CN105717250A - 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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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 Analysis of Natural Products field, particularly relate to a kind of method of topoisomerase I inhibitor in rapid screening natural product；Specifically, the present invention is to utilize ultrafiltration membrane technique and chromatograph, the mass spectrometric hyphenated technique method of rapid screening topoisomerase I inhibitor from natural extracts or monomer whose compound.
Background technology
DNA topoisomerase I, it it is a kind of indispensable enzyme being widely present in protokaryon and eukaryote body, the tyrosinyl residues of its catalytic site attacks DNA strand, enzyme is made to be formed covalently bound with DNA 3'-phosphodiester bond, cause the mono-chain break of DNA, thus by regulation superhelix, chain, go the chain and nucleic acid effect of unhitching, affect DNA topological structure.Targeted drug is the state-of-the-art medicine being applied to treatment of cancer at present, and it stops the growth of tumor cell by there is, grow necessary specific target spot effect with tumor.Research finds, topoisomerase shows the high level expression not being affected by other factors in tumor cell, particularly topoisomerase I content and the activity of colon cancer, cervical cancer and ovarian cancer etc. is far above normal somatic cell, and especially in S phase tumor cell, activity is greatly improved.Therefore, topoisomerase I inhibitor optionally Inhibit proliferaton phase DNA of tumor cell replicates, and stops tumor cell fast breeding, and then kills tumor cell.Owing to topoisomerase I inhibitor has numerous superiority, one of National Cancer Institute drug Mechanism analysis computer network system six big series antineoplastic medicaments that topoisomerase I inhibitor has been classified as primary study, this enzyme has become the important novel targets of design new type anticancer medicine, and the research to its inhibitor can be that treating of tumor provides new clinical medicine and technological means.
The conventional screening method of topoisomerase I inhibitor generally uses spectrographic method, Surface Plasmon Resonance, X-ray dissipate (Chenxi Yao, Na Na, Lingyun Huang, the Dacheng such as color method, calorimetry and nuclear magnetic resonance method
He, Jin Ouyang.Analytica Chimica Acta, 2013,79,60-66；Vanisree
Mulabagal, Angela I. Calderon, Analytical Chemistry, 2010,82,3,616 3621), said method is convenient and swift, can meet screening requirements to a certain extent.But spectrographic method is easily generated false negative and false positive results because there is ambient interferences；Magnetic resonance detection process is not provided that the detailed features etc. that in quick exchange reaction, protein-ligand interacts.Ultrafiltration mass spectrum technology used in conjunction combines the characteristics such as the excellent separation function of ultrafiltration apparatus and mass-spectrometric technique high speed, high sensitivity, high accuracy, have that analysis speed is fast, high specificity and high-throughout feature, the Drug Ligand combined with bio-target molecule can be identified easily, sample analysis consumption is few simultaneously, spectrogram information amount big, has the biggest advantage (Shun in terms of Medicine small molecule part and biomacromolecule acceptor interaction research
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).
Summary of the invention
It is an object of the invention to the shortcoming and defect overcoming prior art to exist, it is provided that a kind of method of topoisomerase I inhibitor in rapid screening natural product, can be used for analyzing natural extracts or monomeric compound to topoisomerase I ex vivo enrichment rate.
The object of the present invention is achieved like this:
After natural extracts of the present invention or monomeric compound refer to that to topoisomerase I ex vivo enrichment rate natural extracts or monomeric compound and topoisomerase I are hatched and combined, receptor-ligand complexes processes through organic solvent and makes smaller ligand discharge, the reactive compound discharged by liquid phase-Mass Spectrometer Method, calculates concentration change and ratio thereof that each composition is combined with topoisomerase I.
Specifically, this method comprises the following steps:
Described ultrafiltration mass spectrometric hyphenated technique refers to a kind of a kind of new research means ultrafiltration membrane technique and chromatograph, mass spectrometric analysis method being used in combination and being formed；
Described topoisomerase I inhibitor is natural extracts or monomer whose compound；The present invention preferred natural product 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；
Accurately weighing potassium acetate 4907 milligrams, trishydroxymethylaminomethane-acetic acid 2422.8 milligrams, magnesium acetate 2144.6 milligrams and dithiothreitol, DTT 154.3 milligrams, using deionized water dissolving is 1 liter to final volume, and after being sufficiently mixed, at 25 DEG C, pH value is 7.9；
2. the preparation of testing sample
Accurately weigh appropriate testing sample, fully dissolve with enzyme association reaction buffer, liquid to be measured final concentration of 1.0-3.0 mg/ml；
Described testing sample is Fructus rhamni (Rhamnus davurica Pall.) extract or maryllidaceous alkaloid extract；
3. the preparation of standard solution
The standard mother solution becoming concentration to be 100 mcg/ml internal standard sample preparation with acetonitrile, adds to analyte sample fluid before carrying out Mass Spectrometer Method, final concentration of 2.0-5.0 mcg/ml；
Described standard substance are Isoschaftoside or nuciferine；
4. testing sample and the normal and association reaction of inactivation topoisomerase I
Experimental group: be sequentially added into the analyte sample fluid of 100 microlitres and DNA topoisomerase I that 10 lli are 0.5 U/ microlitre in the centrifuge tube of 0.2 milliliter, hatch 30 minutes at 37 DEG C；It is in 30,000 daltonian super filter tubes that reaction mixture is transferred to trapped molecular weight, 10000
It is centrifuged 10 minutes under rev/min；Add 200 microliter of buffer liquid and wash away uncombined composition, and be again centrifuged 10 minutes under 10000 revs/min；Repeat the above steps 2 times, merging filtrate；In super filter tube, add 90% acetonitrile of 200 microlitres, after ambient temperatare puts 10 minutes, be centrifuged 10 minutes under 10000 revs/min, discharge part；Repeat the above steps 2 times, merging filtrate；Above-mentioned gained eluent dries up and adds 90% acetonitrile dissolving of 50 microlitres, for Spectrometry；
Matched group: the analyte sample fluid and 10 microlitres that are sequentially added into 100 microlitres in the centrifuge tube of 0.2 milliliter place the DNA topoisomerase I that concentration is 0.5 U/ microlitre inactivated for 10 minutes in boiling water, hatch 30 minutes at 37 DEG C；It is in 30,000 daltonian super filter tubes that reaction mixture is transferred to trapped molecular weight, is centrifuged 10 minutes under 10000 revs/min；Add 200 microliter of buffer liquid and wash away uncombined composition, and be again centrifuged 10 minutes under 10000 revs/min；Repeat the above steps 2 times, merging filtrate；In super filter tube, add 90% acetonitrile of 200 microlitres, after ambient temperatare puts 10 minutes, be centrifuged 10 minutes under 10000 revs/min, discharge part；Repeat the above steps 2 times, merging filtrate；Above-mentioned gained eluent dries up and adds 90% acetonitrile dissolving of 50 microlitres, for Spectrometry；
5. the chromatography-mass spectroscopy detection of response sample is analyzed
With inner mark method ration, to step 4. in two groups of sample solutions carry out efficient liquid phase and Mass Spectrometer Method；Peak area according to peak each in chromatogram and the peak area ratio of internal standard sample, calculate each composition relative concentration；
Extract testing sample, experimental group and control sample are carried out high performance liquid chromatography and Mass Spectrometer Method:
A, the analysis condition of high performance liquid chromatography
High performance liquid chromatograph: Thermo Accela 600；
Chromatographic column: Waters Symmetry C18, specification is 4.6 × 250 millimeters, 5 microns；
Flowing phase: A-0.1% formic acid-water；B-acetonitrile；
Gradient elution program: 0-5 minute, 20%B；5-30 minute, 20-70%B；30-35 minute, 70%B；
Flow velocity: 0.6 ml/min；
Sample size: 10 microlitres；
Detection wavelength: 360 nanometers；
B, mass spectral analysis condition
Mass spectrograph: TSQ Quantum Access MAX；
Ion source: electron spray ionisation source negative ion mode；
Molecular weight sweep limits: 150-1500 dalton；
Two grades of spectrum scan modes: data dependence type scans；
Spray voltage: 3000 volts；
Capillary temperature: 350 DEG C；
Sheath air pressure: 275.8 kPas；
Assist gas pressure, nitrogen: 55.2 kPas；
6. the topoisomerase I accumulation rate to inhibitor
The topoisomerase I accumulation rate to topoisomerase I inhibitor, can carry out Calculation Estimation by the change of each chromatographic peak curve lower section in testing sample before and after enzyme effect long-pending (AUC)；
Amass according to each chromatographic peak curve lower section in the experimental group before and after untreated testing sample and topoisomerase I effect, matched group respectively, according to the equation below calculating topoisomerase I accumulation rate to topoisomerase I inhibitor:
EF=(AExperiment-AComparison)/ATo be measured×100%
In formula, EF is the topoisomerase accumulation rate to I inhibitor, AExperimentFor the sectional area of chromatographic peak, A after sample and topoisomerase I effectComparisonFor the sectional area of chromatographic peak, A after sample and inactivation topoisomerase I effectTo be measuredFor the sectional area of chromatographic peak in the testing sample do not had an effect with topoisomerase I.
The present invention has following advantages and a good effect:
1. ultrafiltration membrane technique and chromatograph, mass spectrometric hyphenated technique method for combined use being applied in the screening of topoisomerase I inhibitor, sample analysis speed is fast, high specificity, it is convenient to identify the Drug Ligand combined with bio-target molecule；
2. sample analysis consumption is few simultaneously, spectrogram information amount big, can realize the rapid screening of lead compound, have the biggest advantage in terms of Medicine small molecule part and biomacromolecule acceptor interaction research；
3. it is applicable to analyze natural extracts or monomeric compound to topoisomerase I ex vivo enrichment rate.
Accompanying drawing explanation
Fig. 1 is the flow chart of steps of the present invention；
Fig. 2 is Fructus rhamni (Rhamnus davurica Pall.) extract liquid chromatogram of gained active component after ultrafiltration step in embodiment 1,
Detection wavelength 360 nanometer, line a represents the Fructus rhamni (Rhamnus davurica Pall.) extract composition liquid chromatogram not having an effect with topoisomerase I, and line b and line c represents experimental group and the liquid chromatogram of matched group gained Fructus rhamni (Rhamnus davurica Pall.) active component respectively, and internal standard product (IS) are Isoschaftoside；
Fig. 3 is liquid chromatogram peak 3(4',5,7-trihydroxyflavone in embodiment 1) the extraction chromatography figure of multiple-reaction monitoring negative ion mode,
Extracting ion used is parent ion 269.04, and signal intensity is 7.14 × 107；
Fig. 4 is liquid chromatogram peak 4(Quercetin in embodiment 1) the extraction chromatography figure of multiple-reaction monitoring negative ion mode,
Extracting ion used is parent ion 301.00, and signal intensity is 3.72 × 106；
Fig. 5 is liquid chromatogram peak 5(rhamnocitrin in embodiment 1) the extraction chromatography figure of multiple-reaction monitoring negative ion mode, extracting ion used is parent ion 298.90, and signal intensity is 1.69 × 106；
Fig. 6 is liquid chromatogram peak 6(sakuranetin in embodiment 1) the extraction chromatography figure of multiple-reaction monitoring negative ion mode,
Extracting ion used is parent ion 284.99, and signal intensity is 1.06 × 107；
Fig. 7 is liquid chromatogram peak 7(monomethyl ether in embodiment 1), peak 8(physcione) the extraction chromatography figure of multiple-reaction monitoring negative ion mode,
Extracting ion used and be respectively parent ion 283.01,282.94, signal intensity is respectively 2.37 × 107、1.76×106；
Fig. 8 is maryllidaceous alkaloid extract liquid chromatogram of gained after ultrafiltration step in embodiment 2,
Detection wavelength 232 nanometer, line d represents the maryllidaceous alkaloid extract component liquid chromatogram not having an effect with topoisomerase I, line e and line f represents experimental group and the liquid chromatogram of matched group gained maryllidaceous alkaloid active component respectively, and internal standard product (IS) are nuciferine；
Fig. 9 is that in embodiment 2, liquid chromatogram peak 4(pacifies Belling) the extraction chromatography figure of multiple-reaction monitoring positive ion mode,
Extracting ion used is parent ion 332.25, and signal intensity is 1.27 × 106；
Figure 10 is liquid chromatogram peak 5(hippeastrine in embodiment 2) the extraction chromatography figure of multiple-reaction monitoring positive ion mode,
Extracting ion used is parent ion 316.21, and signal intensity is 1.79 × 106；
Figure 11 is 6(2 α-hydroxy-6-O-methyloduline in liquid chromatogram peak in embodiment 2) the extraction chromatography figure of multiple-reaction monitoring positive ion mode,
Extracting ion used is parent ion 332.30, and signal intensity is 3.44 × 105；
Figure 12 is liquid chromatogram peak 7((+ in embodiment 2)-8,9-mehtylenedioxylhomolycorine N-oxide) the extraction chromatography figure of multiple-reaction monitoring positive ion mode,
Extracting ion used is parent ion 316.34, and signal intensity is 4.41 × 105；
Figure 13 is Rhizoma Paridis saponin monomer mixture liquid chromatogram of gained after ultrafiltration step in embodiment 3,
Detection wavelength 203 nanometer, solid line and dotted line represent experimental group and the liquid chromatogram of matched group Rhizoma Paridis saponin monomer mixture respectively；
Figure 14 is 1(Rhizoma Paridis saponin VII in liquid chromatogram peak in embodiment 3) the extraction chromatography figure of multiple-reaction monitoring negative ion mode,
Extracting ion used is parent ion 1029.81, and signal intensity is 4.74 × 106；
Figure 15 is 2(Rhizoma Paridis saponin VI in liquid chromatogram peak in embodiment 3) the extraction chromatography figure of multiple-reaction monitoring negative ion mode,
Extracting ion used is parent ion 737.48, and signal intensity is 5.91 × 105；
Figure 16 is 3(Rhizoma Paridis saponin II in liquid chromatogram peak in embodiment 3) the extraction chromatography figure of multiple-reaction monitoring negative ion mode,
Extracting ion used is parent ion 1013.92, and signal intensity is 2.37 × 106；
Figure 17 is 4(Rhizoma Paridis saponin I in liquid chromatogram peak in embodiment 3) the extraction chromatography figure of multiple-reaction monitoring negative ion mode,
Extracting ion used is parent ion 853.74, and signal intensity is 3.97 × 105。
Detailed description of the invention
Describe in detail with embodiment below in conjunction with the accompanying drawings:
One, method
Such as Fig. 1, this method comprises 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 the normal and association reaction-4 of inactivation topoisomerase I；
5. the chromatography-mass spectroscopy detection of response sample analyzes-5；
6. the topoisomerase I accumulation rate-6 to inhibitor.
Two, embodiment
In addition to special remarks, material used all can be bought by commercial sources.
1, embodiment 1
The method of the ultrafiltration mass spectrometric hyphenated technique rapid screening topoisomerase I inhibitor that the present invention provides, comprises the following steps:
Described ultrafiltration mass spectrometric hyphenated technique refers to a kind of a kind of new research means ultrafiltration membrane technique and chromatograph, mass spectrometric analysis method being used in combination and being formed；
Described topoisomerase I inhibitor is natural extracts or monomer whose compound；Topoisomerase I inhibitor described in the present embodiment 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；
Accurately weighing potassium acetate 4907 milligrams, trishydroxymethylaminomethane-acetic acid 2422.8 milligrams, magnesium acetate 2144.6 milligrams and dithiothreitol, DTT 154.3 milligrams, using deionized water dissolving is 1 liter to final volume, and after being sufficiently mixed, at 25 DEG C, pH value is 7.9；
2. the preparation of testing sample
Accurately weigh appropriate testing sample, fully dissolve with step 1. middle prepared buffer, final concentration of 1.0 mg/ml of liquid to be measured；
Described testing sample is Fructus rhamni (Rhamnus davurica Pall.) extract；
3. the preparation of standard solution
The standard mother solution becoming concentration to be 100 mcg/ml internal standard sample preparation with acetonitrile, adds to analyte sample fluid before carrying out Mass Spectrometer Method, final concentration of 2 mcg/ml；
Described standard substance are Isoschaftoside；
4. testing sample and the normal and association reaction of inactivation topoisomerase I
Experimental group: be sequentially added into the Fructus rhamni (Rhamnus davurica Pall.) extract analyte sample fluid of 100 microlitres and DNA topoisomerase I that 10 lli are 0.5 U/ microlitre in the centrifuge tube of 0.2 milliliter, hatch 30 minutes at 37 DEG C；It is in 30,000 daltonian super filter tubes that reaction mixture is transferred to trapped molecular weight, 10000
It is centrifuged 10 minutes under rev/min；Add 200 microliter of buffer liquid and wash away uncombined composition, and be again centrifuged 10 minutes under 10000 revs/min；Repeat the above steps 2 times, merging filtrate；In super filter tube, add 90% acetonitrile of 200 microlitres, after ambient temperatare puts 10 minutes, be centrifuged 10 minutes under 10000 revs/min, discharge part；Repeat the above steps 2 times, merging filtrate；Above-mentioned gained eluent dries up and adds 90% acetonitrile dissolving of 50 microlitres, for Spectrometry；
Matched group: the Fructus rhamni (Rhamnus davurica Pall.) extract analyte sample fluid and 10 microlitres that are sequentially added into 100 microlitres in the centrifuge tube of 0.2 milliliter are placed in boiling water 10 minutes and inactivated the DNA topoisomerase I that concentration is 0.5 U/ microlitre, hatch 30 minutes at 37 DEG C；It is in 30,000 daltonian super filter tubes that reaction mixture is transferred to trapped molecular weight, is centrifuged 10 minutes under 10000 revs/min；Add 200 microliter of buffer liquid and wash away uncombined composition, and be again centrifuged 10 minutes under 10000 revs/min；Repeat the above steps 2 times, merging filtrate；In super filter tube, add 90% acetonitrile of 200 microlitres, after ambient temperatare puts 10 minutes, be centrifuged 10 minutes under 10000 revs/min, discharge part；Repeat the above steps 2 times, merging filtrate；Above-mentioned gained eluent dries up and adds 90% acetonitrile dissolving of 50 microlitres, for Spectrometry；
5. the chromatography-mass spectroscopy detection of response sample is analyzed
With inner mark method ration, to step 4. in two groups of sample solutions carry out efficient liquid phase and chromatograph detection；According to the peak area ratio of peak-to-peak area each in chromatogram Yu internal standard sample, calculate each composition relative concentration；
Extract testing sample, experimental group and control sample are carried out high performance liquid chromatography and Mass Spectrometer Method；
A, the analysis condition of high performance liquid chromatography
High performance liquid chromatograph: Thermo Accela 600；
Chromatographic column: Waters Symmetry C18, specification is 4.6 × 250 millimeters, 5 microns；；
Flowing phase: A-0.1% formic acid-water；B-acetonitrile；
Gradient elution program: 0-5 minute, 20%B；5-30 minute, 20%-70 %B；30-35 minute, 70%B；
Flow velocity: 0.6 ml/min；
Sample size: 10 microlitres；
Detection wavelength: 360 nanometers；
B, mass spectral analysis condition
Mass spectrograph: TSQ Quantum Access MAX；
Ion source: electron spray (ESI) ionization source negative ion mode；
Molecular weight sweep limits: 150-1500 dalton；
Two grades of spectrum scan modes: data dependence type scans；
Spray voltage: 3000 volts；
Capillary temperature: 350 DEG C；
Sheath air pressure: 275.8 kPas；
Assist gas pressure, nitrogen: 55.2 kPas；
6. the topoisomerase I accumulation rate to inhibitor
The topoisomerase I accumulation rate to topoisomerase I inhibitor, can carry out Calculation Estimation by the change of each chromatographic peak curve lower section in testing sample before and after enzyme effect long-pending (AUC)；
Amass according to each chromatographic peak curve lower section in the experimental group before and after untreated testing sample and topoisomerase I effect, matched group respectively, according to the equation below calculating topoisomerase I accumulation rate to topoisomerase I inhibitor:
EF=(AExperiment-AComparison)/ATo be measured×100%
In formula, EF is the topoisomerase accumulation rate to I inhibitor, AExperimentFor the sectional area of chromatographic peak, A after sample and topoisomerase I effectComparisonFor the sectional area of chromatographic peak, A after sample and inactivation topoisomerase I effectTo be measuredFor the sectional area of chromatographic peak in the testing sample do not had an effect with topoisomerase I；
Calculate topoisomerase I and the accumulation rate of compound peaks 1-8 is respectively 0.4%, 0.7%, 5.2%, 3.0%, 5.6%, 1.7%, 3.3% and 5.1%; wherein compound 3(4',5,7-trihydroxyflavone), 4(Quercetin), 5(rhamnocitrin), 6(sakuranetin), 7(monomethyl ether) and 8(rheum emodin) because of higher accumulation rate, infer that it has more preferable potential antitumor drug effect.Result sees Fig. 2-7.
2, embodiment 2
Described topoisomerase I inhibitor is natural extracts or monomer whose compound；The present invention preferred natural product maryllidaceous alkaloid extract, comprises the following steps:
1. the preparation of testing sample
Accurately weigh appropriate testing sample, use prepared buffer in embodiment 1 fully to dissolve, final concentration of 2.0 mg/ml of liquid to be measured；
Described testing sample is maryllidaceous alkaloid extract；
2. the preparation of standard solution
The standard mother solution becoming concentration to be 100 mcg/ml internal standard sample preparation with acetonitrile, adds to analyte sample fluid before carrying out Mass Spectrometer Method, final concentration of 5 mcg/ml；
Described standard substance are nuciferine；
3. testing sample and the normal and association reaction of inactivation topoisomerase I
Experimental group: the maryllidaceous alkaloid extract analyte sample fluid and 10 lli that are sequentially added into 100 microlitres in the centrifuge tube of 0.2 milliliter are 0.5
The DNA topoisomerase I of U/ microlitre, hatches 30 minutes at 37 DEG C；It is in 30,000 daltonian super filter tubes that reaction mixture is transferred to trapped molecular weight, 10000
It is centrifuged 10 minutes under rev/min；Add 200 microliter of buffer liquid and wash away uncombined composition, and be again centrifuged 10 minutes under 10000 revs/min；Repeat the above steps 2 times, merging filtrate；In super filter tube, add 90% acetonitrile of 200 microlitres, after ambient temperatare puts 10 minutes, be centrifuged 10 minutes under 10000 revs/min, discharge part；Repeat the above steps 2 times, merging filtrate；Above-mentioned gained eluent dries up and adds 90% acetonitrile dissolving of 50 microlitres, for Spectrometry；
Matched group: the maryllidaceous alkaloid extract analyte sample fluid and 10 microlitres that are sequentially added into 100 microlitres in the centrifuge tube of 0.2 milliliter place the DNA topoisomerase I that concentration is 0.5 U/ microlitre inactivated for 10 minutes in boiling water, hatch 30 minutes at 37 DEG C；It is in 30,000 daltonian super filter tubes that reaction mixture is transferred to trapped molecular weight, is centrifuged 10 minutes under 10000 revs/min；Add 200 microliter of buffer liquid and wash away uncombined composition, and be again centrifuged 10 minutes under 10000 revs/min；Repeat the above steps 2 times, merging filtrate；In super filter tube, add 90% acetonitrile of 200 microlitres, after ambient temperatare puts 10 minutes, be centrifuged 10 minutes under 10000 revs/min, discharge part；Repeat the above steps 2 times, merging filtrate；Above-mentioned gained eluent dries up and adds 90% acetonitrile dissolving of 50 microlitres, for Spectrometry；
4. the high performance liquid chromatography of response sample, mass spectrographic analysis condition
A, the analysis condition of high performance liquid chromatography
High performance liquid chromatograph: Thermo Accela 600；
Chromatographic column: Phenomenex ODS, specification is 2.0 × 150 millimeters, 5 microns；
Flowing phase: A-40 mM/l of ammonium acetate-water；B-acetonitrile；
Gradient elution program: 0-15 minute, 5%B；15-17 minute, 5%-10%B；17-20 minute, 10%B；20-30 minute, 10%-18%B；30-55 minute, 18%-68%B；
Flow velocity: 0.2 ml/min；
Sample size: 10 microlitres；
Detection wavelength: 232 nanometers；
B, mass spectral analysis condition
Mass spectrograph: TSQ Quantum Access MAX；
Ion source: electron spray (ESI) ionization source positive ion mode；
Molecular weight sweep limits: 200-1000 dalton；
Two grades of spectrum scan modes: data dependence type scans；
Spray voltage: 3000 volts；
Capillary temperature: 250 DEG C；
Sheath air pressure: 275.8 kPas；
Assist gas pressure, nitrogen: 55.2 kPas；
Remaining is with embodiment 1；
Detect according to the detecting step of embodiment 1 and calculate, obtain topoisomerase I and the accumulation rate of compound peaks 1-11 is 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(pacifies Belling), 5(hippeastrine), 6(2 α-hydroxy-6-O-methyloduline) and 7((+)-8,9-mehtylenedioxylhomolycorine
N-oxide) because having higher accumulation rate, infer that these compositions are the main component of anti-tumor activity in maryllidaceous alkaloid.Result sees Fig. 8-12.
3, embodiment 3
In one regulation Rhizoma Paridis medical material of " Chinese Pharmacopoeia " version in 2015, the total amount of dioscin compounds Rhizoma Paridis saponin I, II and bisnosaponin compound Rhizoma Paridis saponin VI, VII must not be less than 0.6% as the index weighing its medical value.In Chinese medicine, Rhizoma Paridis is often combined into the prescription treatment for tumor, such as the esophageal carcinoma, laryngeal carcinoma, rectal cancer, pulmonary carcinoma, cervical cancer, leukemia etc..But, at present the antineoplastic target spot of Rhizoma Paridis medical material and main pharmacodynamics composition thereof is studied less.
Described topoisomerase I inhibitor is natural extracts or monomer whose compound；Topoisomerase I inhibitor described in the present embodiment is monomeric compound Rhizoma Paridis saponin I, II, VI and VII；
1. the preparation of testing sample
Accurately weigh appropriate testing sample, first use a small amount of dmso solution, be subsequently adding prepared buffer in embodiment 1 and fully mix, final concentration of 0.1 mg/ml of liquid to be measured (dimethyl sulfoxide≤1%)；
Described testing sample is monomeric compound Rhizoma Paridis saponin I, II, VI and VII；
2. testing sample and the normal and association reaction of inactivation topoisomerase I
Experimental group: the Rhizoma Paridis saponin mixture analyte sample fluid and 10 lli that are sequentially added into 100 microlitres in the centrifuge tube of 0.2 milliliter are 0.5
The DNA topoisomerase I of U/ microlitre, hatches 30 minutes at 37 DEG C；It is in 30,000 daltonian super filter tubes that reaction mixture is transferred to trapped molecular weight, 10000
It is centrifuged 10 minutes under rev/min；Add 200 microliter of buffer liquid and wash away uncombined composition, and be again centrifuged 10 minutes under 10000 revs/min；Repeat the above steps 2 times, merging filtrate；In super filter tube, add 90% acetonitrile of 200 microlitres, after ambient temperatare puts 10 minutes, be centrifuged 10 minutes under 10000 revs/min, discharge part；Repeat the above steps 2 times, merging filtrate；Above-mentioned gained eluent dries up and adds 90% acetonitrile dissolving of 50 microlitres, for Spectrometry；
Matched group: the Rhizoma Paridis saponin mixture analyte sample fluid and 10 microlitres that are sequentially added into 100 microlitres in the centrifuge tube of 0.2 milliliter place the DNA topoisomerase I that concentration is 0.5 U/ microlitre inactivated for 10 minutes in boiling water, hatch 30 minutes at 37 DEG C；It is in 30,000 daltonian super filter tubes that reaction mixture is transferred to trapped molecular weight, is centrifuged 10 minutes under 10000 revs/min；Add 200 microliter of buffer liquid and wash away uncombined composition, and be again centrifuged 10 minutes under 10000 revs/min；Repeat the above steps 2 times, merging filtrate；In super filter tube, add 90% acetonitrile of 200 microlitres, after ambient temperatare puts 10 minutes, be centrifuged 10 minutes under 10000 revs/min, discharge part；Repeat the above steps 2 times, merging filtrate；Above-mentioned gained eluent dries up and adds 90% acetonitrile dissolving of 50 microlitres, 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 post (4.6 × 150 millimeters, 3.5 microns)；
Flowing phase: 0.15% formic acid-water (A) and acetonitrile (B)；
Gradient elution program: 0-15 minute, 28%-46%B；15-25 minute, 46%-57%B；25-35 minute, 57%B；
Flow velocity: 0.5 ml/min；
Sample size: 10 microlitres；
Detection wavelength: 203 nanometers；
Remaining is with embodiment 1；
Detect according to the detecting step of embodiment 1 and calculate, obtain topoisomerase I and the accumulation rate of Rhizoma Paridis saponin I, II, VI and VII is respectively 12.75%, 3.38%, 9.37% and 4.39%.Because Rhizoma Paridis saponin I and VI and topoisomerase I have higher combination rate, the preliminary active anticancer relatedness maximum inferring itself and Rhizoma Paridis medical material.This selection result is similar to cytotoxicity experimental result in other documents, demonstrates the reliability of the method for the present invention further.Result sees Figure 13-17.
Claims (1)
1. the method for topoisomerase I inhibitor in a rapid screening natural product, it is characterised in that comprise the following steps:
Described ultrafiltration mass spectrometric hyphenated technique refers to a kind of a kind of new research means ultrafiltration membrane technique and chromatograph, mass spectrometric analysis method being used in combination and being formed；
Described topoisomerase I inhibitor is natural extracts or monomer whose compound；The present invention preferred natural product Fructus rhamni (Rhamnus davurica Pall.) extract, maryllidaceous alkaloid extract, monomeric compound Rhizoma Paridis saponin I, II, VI and VII；
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 weighing potassium acetate 4907 milligrams, trishydroxymethylaminomethane-acetic acid 2422.8 milligrams, magnesium acetate 2144.6 milligrams and dithiothreitol, DTT 154.3 milligrams, using deionized water dissolving is 1 liter to final volume, and after being sufficiently mixed, at 25 DEG C, pH value is 7.9；
2. the preparation (2) of testing sample
Accurately weigh appropriate testing sample, fully dissolve with enzyme association reaction buffer, liquid to be measured final concentration of 1.0-3.0 mg/ml；
Described testing sample is Fructus rhamni (Rhamnus davurica Pall.) extract or maryllidaceous alkaloid extract；
3. the preparation (3) of standard solution
The standard mother solution becoming concentration to be 100 mcg/ml internal standard sample preparation with acetonitrile, adds to analyte sample fluid before carrying out Mass Spectrometer Method, final concentration of 2.0-5.0 mcg/ml；
Described standard substance are Isoschaftoside or nuciferine；
4. testing sample and the normal and association reaction (4) of inactivation topoisomerase I
Experimental group: be sequentially added into the analyte sample fluid of 100 microlitres and DNA topoisomerase I that 10 lli are 0.5 U/ microlitre in the centrifuge tube of 0.2 milliliter, hatch 30 minutes at 37 DEG C；It is in 30,000 daltonian super filter tubes that reaction mixture is transferred to trapped molecular weight, 10000
It is centrifuged 10 minutes under rev/min；Add 200 microliter of buffer liquid and wash away uncombined composition, and be again centrifuged 10 minutes under 10000 revs/min；Repeat the above steps 2 times, merging filtrate；In super filter tube, add 90% acetonitrile of 200 microlitres, after ambient temperatare puts 10 minutes, be centrifuged 10 minutes under 10000 revs/min, discharge part；Repeat the above steps 2 times, merging filtrate；Above-mentioned gained eluent dries up and adds 90% acetonitrile dissolving of 50 microlitres, for Spectrometry；
Matched group: the analyte sample fluid and 10 microlitres that are sequentially added into 100 microlitres in the centrifuge tube of 0.2 milliliter place the DNA topoisomerase I that concentration is 0.5 U/ microlitre inactivated for 10 minutes in boiling water, hatch 30 minutes at 37 DEG C；It is in 30,000 daltonian super filter tubes that reaction mixture is transferred to trapped molecular weight, 10000
It is centrifuged 10 minutes under rev/min；Add 200 microliter of buffer liquid and wash away uncombined composition, and be again centrifuged 10 minutes under 10000 revs/min；Repeat the above steps 2 times, merging filtrate；In super filter tube, add 90% acetonitrile of 200 microlitres, after ambient temperatare puts 10 minutes, be centrifuged 10 minutes under 10000 revs/min, discharge part；Repeat the above steps 2 times, merging filtrate；Above-mentioned gained eluent dries up and adds 90% acetonitrile dissolving of 50 microlitres, for Spectrometry；
5. (5) are analyzed in the chromatography-mass spectroscopy detection 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；Peak area according to peak each in chromatogram and the peak area ratio of internal standard sample, calculate each composition relative concentration；
Extract testing sample, experimental group and control sample are carried out high performance liquid chromatography and Mass Spectrometer Method；
6. the topoisomerase I accumulation rate (6) to inhibitor
The topoisomerase I accumulation rate to topoisomerase I inhibitor, can carry out Calculation Estimation by the change that each chromatographic peak curve lower section in testing sample before and after enzyme effect is long-pending；
Amass according to each chromatographic peak curve lower section in the experimental group before and after untreated testing sample and topoisomerase I effect, matched group respectively, according to the equation below calculating topoisomerase I accumulation rate to topoisomerase I inhibitor:
EF=(AExperiment-AComparison)/ATo be measured×100%
In formula, EF is the topoisomerase accumulation rate to I inhibitor, AExperimentFor the sectional area of chromatographic peak, A after sample and topoisomerase I effectComparisonFor the sectional area of chromatographic peak, A after sample and inactivation topoisomerase I effectTo be measuredFor the sectional area of chromatographic peak in the testing sample do not had an effect with topoisomerase I.
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