CN105424821B - A kind of method of composition with genetoxic in screening natural products - Google Patents
A kind of method of composition with genetoxic in screening natural products Download PDFInfo
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Abstract
The invention provides a kind of method for the composition for screening and having genetoxic in natural products, this method is based on DNA adduct technology, detected using high performance liquid chromatography MS, can efficiently screen the composition in natural products with genetoxic.
Description
Technical field
The invention belongs to tcm field, is related to the method for detecting the composition in natural products with genetoxic, specifically
Say that the present invention relates to a kind of method for being detected using DNA adduct and finding there is genetoxic composition in natural products in ground.
Background technology
DNA adduct is that DNA molecular formation is covalently attached in the compound or its metabolite and organism of electrophilicity
Compound, it is the most important and most common form of DNA chemical damages.This adduct once escapes the reparation of itself, so that it may
It can turn into and cause prominent, teratogenesis, carcinogenic the least factor.Therefore the formation of DNA adduct is considered as to cause a weight of neoplastic process
Want initial period.Research shows that mutagenesis, teratogenesis and the carcinogenic effect of many chemicals are relevant with the formation of DNA adduct.It is logical
DNA adduct and mutagenesis and the research of carcinogenic relation are crossed, it was recognized that DNA adduct is probably chemical carcinogen and cancer
Between a molecule bridge.
Researching DNA adduct helps to understand the carcinogenic mutagenic the Molecular Biology Mechanism of chemical toxic thing.The opposing party
Face, the discovery of DNA adduct may have the meaning of early detection and diagnosis for the carcinogenic of medicine, mutagenicity.Mesh
Before have been observed that produce DNA adduct composition include aristolochic acid (AA) and Pyrrolizidine alkaloid (PAs) etc..Natural production
There is various component in thing, and most of constituent content is seldom, it is difficult to have by the method for routine to detect or screen
The composition of genetoxic.Therefore, those skilled in the art, which are directed to exploitation, can efficiently screen in natural products with heredity
The method of toxic component.
The content of the invention
It is an object of the invention to provide a kind of method of composition for screening and there is genetoxic in natural products and its answer
With.
The first aspect of the present invention, there is provided a kind of method for detecting the composition in natural products with genetoxic, bag
Include step：
Prepare reaction system and carry out temperature and incubate reaction, the reaction system includes：Sample to be tested, deoxyribonucleoside and isotope
The deoxyribonucleoside of mark；
After terminating reaction, the formation of DNA adduct in reaction system is detected using tablets by HPLC-MS.
Methods described includes, and the molecular weight of various materials in reaction system is obtained using tablets by HPLC-MS；If
Material X and material X ' (that is, n=material X' quasi-molecular ion-material X accurate point of the quasi-molecular ion difference for n in mass spectrogram be present
Daughter ion), n is the molecular weight difference of the deoxyribonucleoside of deoxyribonucleoside and isotope marks, then preliminary judgement material X is can shape
Into the composition (that is, the composition for indicating genetoxic) of DNA adduct.Further, methods described includes step：Obtaining should
The quasi-molecular ion of material X and material X ' daughter ion, material X daughter ion and material X ' son if there is difference for n
Ion, then it is the composition with genetoxic that can further judge the material X.
In another preference, methods described includes step：
(1) preliminary screening of DNA adduct
Reaction system is prepared, the reaction system includes：It is sample to be tested, hepatomicrosome (including liver S9), deoxyribonucleoside, same
The deoxyribonucleoside and reduced Coenzyme II of position element mark；
The reaction system is incubated under the conditions of 37 DEG C and reacted；
Isometric ice acetonitrile terminating reaction is added after the completion of reaction；
HPLC-MS/MS detects the formation of DNA adduct.
In another preference, the sample to be tested is natural extract, is included in the natural extract at least one kind of
(preferably >=3 kind；More preferably >=5 kind；Most preferably >=8 kind) composition (compound).
In another preference, after adding isometric ice acetonitrile terminating reaction in step (1), centrifugation, filtering, or by solid
The methods of phase extraction column, is handled.
In another preference, methods described also includes step (2)：
(2) preparing includes sample to be tested, hepatomicrosome (or Animal Liver S9), calf thymus DNA and reduced Coenzyme II
Reaction system；The reaction system is incubated under the conditions of 37 DEG C and reacted；DNA is extracted after the completion of reaction；Enzymolysis or sour water
Solution, SPE, HPLC-MS/MS detect the formation of DNA adduct；
The sample to be tested is the material (material X) that can form DNA adduct that step (1) detection obtains.
In another preference, in the reaction system of the step (1), the content of each component is as follows：
The μ g/ml of sample to be tested concentration >=20；Hepatomicrosome protein concentration >=0.1mg/ml；The μ g/ of deoxyribonucleoside content >=20
ml；The μ g/ml of the deoxyribonucleoside content of isotope marks >=20；Reduced Coenzyme II or the content of oxidized coenzyme-II 0.5-
2.0mmol/L。
In another preference, in the reaction system of the step (1), the content of each component is as follows：
Sample to be tested concentration is 20 μ g/ml~5mg/ml (being preferably, 0.4mg/ml~1.0mg/ml)；Hepatomicrosome albumen
Concentration is 0.1mg/ml~5mg/ml (being preferably 1.0mg/ml~3.5mg/ml)；Deoxyribonucleoside content is 20 μ g/ml~5mg/
Ml (preferably, 1.0mg/ml~3mg/ml)；The deoxyribonucleoside content of isotope marks be 20 μ g/ml~3mg/ml (be preferably,
0.5mg/ml~2mg/ml)；Reduced Coenzyme II or the content of oxidized coenzyme-II 0.8mmol/L~1.5mmol/L are (preferably,
1mmol/L)。
In another preference, in the step (1), the incubation reaction time is 1-24h.
In another preference, in the reaction system of the step (2), the content of each component is as follows：
The μ g/ml of sample to be tested concentration >=20；Hepatomicrosome protein concentration >=0.1mg/ml；Calf thymus DNA content >=
0.5mg/ml；Reduced Coenzyme II content is 1mmol/L.
In another preference, in the reaction system of the step (2), the content of each component is as follows：
Sample to be tested concentration is 20 μ g/ml~5mg/ml (being preferably, 0.4mg/ml~1.0mg/ml)；Hepatomicrosome albumen
Concentration is 0.1mg/ml~5mg/ml (being preferably, 1.0mg/ml~3.5mg/ml)；Calf thymus DNA content be 0.1mg/ml~
5mg/ml (preferably, 0.5mg/ml~3mg/ml)；Reduced Coenzyme II or the content of oxidized coenzyme-II 0.8mmol/L~
1.5mmol/L (preferably, 1mmol/L).
In another preference, in the step (2), the incubation reaction time is 1-24h.
In another preference, in the step (2), the enzyme used in enzymolysis is selected from：Deoxyribonuclease, nuclease
P1, alkaline phosphatase, phosphodiesterase or its combination；Preferably, the acid that sour water solution uses is selected from formic acid, hydrochloric acid or its combination.
In another preference, the tablets by HPLC-MS includes high performance liquid chromatography-triple level Four bar
Mass spectrum and high performance liquid chromatography-high resolution mass spectrum；Preferably, the high performance liquid chromatography-triple level Four bar mass spectrum is using neutral
Lose Scanning Detction DNA adduct；Preferably, using the mass deficit filtering technique of high performance liquid chromatography-high resolution mass spectrum, or
Accurate neutral loss detection DNA adduct；Preferably, high performance liquid chromatography include high performance liquid chromatography, ultra performance liquid chromatography,
Nano- high performance liquid chromatography and two-dimensional highly effective liquid phase chromatographic etc.；Preferably, in the reaction with calf thymus DNA, HPLC-MS
High performance liquid chromatography-mass spectrographic multiple-reaction monitoring of triple level Four bar (MRM) or high performance liquid chromatography-high resolution mass spectrum are used with essence
True molecular weight is detected.
In another preference, the isotope is selected from：Carbon isotope, nitrogen isotope, oxygen isotope, hydrogen isotope or its
Combination.
In another preference, one or more isotope marks are contained in the deoxyribonucleoside of the isotope marks.
In another preference, the reduced Coenzyme II includes NADPH, or NADP+And its regenerative system.
In another preference, the deoxyribonucleoside include desoxyadenossine, deoxyguanosine, deoxycytidine, AZT and
Its triphosphoric acid thing (including its metal salt).
In another preference, the sample to be tested is natural products.
In another preference, methods described also includes step：
(3) extraction separation is carried out to the composition that can form DNA adduct.
In another preference, the sample to be tested is plant extracts, preferably Chinese medicinal plant extract.
In another preference, the sample to be tested is Chinese medical extract.
In another preference, the plant is selected from the group：Levisticum and the red sage root.
In another preference, the composition with genetoxic is Osthole or Tanshinone I I A.
The second aspect of the present invention, there is provided it is a kind of to detect the kit for having genetoxic composition in Chinese medicine or extract,
The kit includes：Hepatomicrosome (or liver S9), deoxyribonucleoside, the deoxyribonucleoside of isotope marks and reduced Coenzyme II
Reagent needed for (or oxidized coenzyme Ⅱ) and reduced Coenzyme II regenerative system.
In another preference, also include in the kit：Calf thymus DNA.
In another preference, enzymolysis reagent is also included in the kit, the enzymolysis reagent is selected from：Deoxyribose core
Sour enzyme, nuclease P 1, alkaline phosphatase, phosphodiesterase or its combination.
The third aspect of the present invention, there is provided a kind of method for the genetoxic for detecting extractive of pubescent angelica root coumarin, methods described bag
Include step：Detect the content of Osthole in extractive of pubescent angelica root coumarin.
In another preference, methods described includes step：
(a) the content C2 of Osthole in the extractive of pubescent angelica root coumarin is determined, and compared with predetermined value C0；
(b) when the content C2 is higher than C0, then the off quality of the product is judged；Be less than as the content C2 or
During equal to C0, then the up-to-standard of the product is judged.
In another preference, the C2 is percentage by weight of the Osthole in the extractive of pubescent angelica root coumarin.
In another preference, C0≤10% (w/w), it is preferable that C0≤5% (w/w), it is highly preferred that institute
C0≤1% is stated, the C0 can be 0.01%, 0.05%, 0.1%, 0.2%, 0.5%, 1.0%, 5%, 10%.
The fourth aspect of the present invention, there is provided a kind of method for the genetoxic for detecting Salvia root P.E, methods described bag
Include step：Detect the content of Tanshinone I I A in Salvia root P.E.
In another preference, methods described includes step：
(a) the content D2 of Tanshinone I I A in the Salvia root P.E is determined, and compared with predetermined value D0；
(b) when the content D2 is higher than D0, then the off quality of the product is judged；Be less than as the content D2 or
During equal to D0, then the up-to-standard of the product is judged.
In another preference, the D2 is percentage by weights of the Tanshinone I I A in the Salvia root P.E.
In another preference, D0≤10% (w/w), it is preferable that D0≤5% (w/w), it is highly preferred that institute
D0≤1% is stated, the D0 can be 0.01%, 0.05%, 0.1%, 0.2%, 0.5%, 1.0%, 5%, 10%.It should be understood that
In within the scope of the present invention, above-mentioned each technical characteristic of the invention and each technology specifically described in below (eg embodiment) are special
It can be combined with each other between sign, so as to form new or preferable technical scheme.As space is limited, no longer tire out one by one herein and state.
Brief description of the drawings
Fig. 1 shows the result of Osthole DNA adduct HPLC-MS detections.A. Osthole+deoxyribonucleoside neutrality is lost
Lose scanning mass spectrogram；B. the doubtful adduct of Osthole [M+H]+Product ion figure；C. the doubtful adduct of Osthole [M+Na]+
Product ion figure；D. the doubtful adduct of Osthole [M+Na]+Isotopic peak product ion figure；E. Osthole and calf thymus
DNA adduct multiple-reaction monitoring (MRM) figure in DNA reaction systems.
Fig. 2 shows the result of tanshinone IIA-DNA adduct HPLC-MS detections.A. in tanshinone IIA+deoxyribonucleoside
Property lose scanning mass spectrogram；B. the doubtful adduct of tanshinone IIA [M+H]+Product ion figure；C. the doubtful adduct of tanshinone IIA
[M+H]+Isotopic peak product ion figure；D. tanshinone IIA reacts prison more with DNA adduct in calf thymus DNA reaction system
Survey (MRM) figure；E.DNA adduct multiple-reaction monitoring negative controls.
Fig. 3 shows Comet Assay research tanshinone IIA to HepG2 cytogenetic toxicity results.A is that tanshinone IIA is real
Test group；B. negative control group.
Embodiment
The present inventor and in-depth study, obtains a kind of composition for screening and having genetoxic in natural products by extensive
Method, difference of deoxyribonucleoside of this method based on deoxyribonucleoside and isotope marks on molecular weight, utilize efficient liquid phase
Chromatograph-mass spectrometer coupling method is detected, and can efficiently screen the composition in natural products with genetoxic, on this basis
Complete the present invention.
The invention aims to solve problems of the prior art, there is provided one kind using high performance liquid chromatography-
MS (HPLC-MS) detects DNA adduct, so as to screen the composition in natural products with genetoxic.
The invention provides a kind of formation by detecting DNA adduct to have genetoxic to screen in natural products
The method of composition.DNA adduct detection, high-efficient liquid phase color are carried out using tablets by HPLC-MS (HPLC-MS)
Spectrum-MS includes high performance liquid chromatography-triple level Four bar mass spectrum and high performance liquid chromatography-high resolution mass spectrum, preferably
The high performance liquid chromatography-triple level Four bar mass spectrum utilizes high-efficient liquid phase color using neutral loss scan detection DNA adduct
The mass deficit filtering technique of spectrum-high resolution mass spectrum, or accurate neutral loss detection DNA adduct.To forming DNA adduct
Composition is separated, and obtains the composition with genetoxic.
As used herein, term " tablets by HPLC-MS " includes high performance liquid chromatography-triple level Four bar matter
Spectrum and high performance liquid chromatography-high resolution mass spectrum, wherein the high performance liquid chromatography-triple level Four bar mass spectrum is swept using neutral loss
Detection DNA adduct is retouched, is lost using the mass deficit filtering technique of high performance liquid chromatography-high resolution mass spectrum, or accurate neutral
Detect DNA adduct.And added according to the quasi-molecular ion of the DNA adduct detected and the product ion information analysis DNA
The structure of compound, and the constituent structure information of DNA adduct is formed, and the composition is separated.
There is the method for the composition of genetoxic in the screening natural products of the present invention, comprise the following steps：1. doubtful DNA
The discovery of adduct：Hepatomicrosome is added in incubation system, the natural products to be detected, deoxyribonucleoside, isotope marks it is de-
Oxygen nucleosides, reduced Coenzyme II, 37 DEG C of incubations, isometric ice acetonitrile terminating reaction is added, is centrifuged, filtering, HPLC-MS detections,
Doubtful adduct should have corresponding isotopic peak simultaneously, and Product ion scans are carried out to the ion of doubtful adduct；2. verify：
Incubation method is same as above, and replaces deoxyribonucleoside and the deoxyribonucleoside of isotope marks with calf thymus DNA, after 37 DEG C are incubated, at a high speed
Centrifugation, supernatant is taken, extract DNA, enzymolysis, SPE, HPLC-MS detections, compared with 1. sample that step is prepared, divide
Analyse whether target component or extract can form DNA adduct with calf thymus DNA in vitro, if in natural extracts
In detect DNA adduct, just extract is separated, 1. each composition after separation is operated by step (be not added with isotope
The deoxyribonucleoside of mark), until the isolated composition that can form doubtful adduct.And genetoxic is carried out to the composition and ground
Study carefully.
Main advantages of the present invention are：
(1) composition in natural products with genetoxic can be efficiently detected using the method for the present invention；
(2) for natural products complicated component, active component content is low the characteristics of, detection method of the invention, sample need
The amount of asking is few, simple to operate, and testing result is accurate.
(3) method of the invention can be used for efficient discovery DNA adduct.
With reference to specific embodiment, the further old present invention in detail.It should be understood that these embodiments are merely to illustrate the present invention
Rather than limitation the scope of the present invention.Unless otherwise indicated, otherwise percentage and number are calculated by weight.In following examples
Experiment material used and reagent can obtain from commercially available channel unless otherwise instructed.
The screening of genetoxic composition in the levisticum of embodiment 1
Levisticum is samphire Angelica pubescens Angelica pubescens Maxim.f.biserrata Shan
Et Yuan dry root, there is the effect of dispelling wind and eliminating dampness, numbness relieving and pain relieving, be clinically used for wind-cold-dampness arthralgia, lumbocrural pain, few cloudy volt
Wind headache, chill hold wet headache etc. under the arm.The present inventor is to asiatic moonseed extract and its contained Osthole, xanthotoxin, psoralea corylifolia
Element, umbelliferone, Columbianedin angelate are studied.Osthole, xanthotoxin, psoralen, umbrella shape
Flower lactone, Columbianedin angelate structure are as follows：
A. Osthole；B. xanthotoxin；C. psoralen；D. umbelliferone；E. Columbianedin angelate.
1.1 extractive of pubescent angelica root coumarin and the doubtful DNA adduct detection of reference substance
Levisticum medicinal material 100g, crush, sieving, 80% ethanol of 10 times of amounts extracts 2 times, filtering, merging filtrate, is concentrated under reduced pressure
To without alcohol taste, add distilled water to dilute, filter, filtrate adds to the D101 macroporous resin columns activated, distilled water elution, and eluent is abandoned
Go, then respectively with 20%, 40%, 60%, 80%, 95% ethanol elution, be concentrated under reduced pressure eluent.
Each several part is eluted into concentrate and Osthole, xanthotoxin, psoralen, umbelliferone, dihydro Ou Shan celerys
Plain angelate, as sample to be tested, detected respectively：
Analysis condition chromatographic condition Agilent Proshell C18 chromatographic columns (4.6mm*50mm, 2.7 μm)；Mobile phase
For the acetic acid of acetonitrile -0.2% (25：75)；Volume flow 0.4mL/min；30 DEG C of column temperature；The μ L of sample size 2.
Mass Spectrometry Conditions electric spray ion source (ESI), cation scanning；Detection mode：Neutral loss scan (116u, ce value
For 10,20,30ev) and Product ion scans；Dry 350 DEG C of temperature degree；Dry gas product flow 10L/min；Nebulizer pressure
45psi (1psi=6.895kPa)；400 DEG C of sheath temperature degree；Sheath gas volumetric flow 11L/min.
Incubation system 0.5ml, rat liver microsomes 2mg/ml is added, is separately added into sample to be tested 0.5-1mg, desoxyadenossine
2mg, isotope marks desoxyadenossine (15N5) 0.5mg, reduced Coenzyme II 1mmol/L, phosphate buffer supplies volume
To 0.5ml, organic solvent content is no more than 5% in system, 37 DEG C of incubation 3h, adds androgynous accumulated ice acetonitrile and stops reaction, be vortexed
Vibration, 3000r centrifugation 10min, 0.22 μm of filtering with microporous membrane, high performance liquid chromatography-triple level Four bar mass spectrum are lost using neutrality
Scanning is lost to be detected.Control group is set up simultaneously, does not add deoxyribonucleoside and the deoxyribonucleoside of isotope marks in control group.
Testing result is as shown in Figure 1, the results showed that, in 60%, 80%, 95% eluent and Osthole incubation system
Detect doubtful DNA adduct.In xanthotoxin, psoralen, umbelliferone, Columbianedin incubation system not
Detect DNA adduct.In the eluent incubation system of levisticum 60%, 80%, 95%, the doubtful DNA adduct [M+H that detects
]+M/z493.9, isotopic peak m/z498.0, [M+Na]+M/z515.8, isotopic peak m/z520.9, body is incubated in Osthole
Doubtful DNA adduct [M+H] is also detected that in system+M/z493.9, isotopic peak m/z498.0, [M+Na]+M/z515.8, same to position
Plain peak m/z520.9.
That sets up at the same time is not detected by corresponding doubt plus in deoxyribonucleoside and the deoxyribonucleoside control group of isotope marks
Like adduct, not plus in the deoxyribonucleoside control group of isotope marks, corresponding isotopic peak is not detected by.
By contrasting retention time and product ion information, inventor has found to detect in 3 eluent incubation systems of levisticum
To doubtful DNA adduct and the doubtful DNA adduct in Osthole incubation system retention time and product ion it is homogeneous
Together, it is thus determined that the doubtful DNA adduct detected in extractive of pubescent angelica root coumarin incubation system is by Osthole or its metabolite shape
Into.[M+H]+M/z493.9 product ion m/z377.8, it is [M+H]+Slough the fragment ion of deoxyribose；M/z135.9, it is
The fragment ion of deoxyribonucleoside base；M/z242.9, it is the fragment ions of Osthole.[M+Na]+M/z515.8 product from
Sub- m/z400.2, it is [M+Na]+M/z515.8 sloughs the fragment ion of deoxyribose；[M+H]+M/z493.9 product ion is
m/z377.8；M/z157.9, add sodium peak for deoxyribonucleoside base fragment ion, m/z265.1, be Osthole fragment ion
Plus sodium peak.[M+Na]+Isotopic peak m/z521.5 product ion m/z405.2, than [M+Na]+M/z515.8 sloughs deoxidation core
The more 5u of m/z400.2 that the fragment ion of sugar is formed, are its isotopic peak, and m/z243.0 is the fragment ions of Osthole, m/
Z265.1, add sodium peak for Osthole fragment ion, m/z163.0 is the same of deoxyribonucleoside base m/z157.9 fragment ions
The plain peak in position.Therefore the doubtful adduct is made up of deoxyribonucleoside and Osthole two parts.
The reaction of 1.2 Ostholes and calf thymus DNA
Incubation system 0.5ml, calf thymus DNA 0.5mg, reduced Coenzyme II 1mmol/L, Osthole 0.5mg are added,
Phosphate buffer supplies volume, and organic solvent content is no more than 5% in system, 37 DEG C of incubation 3h, by tissue/cell genome
DNA extraction kit (Beijing Ai Delai bio tech ltd) specification record method extracts DNA.100 μ g DNA are taken, are added
Entering the μ L of deoxyribonuclease I (Dnase I) 15, (20mg/Ml, 4 DEG C, matching while using, raw work bioengineering (Shanghai) share has
Limit company, 600U), 35 μ L 10mM MgCl2 and 10mM Tris buffer solutions, with 10mM Tris buffer solution complement products to 200 μ L.
Adding 197 μ L 0.2M Tris and 2 μ L phosphodiesterases I after 37 DEG C of incubation 2h into enzymatic hydrolysis system, (100mU/ μ L, give birth to by -20 DEG C
Work) and 1.3 μ L alkaline phosphatases (1.25U/ μ L, 4 degree of refrigerators, sigma).Taken out after being incubated 24h, carry out SPE.
Cleanert PEP-2 solid-phase extraction columns (1ml), activation：After solid-phase extraction column adds 1ml methanol to elute, then 1ml distilled waters are added to wash
It is de-, by, into the solid-phase extraction column after activation, the elution of 1ml distilled waters, eluent discards, then uses 1ml in the DNA sample after enzymolysis
Methanol elutes, and eluent drying, adds 100 μ l methanol constant volumes, filters, HPLC-MS is detected using multiple-reaction monitoring (MRM).
Control group is set up simultaneously, Osthole is not added in control group.
Testing result detects corresponding add as shown in figure 1, in the reaction system of Osthole and calf thymus DNA
Compound.Show that Osthole can form DNA adduct in incubation system with calf thymus DNA.
1.3 Salmonella reversion tests (Ames test) study the mutagenesis of Osthole
Choose two bacterial strains of TA98 and TA100.It is identified qualified before experiment.Using Ames micro fluctuation experiment investigation snakes
The mutagenesis of machine tool element.Under the conditions of-S9, positive control difference 2-Nitroflucrene 4 μ of TA98 and TA100 bacterial strains
The g/mL and μ g/mL of Nitrofurantoin 0.25；Under the conditions of+S9, the positive control of TA98 and TA100 bacterial strains is 2-
Aminoanthracene, concentration are respectively 2 and 5 μ g/mL.Take the mixed of 10 μ L test samples liquid, 240 μ L exposure culture mediums and bacterium solution
Close liquid to add in 24 orifice plates, each multiple holes of dosage 3, be placed in 37 DEG C of constant-temperature table 150rpm/min, be incubated 90min.Incubation terminates
Afterwards, 2.8mL indicator mediums are added per hole, are well mixed, take 50 μ L to be added in 384 orifice plates per hole, corresponding plus 48 holes per hole
(384 plate hole).384 well culture plates are placed in and are set in 37 DEG C of constant incubator culture about 48-72h.After culture expires, training is taken out
Plate is supported, the bacterium colony that detects by an unaided eye is counted, and yellow hole is the colony growth hole of back mutation, and purple hole is without mutation bacterium colony
Grow hole.(there have under 1000 and 5000 μ g/mL dosage to be serious heavy using 1.3,10.30,100,300 μ g/ml concentration for Osthole
Form sediment, have a small amount of precipitation in 300 μ g/mL.Therefore 1000 and 5000 μ g/mL dosage are not set),
Efficiency test standard：4 dosage that can be used for interpretation of result should at least be included；The reply of TA98 bacterial strain negative controls
It is mutated the hole of hole count average value≤10/48, the hole of back mutation hole count average value≤12 of TA100 bacterial strain negative controls/48, sun
Property control the hole of back mutation hole count average value >=25/48.
Evaluate mutation effect standard
(1) the Mean+SD values of the back mutation hole count of negative control group are set to baseline, the reply of all test groups
Mutation hole count average value is less than 2 times of baseline, is judged to feminine gender.
(2) the back mutation hole count average value of test group is more than 2 times of baseline, then carries out Student's t-test
(1 sided, unpaired) is detected, still nonsignificance, then can be judged to feminine gender.If there is significant difference (P<0.05) can join
According to dose relationship and biological significance, there is the increase of concentration dependent or multiple in the back mutation hole count that test sample is induced
Occur the increase of repeatability in concentration group, can determine whether the positive.
As a result
(1) the back mutation hole count average value of negative control group and positive controls meets the requirements.
(2) it is loaded in test sample in processing procedure, Osthole has a small amount of precipitation to produce under 300 μ g/mL dosage, >=
There is more precipitation to produce under 1000 μ g/mL dosage.
(3) Osthole TA100 bacterial strains does not occur at each dose the increase of back mutation hole count, not less than
2 times of baseline；In+S9Under the conditions of TA98 bacterial strains returned by parameters dose dependents are presented in 100 and 300 μ g/mL bacterium colonies increase
Add, and more than 2 times, respectively the 3.7 of baseline times and 7.6 times of baseline.
Conclusion：Under this experiment condition, it is believed that Osthole has under the conditions of+S9 to salmonella typhimurium TA98 bacterial strains
There is Mutation induction.
Genetoxic composition selection in the red sage root of embodiment 2
The red sage root is labiate red sage root Salvia miltiorrhiza Bge. dry root and rhizome, is dispelled with promoting blood circulation
The stasis of blood, inducing meastruation to relieve menalgia, relieving restlessness that clears away heart-fire, cool blood to disappear carbuncle.For chest impediment and cardialgia, gastral cavity abdomen hypochondriac pain , lumps in the chest and abdomen, hot numbness pain, it is vexed not
Sleep, irregular menstruation, dysmenorrhoea Amenorrhea, sore swell and ache curative.The present inventor is studied Salvia root P.E.
The doubtful DNA adduct detection of 2.1 Salvia root P.Es
Red rooted salvia 50g, crushing, sieving, 50% ethanol extracts 2 times, filtering, merging filtrate, is concentrated under reduced pressure into no alcohol taste,
With ethyl acetate, extracting n-butyl alcohol, extract is concentrated under reduced pressure.By the water layer after extraction, ethyl acetate portion, n-butanol fraction and
Sodium Danshensu, tanshinone IIA, Cryptotanshinone are detected respectively as sample to be tested：
Analysis condition chromatographic condition Agilent Proshell C18 chromatographic columns (4.6mm*50mm, 2.7 μm)；Mobile phase
It is mobile phase for acetonitrile (A)-water (B), gradient elution (0-15min, 10%-90%B；Volume flow 0.4mL/min；Column temperature 30
℃；The μ L of sample size 2.
Mass Spectrometry Conditions electric spray ion source (ESI), cation scanning；Detection mode：Neutral loss scan (116u, ce value
For 10,20,30ev) and Product ion scans；Dry 350 DEG C of temperature degree；Dry gas product flow 10L/min；Nebulizer pressure
45psi (1psi=6.895kPa)；400 DEG C of sheath temperature degree；Sheath gas volumetric flow 11L/min.
Incubation system specifically refers to the 1.1 of embodiment 1.
Testing result is as shown in Figure 2, the results showed that, detected in ethyl acetate portion and tanshinone IIA incubation system
Doubtful DNA adduct, the doubtful DNA adduct m/z544.1, isotopic peak m/z549.4 detected.That sets up at the same time does not add
Corresponding doubtful adduct is not detected by deoxyribonucleoside control group, in the deoxyribonucleoside control group of isotope marks is not added, not
Detect corresponding isotopic peak.
By contrasting retention time and product ion information, the inventors discovered that being detected in Salvia root P.E incubation system
Doubtful DNA adduct m/z544.1, isotopic peak m/z549.4 with it is doubtful in tanshinone IIA and Cryptotanshinone incubation system
DNA adduct retention time and product ion all same, through consulting literatures, tanshinone IIA is the metabolite of Cryptotanshinone, because
The doubtful DNA adduct m/z544.1 detected in this determination Salvia root P.E incubation system is produced by tanshinone IIA or its metabolism
Thing is formed.M/z544.1 product ion m/z427.9, the fragment ion of deoxyribose is sloughed for m/z544.1.Isotopic peak m/
Z549.4 product ion m/z433.2, the fragment ion of deoxyribose is sloughed for m/z549.4, be m/z427.9 isotopic peak.
The reaction of 2.2 tanshinone IIAs and calf thymus DNA
Using 1.2 method of embodiment 1, the reaction of tanshinone IIA and calf thymus DNA is detected, HPLC-MS uses more
Reaction monitoring (MRM) is detected.Control group is set up simultaneously, tanshinone IIA is not added in control group.Tanshinone IIA with it is small
In the reaction system of bovine chest gland DNA, corresponding adduct is detected, as a result as shown in Figure 3.It is not detected by blank control group corresponding
Adduct.Show that tanshinone IIA and calf thymus DNA can form DNA adduct.
Alkaline comet assay studies the genetoxic of tanshinone IIA
HepG2 cells contain 10% calf serum and dual anti-(1 × 105U/L penicillin, 100mg/L streptomysins) MEM/
In EBSS culture mediums, in atmosphere containing 5%CO2, culture, passage in 37 DEG C of incubator, growth period cell of taking the logarithm is used to try
Test.
Logarithmic phase grows cell and digests harvest with pancreatin, adds the tanshinone IIA and positive control H of various concentrations2O2, 37
DEG C suspend culture 1h, Trypan Blue detection cell survival rate, Hoechst 33342 dyeing detection cell apoptosis rate, centrifugation
Supernatant is removed, adds PBS washings cell twice, cell is finally adjusted to 106/mL。
Film-making：It is prepared by first layer gel：The μ l of 1.5%NMA 200 of melting are dropped on frosted slide, close the lid glass
Piece, 4 DEG C of refrigerators place 10min.It is prepared by second layer gel：By 1：8 ratio is mixed at 37 DEG C by above-mentioned cell suspension and 1%LMA
It is even, the above-mentioned μ L of mixed liquor 80 are added dropwise on first layer gel rapidly, covered, 4 DEG C of refrigerators place 10min.
Cracking：Remove cover glass, by slide it is horizontal immerse Fresh cell cracking (NaCL containing 2.5mol/L,
100mol/L EDTA, 10mol/L Tris, 1%Tri tonX-100 and 10%DMSO, pH=10) in, crack 1.5h.
Electrophoresis：Slide is taken out from lysate, lysate is blotted and is placed in Horizontal electrophoresis tank anode tap, in electrophoresis tank
The alkaline electrophoresis buffer (NaOH 300mmol/L, EDTA 1mmol/L, pH=13) of Fresh is added, places 20min solutions
Spiral.Under voltage 18V, current strength 200mA, electrophoresis 30min.
Neutralize and dye：Film is embathed 3 times in 0.4mol/L Tris-HCL (pH7.5) buffer solution after electrophoresis, every time
5min, to film on the μ l of 20g/mL Ethidum Eremides (EB) dye liquor 50 are added dropwise, covered, observe result after 5min.
Observation and evaluation：The diagosis under fluorescence microscope (excitation wavelength 549nm, launch wavelength 590nm).Image is directly defeated
Enter computer, save as tif files.50 randomly selected cells are observed, utilize CAPS software analysis.
In Comet Assay, 10,50,100 μm of ol/L tanshinone IIA and positive control H2O2 (20 μm of ol/L) with
After HepG2 cells contactings 1h, each group cell survival rate is more than 90%, the cell of no apoptosis.After EB dyeing, in fluorescence microscopy
Under mirror, the unbroken DNA of HepG2 cells only has a complete head, and the DNA of fracture forms hangover, with tanshinone IIA agent
The increase of amount, hangover are more and more obvious.It is shown in Table 1.
Influence of the tanshinone IIA of table 1 to HepG2 DNA chain breakage degree
| Tested material | DNA hangovers rate (%) | Tail length (μm) |
| 0 | 12.44±1.36 | 3.16±0.35 |
| 10 μm of ol/L of tanshinone IIA | 16.54±1.01* | 4.10±0.10** |
| 50 μm of ol/L of tanshinone IIA | 21.66±2.55** | 17.17±3.39** |
| 100 μm of ol/L of tanshinone IIA | 34.35±11.63** | 29.50±13.55 |
| H2O2(20μmol/L) | 28.68±7.58** | 21.31±3.95** |
Note：**With comparing P ＜ 0.01,*P ＜ 0.05
Fig. 3 shows Comet Assay research tanshinone IIA to HepG2 cytogenetic toxicity results.A is that tanshinone IIA is real
Test group；B. negative control group.It can be seen that tanshinone IIA has significant Genotoxic Effect to HepG2.
All it is incorporated as referring in this application in all documents that the present invention refers to, it is independent just as each document
It is incorporated as with reference to such.In addition, it is to be understood that after the above-mentioned instruction content of the present invention has been read, those skilled in the art can
To be made various changes or modifications to the present invention, these equivalent form of values equally fall within the model that the application appended claims are limited
Enclose.
Claims (10)
- A kind of 1. method for detecting the composition in natural products with genetoxic, it is characterised in that including step：Prepare reaction system and carry out temperature and incubate reaction, the reaction system includes：Sample to be tested, deoxyribonucleoside and isotope marks Deoxyribonucleoside；After terminating reaction, the formation of DNA adduct in reaction system is detected using tablets by HPLC-MS；Wherein Including using the molecular weight of various materials in tablets by HPLC-MS acquisition reaction system；If in mass spectrogram Quasi-molecular ion difference be present for n material X and material X ', n is the molecule of deoxyribonucleoside and the deoxyribonucleoside of isotope marks Difference is measured, then preliminary judgement material X is the composition that can form DNA adduct.
- 2. the method as described in claim 1, it is characterised in that methods described includes step：(1) preliminary screening of DNA adductReaction system is prepared, the reaction system includes：Sample to be tested, hepatomicrosome or liver S9, deoxyribonucleoside, isotope marks Deoxyribonucleoside and reduced Coenzyme II；The reaction system is incubated under the conditions of 37 DEG C and reacted；Isometric ice acetonitrile terminating reaction is added after the completion of reaction；HPLC-MS/MS detects the formation of DNA adduct.
- 3. method as claimed in claim 2, it is characterised in that methods described also includes step (2)：(2) preparing includes the reactant of sample to be tested, hepatomicrosome or Animal Liver S9, calf thymus DNA and reduced Coenzyme II System；The reaction system is incubated under the conditions of 37 DEG C and reacted；DNA is extracted after the completion of reaction；Enzymolysis or sour water solution, solid phase Extraction, HPLC-MS/MS detect the formation of DNA adduct；The sample to be tested is the material that can form DNA adduct that step (1) detection obtains.
- 4. method as claimed in claim 2, it is characterised in that in the reaction system of the step (1), the content of each component is such as Under：The μ g/ml of sample to be tested concentration >=20；Hepatomicrosome protein concentration >=0.1mg/ml；The μ g/ml of deoxyribonucleoside content >=20；Together The μ g/ml of deoxyribonucleoside content >=20 of position element mark；Reduced Coenzyme II or the content of oxidized coenzyme-II 0.5-2.0mmol/L.
- 5. method as claimed in claim 3, it is characterised in that in the reaction system of the step (2), the content of each component is such as Under：The μ g/ml of sample to be tested concentration >=20；Hepatomicrosome protein concentration >=0.1mg/ml；Calf thymus DNA content >=0.5mg/ ml；Reduced Coenzyme II content is 1mmol/L.
- 6. the method as described in claim 1, it is characterised in that the isotope is selected from：Carbon isotope, nitrogen isotope, oxygen are same Position element, hydrogen isotope or its combination；Preferably, the deoxyribonucleoside includes desoxyadenossine, deoxyguanosine, deoxycytidine, AZT and its triphosphoric acid thing.
- 7. method as claimed in claim 6, it is characterised in that the triphosphoric acid thing includes its metal salt.
- 8. the method as described in claim 1, it is characterised in that the sample to be tested is plant extracts.
- 9. method as claimed in claim 8, it is characterised in that the plant extracts is Chinese medicinal plant extract.
- 10. there is the kit of genetoxic composition in the method detection Chinese medicine or extract in a kind of usage right requirement 1, it is special Sign is that the kit includes：Hepatomicrosome or liver S9, deoxyribonucleoside, the deoxyribonucleoside and reduced form of isotope marks Reagent needed for codehydrogenase Ⅱ or oxidized coenzyme Ⅱ and reduced Coenzyme II regenerative system.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1420984A (en) * | 2000-03-16 | 2003-05-28 | 有限会社环境技术研究所 | Simple method of biologically evaluating natural and artificial chemicals by using DNA injury index and apparatus therefor |
| CN101568548A (en) * | 2006-09-29 | 2009-10-28 | 詹森药业有限公司 | Isotopically labeled trapping agent and method for identifying reactive metabolites |
| CN103808836A (en) * | 2014-03-14 | 2014-05-21 | 国家烟草质量监督检验中心 | Determination method of 3-alkylated adenine DNA adducts in urine |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004109279A2 (en) * | 2003-06-09 | 2004-12-16 | Pfizer Products Inc. | Sensitive and selective in vitro assay for the detection of reactive drug intermediates |
-
2015
- 2015-08-17 CN CN201510504987.5A patent/CN105424821B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1420984A (en) * | 2000-03-16 | 2003-05-28 | 有限会社环境技术研究所 | Simple method of biologically evaluating natural and artificial chemicals by using DNA injury index and apparatus therefor |
| CN101568548A (en) * | 2006-09-29 | 2009-10-28 | 詹森药业有限公司 | Isotopically labeled trapping agent and method for identifying reactive metabolites |
| CN103808836A (en) * | 2014-03-14 | 2014-05-21 | 国家烟草质量监督检验中心 | Determination method of 3-alkylated adenine DNA adducts in urine |
Non-Patent Citations (6)
| Title |
|---|
| Development of a Method for Quantification of Acrolein-Deoxyguanosine Adducts in DNA Using Isotope Dilution-Capillary LC/MS/MS and Its Application to Human Brain Tissue;Xinli Liu et al;《Anal.Chem.》;20050915;第77卷(第18期);第5983页右栏第2段，第5984页右栏第4段-第5985页右栏第1段 * |
| Metabolism of Methyleugenol in Liver Microsomes and Primary Hepatocytes: Pattern of Metabolites, Cytotoxicity, and DNA-Adduct Formation;Alexander T. Cartus et al;《Toxicological Sciences 》;20120518;摘要，第10页第16行-第11页第35行 * |
| N‑Hydroxycinnamide Derivatives of Osthole Presenting Genotoxicity and Cytotoxicity against Human Colon Adenocarcinoma Cells in Vitro and in Vivo;Ling-Yu Liu et al;《Chem.Res.Toxicol.》;20131015;第26卷;摘要 * |
| Potential Genotoxicity of Traditional Chinese Medicinal Plants and Phytochemicals:An Overview;Jue Zhou et al;《PHYTOTHERAPY RESEARCH》;20130219;第27卷;第1745-1755页 * |
| Quantitation of DNA Adductsby Stable Isotope Dilution Mass Spectrometry;Natalia Tretyakova et al;《Chem.Res.Toxicol.》;20120724;第25卷;第2007-2035页 * |
| 丹参酮ⅡA对大鼠肝癌细胞CBRH7919的抑制及凋亡诱导作用;周静 等;《中药材》;20100630;第33卷(第6期);摘要 * |
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