention to overcome the shortcoming and defect that prior art is present, there is provided a kind of rapid screening natural product
The method of middle topoisomerase I inhibitor, can be used to analyze natural extracts or monomeric compound to topoisomerase I
Ex vivo enrichment rate.
The object of the present invention is achieved like this：
Natural extracts of the present invention or monomeric compound refer to day to topoisomerase I ex vivo enrichment rate
After so product extract or monomeric compound are hatched and combined with topoisomerase I, receptor-ligand complexes Jing is organic
Solvent process discharges smaller ligand, the reactive compound discharged with liquid phase-Mass Spectrometer Method, calculate each composition with
Concentration change and its ratio that topoisomerase I is combined.
Specifically, this method is comprised the following steps：
Described ultrafiltration mass spectrometric hyphenated technique refers to that ultrafiltration membrane technique is used in combination by one kind with chromatograph, mass spectrometric analysis method
A kind of new research meanses for being formed；
Described topoisomerase I inhibitor is natural extracts or monomer compound；Natural product of the present invention
Fructus rhamni (Rhamnus davurica Pall.) extract, maryllidaceous alkaloid extract, monomeric compound Rhizoma Paridis saponin I, II, VI and VII；
1. the preparation of enzyme association reaction buffer
Enzyme association reaction buffer includes：
Potassium acetate：50 mM/ls, trishydroxymethylaminomethane-acetic acid：20 mM/ls,
Magnesium acetate：10 mM/ls, dithiothreitol, DTT：1 mM/l；
4907 milligrams of potassium acetate, 2422.8 milligrams of trishydroxymethylaminomethane-acetic acid, 2144.6 milli of magnesium acetate are weighed accurately
Gram and 154.3 milligrams of dithiothreitol, DTT, using deionized water dissolving to final volume be 1 liter, after being sufficiently mixed, at 25 DEG C, pH value is
7.9；
2. the preparation of testing sample
Appropriate testing sample is accurately weighed, is fully dissolved with enzyme association reaction buffer, the final concentration of 1.0-3.0 of prepare liquid
Mg/ml；
Described testing sample is Fructus rhamni (Rhamnus davurica Pall.) extract or maryllidaceous alkaloid extract；
3. the preparation of standard solution
With acetonitrile by internal standard sample preparation into the standard mother solution that concentration is 100 mcg/mls, add before carrying out Mass Spectrometer Method
Into analyte sample fluid, final concentration of 2.0-5.0 mcg/mls；
Described internal standard sample is Isoschaftoside or nuciferine；
4. testing sample and association reaction that is normal and inactivating topoisomerase I
Experimental group：The analyte sample fluid and 10 microlitres of concentration that 100 microlitres are sequentially added in 0.2 milliliter of centrifuge tube be
0.5U/ microlitre of DNA topoisomerase Is, are incubated 30 minutes at 37 DEG C；Reaction mixture is transferred to into trapped molecular weight for 3
In the super filter tube of ten thousand dalton, it is centrifuged 10 minutes under 10000 revs/min；Add 200 microlitres of buffer to wash away to be not associated with into
Point, and be centrifuged 10 minutes under 10000 revs/min again；Repeat the above steps 2 times, merging filtrate；Add in super filter tube
200 microlitres of 90% acetonitrile, after placing 10 minutes, is centrifuged 10 minutes under 10000 revs/min, discharges part under room temperature；In repetition
State step 2 time, merging filtrate；Above-mentioned gained filtrate is dried up and adds 50 microlitres of 90% acetonitrile to dissolve, for chromatography-mass spectroscopy
Analysis；
Matched group：Sequentially add in 0.2 milliliter of centrifuge tube 100 microlitres analyte sample fluid and 10 microlitres in boiling water
The concentration for placing inactivation in 10 minutes is 0.5U/ microlitre of DNA topoisomerase Is, is incubated 30 minutes at 37 DEG C；By reaction mixture
It is transferred in the super filter tube that trapped molecular weight is 30,000 dalton, is centrifuged 10 minutes under 10000 revs/min；Add 200 micro-
Rise buffer and wash away uncombined composition, and be centrifuged 10 minutes under 10000 revs/min again；Repeat the above steps 2 times, merge
Filtrate；200 microlitres of 90% acetonitrile is added in super filter tube, after placing 10 minutes under room temperature, under 10000 revs/min, 10 is centrifuged
Minute, discharge part；Repeat the above steps 2 times, merging filtrate；Above-mentioned gained filtrate is dried up and 50 microlitres of 90% second is added
Nitrile dissolves, for Spectrometry；
5. the chromatography-mass spectroscopy detection and analysis of response sample
With inner mark method ration, to step 4. in two groups of sample solutions carry out efficient liquid phase and Mass Spectrometer Method；According to chromatogram
In each peak peak area and internal standard sample peak area ratio, calculate each composition relative concentration；
High performance liquid chromatography and Mass Spectrometer Method are carried out to extract testing sample, experimental group and control sample：
The analysis condition of A, high performance liquid chromatography
High performance liquid chromatograph：Thermo Accela 600；
Chromatographic column：Waters Symmetry C18, specification are 4.6 × 250 millimeters, 5 microns；
Mobile phase：A-0.1% formic acid-water；B- acetonitriles；
Gradient elution program：0-5 minutes, 20%B；5-30 minutes, 20-70%B；30-35 minutes, 70%B；
Flow velocity：0.6 ml/min；
Sample size：10 microlitres；
Detection wavelength：360 nanometers；
B, mass spectral analyses condition
Mass spectrograph：TSQ Quantum Access MAX；
Ion source：Electron spray ionisation source negative ion mode；
Molecular weight sweep limitss：150-1500 dalton；
Two grades of spectrum scan modes：Data dependence type is scanned；
Spray voltage：3000 volts；
Capillary temperature：350℃；
Sheath air pressure：275.8 kPa；
Assist gas pressure, nitrogen：55.2 kPas；
6. accumulation rate of the topoisomerase I to inhibitor
Accumulation rate of the topoisomerase I to topoisomerase I inhibitor, can by testing sample before and after enzyme effect each
The change of chromatographic peak curve lower section product (AUC) carries out Calculation Estimation；
Respectively according to each chromatograph in the experimental group before and after untreated testing sample and topoisomerase I effect, matched group
Peak curve lower section product, calculates accumulation rate of the topoisomerase I to topoisomerase I inhibitor according to equation below：
EF=(AExperiment-AControl)/AIt is to be measured× 100%
In formula, EF be accumulation rate of the topoisomerase to I inhibitor, AExperimentChromatograph after acting on topoisomerase I for sample
The sectional area at peak, AControlThe sectional area of chromatographic peak, A after acting on inactivation topoisomerase I for sampleIt is to be measuredFor not with topoisomerase
The sectional area of chromatographic peak in the testing sample that I has an effect.
The present invention has following advantages and good effect：
1. ultrafiltration membrane technique and chromatograph, mass spectrometric hyphenated technique method for combined use are applied to the sieve of topoisomerase I inhibitor
Choose, sample analysis speed is fast, high specificity, it is convenient to the Drug Ligand that identification is combined with bio-target molecule；
2. simultaneously sample analysis consumption is few, spectrogram information amount is big, is capable of achieving the rapid screening of lead compound, little in medicine
There is in terms of molecule ligand and biomacromolecule acceptor interaction research very big advantage；
3. it is applied to analysis natural extracts or monomeric compound to topoisomerase I ex vivo enrichment rate.
Description of the drawings
Fig. 1 is the step flow chart of the present invention；
Fig. 2 is the liquid chromatogram of Fructus rhamni (Rhamnus davurica Pall.) extract gained active component Jing after ultrafiltration step in embodiment 1,
360 nanometers of Detection wavelength, line a represent the Fructus rhamni (Rhamnus davurica Pall.) extract composition liquid phase color not having an effect with topoisomerase I
Spectrogram, line b and line c represent the liquid chromatogram of Fructus rhamni (Rhamnus davurica Pall.) active component obtained by experimental group and matched group respectively, and internal standard product (IS) are
Isoschaftoside；
Fig. 3 is the extraction chromatography of the multiple-reaction monitoring negative ion mode at liquid chromatogram peak 3 (4',5,7-trihydroxyflavone) in embodiment 1
Figure,
Ion used by extracting is parent ion 269.04, and signal intensity is 7.14 × 107；
Fig. 4 is the extraction chromatography of the multiple-reaction monitoring negative ion mode at liquid chromatogram peak 4 (Quercetin) in embodiment 1
Figure,
Ion used by extracting is parent ion 301.00, and signal intensity is 3.72 × 106；
Fig. 5 is the extraction color of the multiple-reaction monitoring negative ion mode at liquid chromatogram peak 5 (rhamnocitrin) in embodiment 1
Spectrogram,
Ion used by extracting is parent ion 298.90, and signal intensity is 1.69 × 106；
Fig. 6 is the extraction chromatography of the multiple-reaction monitoring negative ion mode at liquid chromatogram peak 6 (sakuranetin) in embodiment 1
Figure,
Ion used by extracting is parent ion 284.99, and signal intensity is 1.06 × 107；
Fig. 7 is liquid chromatogram peak 7 (monomethyl ether), the multiple-reaction monitoring anion at peak 8 (physcione) in embodiment 1
The extraction chromatography figure of pattern,
Ion used by extracting is respectively parent ion 283.01,282.94, and signal intensity is respectively 2.37 × 107、1.76×
106；
Fig. 8 is the liquid chromatogram of maryllidaceous alkaloid extract gained Jing after ultrafiltration step in embodiment 2,
232 nanometers of Detection wavelength, line d represent the maryllidaceous alkaloid extract component not having an effect with topoisomerase I
Liquid chromatogram, line e and line f represent the liquid chromatogram of maryllidaceous alkaloid active component obtained by experimental group and matched group respectively,
Internal standard product (IS) are nuciferine；
Fig. 9 is the extraction chromatography of the multiple-reaction monitoring positive ion mode at liquid chromatogram peak 4 (peace Behring) in embodiment 2
Figure,
Ion used by extracting is parent ion 332.25, and signal intensity is 1.27 × 106；
Figure 10 is the extraction color of the multiple-reaction monitoring positive ion mode at liquid chromatogram peak 5 (hippeastrine) in embodiment 2
Spectrogram,
Ion used by extracting is parent ion 316.21, and signal intensity is 1.79 × 106；
Figure 11 is many reaction prisons at liquid chromatogram peak 6 (2 α-hydroxy-6-O-methyloduline) in embodiment 2
The extraction chromatography figure of positive ion mode is surveyed,
Ion used by extracting is parent ion 332.30, and signal intensity is 3.44 × 105；
Figure 12 is 7 ((+) -8,9-mehtylenedioxylhomolycorine N- of liquid chromatogram peak in embodiment 2
The extraction chromatography figure of multiple-reaction monitoring positive ion mode oxide),
Ion used by extracting is parent ion 316.34, and signal intensity is 4.41 × 105；
Figure 13 is the liquid chromatogram of Rhizoma Paridis saponin monomer mixture gained Jing after ultrafiltration step in embodiment 3,
203 nanometers of Detection wavelength, solid line and dotted line represent experimental group and matched group Rhizoma Paridis saponin monomer mixture respectively
Liquid chromatogram；
Figure 14 is the extraction of the multiple-reaction monitoring negative ion mode at liquid chromatogram peak 1 (Rhizoma Paridis saponin VII) in embodiment 3
Chromatogram,
Ion used by extracting is parent ion 1029.81, and signal intensity is 4.74 × 106；
Figure 15 is the extraction of the multiple-reaction monitoring negative ion mode at liquid chromatogram peak 2 (Rhizoma Paridis saponin VI) in embodiment 3
Chromatogram,
Ion used by extracting is parent ion 737.48, and signal intensity is 5.91 × 105；
Figure 16 is the extraction of the multiple-reaction monitoring negative ion mode at liquid chromatogram peak 3 (Rhizoma Paridis saponin II) in embodiment 3
Chromatogram,
Ion used by extracting is parent ion 1013.92, and signal intensity is 2.37 × 106；
Figure 17 is the extraction color of the multiple-reaction monitoring negative ion mode at liquid chromatogram peak 4 (Rhizoma Paridis saponin I) in embodiment 3
Spectrogram,
Ion used by extracting is parent ion 853.74, and signal intensity is 3.97 × 105。
Specific embodiment
Describe in detail with reference to the accompanying drawings and examples：
First, method
Such as Fig. 1, this method comprise the following steps：
1. the preparation -1 of enzyme association reaction buffer；
2. the preparation -2 of testing sample；
3. the preparation -3 of standard solution；
4. testing sample and association reaction -4 that is normal and inactivating topoisomerase I；
5. the chromatography-mass spectroscopy detection and analysis -5 of response sample；
6. accumulation rate -6 of the topoisomerase I to inhibitor.
2nd, embodiment
In addition to special remarks, material used can be bought by commercial sources.
1st, embodiment 1
The method of the ultrafiltration mass spectrometric hyphenated technique rapid screening topoisomerase I inhibitor that the present invention is provided, including it is following
Step：
Described ultrafiltration mass spectrometric hyphenated technique refers to that ultrafiltration membrane technique is used in combination by one kind with chromatograph, mass spectrometric analysis method
A kind of new research meanses for being formed；
Described topoisomerase I inhibitor is natural extracts or monomer compound；Described in the present embodiment
Topoisomerase I inhibitor is Fructus rhamni (Rhamnus davurica Pall.) extract；
1. the preparation of enzyme association reaction buffer
Enzyme association reaction buffer includes：
Potassium acetate：50 mM/ls, trishydroxymethylaminomethane-acetic acid：20 mM/ls,
Magnesium acetate：10 mM/ls, dithiothreitol, DTT：1 mM/l；
4907 milligrams of potassium acetate, 2422.8 milligrams of trishydroxymethylaminomethane-acetic acid, 2144.6 milli of magnesium acetate are weighed accurately
Gram and 154.3 milligrams of dithiothreitol, DTT, using deionized water dissolving to final volume be 1 liter, after being sufficiently mixed, at 25 DEG C, pH value is
7.9；
2. the preparation of testing sample
Appropriate testing sample is weighed accurately, with step, 1. middle prepared buffer fully dissolves, prepare liquid final concentration of 1.0
Mg/ml；
Described testing sample is Fructus rhamni (Rhamnus davurica Pall.) extract；
3. the preparation of standard solution
With acetonitrile by internal standard sample preparation into the standard mother solution that concentration is 100 mcg/mls, add before carrying out Mass Spectrometer Method
Into analyte sample fluid, final concentration of 2 mcg/ml；
Described internal standard sample is Isoschaftoside；
4. testing sample and association reaction that is normal and inactivating topoisomerase I
Experimental group：The Fructus rhamni (Rhamnus davurica Pall.) extract analyte sample fluid for sequentially adding 100 microlitres in 0.2 milliliter of centrifuge tube is micro- with 10
The DNA topoisomerase Is that concentration is for 0.5U/ microlitre are risen, is incubated 30 minutes at 37 DEG C；Reaction mixture is transferred to into retention molecule
During quality is the super filter tube of 30,000 dalton, it is centrifuged 10 minutes under 10000 revs/min；200 microlitres of buffer are added to wash away not
Binding constituents, and be centrifuged 10 minutes under 10000 revs/min again；Repeat the above steps 2 times, merging filtrate；To in super filter tube
200 microlitres of 90% acetonitrile is added, after placing 10 minutes under room temperature, is centrifuged 10 minutes under 10000 revs/min, is discharged part；Weight
Multiple above-mentioned steps 2 times, merging filtrate；Above-mentioned gained filtrate is dried up and adds 50 microlitres of 90% acetonitrile to dissolve, for chromatograph-
Mass spectral analyses；
Matched group：The Fructus rhamni (Rhamnus davurica Pall.) extract analyte sample fluid for sequentially adding 100 microlitres in 0.2 milliliter of centrifuge tube is micro- with 10
Rise the DNA topoisomerase Is for placing that in boiling water 10 minutes inactivation concentration is for 0.5U/ microlitre, incubation 30 minutes at 37 DEG C；Will be anti-
Answer mixed liquor to be transferred in the super filter tube that trapped molecular weight is 30,000 dalton, be centrifuged 10 minutes under 10000 revs/min；Plus
Enter 200 microlitres of buffer and wash away uncombined composition, and be centrifuged 10 minutes under 10000 revs/min again；Repeat the above steps 2
It is secondary, merging filtrate；200 microlitres of 90% acetonitrile is added in super filter tube, after placing 10 minutes under room temperature, under 10000 revs/min
Centrifugation 10 minutes, discharges part；Repeat the above steps 2 times, merging filtrate；Above-mentioned gained filtrate is dried up and 50 microlitres are added
90% acetonitrile dissolves, for Spectrometry；
5. the chromatography-mass spectroscopy detection and analysis of response sample
With inner mark method ration, to step 4. in two groups of sample solutions carry out efficient liquid phase and chromatograph detection；According to chromatogram
In each peak-to-peak area and internal standard sample peak area ratio, calculate each composition relative concentration；
High performance liquid chromatography and Mass Spectrometer Method are carried out to extract testing sample, experimental group and control sample；
The analysis condition of A, high performance liquid chromatography
High performance liquid chromatograph：Thermo Accela 600；
Chromatographic column：Waters Symmetry C18, specification are 4.6 × 250 millimeters, 5 microns；；
Mobile phase：A-0.1% formic acid-water；B- acetonitriles；
Gradient elution program：0-5 minutes, 20%B；5-30 minutes, 20%-70%B；30-35 minutes, 70%B；
Flow velocity：0.6 ml/min；
Sample size：10 microlitres；
Detection wavelength：360 nanometers；
B, mass spectral analyses condition
Mass spectrograph：TSQ Quantum Access MAX；
Ion source：Electron spray (ESI) ionization source negative ion mode；
Molecular weight sweep limitss：150-1500 dalton；
Two grades of spectrum scan modes：Data dependence type is scanned；
Spray voltage：3000 volts；
Capillary temperature：350℃；
Sheath air pressure：275.8 kPa；
Assist gas pressure, nitrogen：55.2 kPas；
6. accumulation rate of the topoisomerase I to inhibitor
Accumulation rate of the topoisomerase I to topoisomerase I inhibitor, can by testing sample before and after enzyme effect each
The change of chromatographic peak curve lower section product (AUC) carries out Calculation Estimation；
Respectively according to each chromatograph in the experimental group before and after untreated testing sample and topoisomerase I effect, matched group
Peak curve lower section product, calculates accumulation rate of the topoisomerase I to topoisomerase I inhibitor according to equation below：
EF=(AExperiment-AControl)/AIt is to be measured× 100%
In formula, EF be accumulation rate of the topoisomerase to I inhibitor, AExperimentChromatograph after acting on topoisomerase I for sample
The sectional area at peak, AControlThe sectional area of chromatographic peak, A after acting on inactivation topoisomerase I for sampleIt is to be measuredFor not with topoisomerase
The sectional area of chromatographic peak in the testing sample that I has an effect；
Calculate topoisomerase I be respectively 0.4% to the accumulation rate of compound peaks 1-8,0.7%, 5.2%,
3.0%th, 5.6%, 1.7%, 3.3% and 5.1%, wherein compound 3 (4',5,7-trihydroxyflavone), 4 (Quercetins), 5 (rhamnocitrins), 6
The accumulation rate of (sakuranetin), 7 (monomethyl ethers) and 8 (rheum emodin) Yin Genggao, infers which has more preferable potential antitumor drug effect.Knot
Fruit is referring to Fig. 2-7.
2nd, embodiment 2
Described topoisomerase I inhibitor is natural extracts or monomer compound；Natural product of the present invention
Maryllidaceous alkaloid extract, comprises the following steps：
1. the preparation of testing sample
Appropriate testing sample is accurately weighed, is fully dissolved using prepared buffer in embodiment 1, prepare liquid is final concentration of
2.0 mg/ml；
Described testing sample is maryllidaceous alkaloid extract；
2. the preparation of standard solution
With acetonitrile by internal standard sample preparation into the standard mother solution that concentration is 100 mcg/mls, add before carrying out Mass Spectrometer Method
Into analyte sample fluid, final concentration of 5 mcg/ml；
Described internal standard sample is nuciferine；
3. testing sample and association reaction that is normal and inactivating topoisomerase I
Experimental group：100 microlitres of maryllidaceous alkaloid extract analyte sample fluid is sequentially added in 0.2 milliliter of centrifuge tube
It is 0.5U/ microlitre of DNA topoisomerase Is with 10 microlitres of concentration, is incubated 30 minutes at 37 DEG C；Reaction mixture is transferred to and is cut
In staying the super filter tube that molecular mass is 30,000 dalton, it is centrifuged 10 minutes under 10000 revs/min；Add 200 microlitres of buffer
Uncombined composition is washed away, and is centrifuged 10 minutes under 10000 revs/min again；Repeat the above steps 2 times, merging filtrate；Xiang Chao
200 microlitres of 90% acetonitrile is added in chimney filter, after placing 10 minutes under room temperature, is centrifuged 10 minutes under 10000 revs/min, release
Part；Repeat the above steps 2 times, merging filtrate；Above-mentioned gained filtrate is dried up and adds 50 microlitres of 90% acetonitrile to dissolve, used
In Spectrometry；
Matched group：100 microlitres of maryllidaceous alkaloid extract analyte sample fluid is sequentially added in 0.2 milliliter of centrifuge tube
It is 0.5U/ microlitre of DNA topoisomerase Is with 10 microlitres of concentration that inactivation in 10 minutes is placed in boiling water, at 37 DEG C, is incubated 30
Minute；Reaction mixture is transferred in the super filter tube that trapped molecular weight is 30,000 dalton, is centrifuged under 10000 revs/min
10 minutes；Add 200 microlitres of buffer to wash away uncombined composition, and be centrifuged 10 minutes under 10000 revs/min again；Repeat
Above-mentioned steps 2 times, merging filtrate；200 microlitres of 90% acetonitrile, after placing 10 minutes under room temperature, 10000 are added in super filter tube
It is centrifuged 10 minutes under rev/min, discharges part；Repeat the above steps 2 times, merging filtrate；Above-mentioned gained filtrate is dried up and added
Enter 50 microlitres of 90% acetonitrile dissolving, for Spectrometry；
4. the high performance liquid chromatography of response sample, mass spectrographic analysis condition
The analysis condition of A, high performance liquid chromatography
High performance liquid chromatograph：Thermo Accela 600；
Chromatographic column：Phenomenex ODS, specification are 2.0 × 150 millimeters, 5 microns；
Mobile phase：A-40 mM/l of ammonium acetate-water；B- acetonitriles；
Gradient elution program：0-15 minutes, 5%B；15-17 minutes, 5%-10%B；17-20 minutes, 10%B；20-30
Minute, 10%-18%B；30-55 minutes, 18%-68%B；
Flow velocity：0.2 ml/min；
Sample size：10 microlitres；
Detection wavelength：232 nanometers；
B, mass spectral analyses condition
Mass spectrograph：TSQ Quantum Access MAX；
Ion source：Electron spray (ESI) ionization source positive ion mode；
Molecular weight sweep limitss：200-1000 dalton；
Two grades of spectrum scan modes：Data dependence type is scanned；
Spray voltage：3000 volts；
Capillary temperature：250℃；
Sheath air pressure：275.8 kPa；
Assist gas pressure, nitrogen：55.2 kPas；
Remaining is with embodiment 1；
Detecting step according to embodiment 1 detects and calculates, and obtains accumulation rate of the topoisomerase I to compound peaks 1-11
Respectively 0.4%, 1.3%, 2.3%, 12.7%, 49.3%, 11.1%, 24.2%, 4.1%, 2.6%, 8.3% and 6.1%,
Wherein compound 4 (peace Behring), 5 (hippeastrines), 6 (2 α-hydroxy-6-O-methyloduline) and 7 ((+) -8,9-
Mehtylenedioxylhomolycorine N-oxide) because with higher accumulation rate, inferring these compositions for Bulbus Lycoridis Radiatae biology
The main component of anti-tumor activity in alkali.As a result referring to Fig. 8-12.
3rd, embodiment 3
《Chinese Pharmacopoeia》Dioscin class compound Rhizoma Paridis saponin I, II and partially in the one regulation Rhizoma Paridis medical material of version in 2015
The total amount of promise saponins compound Rhizoma Paridis saponin VI, VII must not be less than 0.6% as the index for weighing its medical value.The traditional Chinese medical science
In, Rhizoma Paridis are often combined into prescription is used for the treatment of tumor, the such as esophageal carcinoma, laryngeal carcinoma, rectal cancer, pulmonary carcinoma, cervical cancer, leukemia etc..
However, the antineoplastic target spot research at present to Rhizoma Paridis medical material and its main pharmacodynamics composition is less.
Described topoisomerase I inhibitor is natural extracts or monomer compound；Described in the present embodiment
Topoisomerase I inhibitor is monomeric compound Rhizoma Paridis saponin I, II, VI and VII；
1. the preparation of testing sample
Appropriate testing sample is accurately weighed, first using a small amount of dmso solution, is prepared in being subsequently adding embodiment 1
Buffer is fully mixed, final concentration of 0.1 mg/ml (dimethyl sulfoxide≤1%) of prepare liquid；
Described testing sample is monomeric compound Rhizoma Paridis saponin I, II, VI and VII；
2. testing sample and association reaction that is normal and inactivating topoisomerase I
Experimental group：Sequentially add in 0.2 milliliter of centrifuge tube 100 microlitres of Rhizoma Paridis saponin mixture analyte sample fluid with
10 microlitres of concentration are 0.5U/ microlitre of DNA topoisomerase Is, are incubated 30 minutes at 37 DEG C；Reaction mixture is transferred to into retention
During molecular mass is the super filter tube of 30,000 dalton, it is centrifuged 10 minutes under 10000 revs/min；200 microlitres of buffer are added to wash
Uncombined composition is removed, and is centrifuged 10 minutes under 10000 revs/min again；Repeat the above steps 2 times, merging filtrate；To ultrafiltration
200 microlitres of 90% acetonitrile is added in pipe, after placing 10 minutes under room temperature, is centrifuged 10 minutes under 10000 revs/min, release is matched somebody with somebody
Body；Repeat the above steps 2 times, merging filtrate；Above-mentioned gained filtrate is dried up and adds 50 microlitres of 90% acetonitrile to dissolve, be used for
Spectrometry；
Matched group：Sequentially add in 0.2 milliliter of centrifuge tube 100 microlitres of Rhizoma Paridis saponin mixture analyte sample fluid with
10 microlitres of concentration that inactivation in 10 minutes is placed in boiling water are 0.5U/ microlitre of DNA topoisomerase Is, are incubated 30 points at 37 DEG C
Clock；Reaction mixture is transferred in the super filter tube that trapped molecular weight is 30,000 dalton, 10 is centrifuged under 10000 revs/min
Minute；Add 200 microlitres of buffer to wash away uncombined composition, and be centrifuged 10 minutes under 10000 revs/min again；In repetition
State step 2 time, merging filtrate；200 microlitres of 90% acetonitrile, after placing 10 minutes under room temperature, 10000 are added in super filter tube
It is centrifuged 10 minutes under rev/min, discharges part；Repeat the above steps 2 times, merging filtrate；Above-mentioned gained filtrate is dried up and added
Enter 50 microlitres of 90% acetonitrile dissolving, for Spectrometry；
3. the analysis condition of the high performance liquid chromatography of response sample
High performance liquid chromatograph：Thermo Accela 600；
Chromatographic column：SunFireTMC18 posts (4.6 × 150 millimeters, 3.5 microns)；
Mobile phase：0.15% formic acid-water (A) and acetonitrile (B)；
Gradient elution program：0-15 minutes, 28%-46%B；15-25 minutes, 46%-57%B；25-35 minutes, 57%
B；
Flow velocity：0.5 ml/min；
Sample size：10 microlitres；
Detection wavelength：203 nanometers；
Remaining is with embodiment 1；
Detecting step according to embodiment 1 detects and calculates, and obtains topoisomerase I to Rhizoma Paridis saponin I, II, VI and VII
Accumulation rate be respectively 12.75%, 3.38%, 9.37% and 4.39%.Because Rhizoma Paridis saponin I and VI is had with topoisomerase I
Higher combination rate, tentatively infers which is maximum with the active anticancer relatedness of Rhizoma Paridis medical material.This selection result is similar to other
Cytotoxicity experimental result in document, further demonstrates the reliability of the method for the present invention.As a result referring to Figure 13-17.
Claims (1)
1. in a kind of rapid screening natural product topoisomerase I inhibitor method, it is characterised in that comprise the following steps：
Ultrafiltration mass spectrometric hyphenated technique is referred to and a kind of ultrafiltration membrane technique and chromatograph, mass spectrometric analysis method is used in combination formed one
Plant new research meanses；
1. the preparation (1) of enzyme association reaction buffer
Enzyme association reaction buffer includes：
Potassium acetate：50 mM/ls, trishydroxymethylaminomethane-acetic acid：20 mM/ls,
Magnesium acetate：10 mM/ls, dithiothreitol, DTT：1 mM/l；
Accurately weigh 4907 milligrams of potassium acetate, 2422.8 milligrams of trishydroxymethylaminomethane-acetic acid, 2144.6 milligrams of magnesium acetate and
154.3 milligrams of dithiothreitol, DTT, is 1 liter using deionized water dissolving to final volume, and after being sufficiently mixed, at 25 DEG C, pH value is 7.9；
2. the preparation (2) of testing sample
Appropriate testing sample is accurately weighed, is fully dissolved with enzyme association reaction buffer, the final concentration of 1.0-3.0 millis of prepare liquid
Grams per milliliter；
Described testing sample is Fructus rhamni (Rhamnus davurica Pall.) extract or maryllidaceous alkaloid extract；
3. the preparation (3) of standard solution
With acetonitrile by internal standard sample preparation into the standard mother solution that concentration is 100 mcg/mls, add before carrying out Mass Spectrometer Method to treating
Survey in sample liquid, final concentration of 2.0-5.0 mcg/mls；
Described internal standard sample is Isoschaftoside or nuciferine；
4. testing sample and association reaction (4) that is normal and inactivating topoisomerase I
Experimental group：The analyte sample fluid and 10 microlitres of concentration that 100 microlitres are sequentially added in 0.2 milliliter of centrifuge tube is 0.5U/
Microlitre DNA topoisomerase Is, at 37 DEG C be incubated 30 minutes；Reaction mixture is transferred to into trapped molecular weight for 30,000 dongles
In the super filter tube for pausing, it is centrifuged 10 minutes under 10000 revs/min；200 microlitres of buffer are added to wash away uncombined composition, and again
It is secondary to be centrifuged 10 minutes under 10000 revs/min；Repeat the above steps 2 times, merging filtrate；200 microlitres are added in super filter tube
90% acetonitrile, after placing 10 minutes, is centrifuged 10 minutes under 10000 revs/min, discharges part under room temperature；Repeat the above steps 2
It is secondary, merging filtrate；Above-mentioned gained filtrate is dried up and adds 50 microlitres of 90% acetonitrile to dissolve, for Spectrometry；
Matched group：Sequentially add in 0.2 milliliter of centrifuge tube 100 microlitres analyte sample fluid and 10 microlitres in boiling water place
The concentration of inactivation in 10 minutes is 0.5U/ microlitre of DNA topoisomerase Is, is incubated 30 minutes at 37 DEG C；Reaction mixture is shifted
Into the super filter tube that trapped molecular weight is 30,000 dalton, it is centrifuged 10 minutes under 10000 revs/min；200 microlitres are added to delay
Rush liquid and wash away uncombined composition, and be centrifuged 10 minutes under 10000 revs/min again；Repeat the above steps 2 times, merging filtrate；
200 microlitres of 90% acetonitrile is added in super filter tube, after placing 10 minutes under room temperature, is centrifuged 10 minutes under 10000 revs/min,
Release part；Repeat the above steps 2 times, merging filtrate；Above-mentioned gained filtrate is dried up and adds 50 microlitres of 90% acetonitrile molten
Solution, for Spectrometry；
5. the chromatography-mass spectroscopy of response sample tests and analyzes (5)
With inner mark method ration, to step 4. in two groups of sample solutions carry out efficient liquid phase and Mass Spectrometer Method；According to each in chromatogram
The peak area at peak and the peak area ratio of internal standard sample, calculate each composition relative concentration；
High performance liquid chromatography and Mass Spectrometer Method are carried out to extract testing sample, experimental group and control sample；
6. accumulation rate (6) of the topoisomerase I to inhibitor
Accumulation rate of the topoisomerase I to topoisomerase I inhibitor, can be by each chromatograph in testing sample before and after enzyme effect
The change of peak curve lower section product carries out Calculation Estimation；
It is bent according to each chromatographic peak in the experimental group before and after untreated testing sample and topoisomerase I effect, matched group respectively
Line lower section is accumulated, and calculates accumulation rate of the topoisomerase I to topoisomerase I inhibitor according to equation below：
EF=(AExperiment-AControl)/AIt is to be measured× 100%
In formula, EF be accumulation rate of the topoisomerase to I inhibitor, AExperimentChromatographic peak after acting on topoisomerase I for sample
Sectional area, AControlThe sectional area of chromatographic peak, A after acting on inactivation topoisomerase I for sampleIt is to be measuredNot send out with topoisomerase I
The sectional area of chromatographic peak in the testing sample of raw effect.
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