CN104931326A - Method for extracting zebra fish metabolite and application thereof - Google Patents
Method for extracting zebra fish metabolite and application thereof Download PDFInfo
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- CN104931326A CN104931326A CN201510387669.5A CN201510387669A CN104931326A CN 104931326 A CN104931326 A CN 104931326A CN 201510387669 A CN201510387669 A CN 201510387669A CN 104931326 A CN104931326 A CN 104931326A
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Abstract
A method for extracting zebra fish metabolite comprises the following steps: 1 washing a zebra fish sample with normal saline, and immersing the sample into liquid nitrogen for storage and standby application; 2 grinding the sample at the temperature of minus 80 DEG C into homogeneous slurry; 3 adding the slurry sample into an extracting solution which is precooled at the temperature of minus 20 DEG C, conducting microwave-assisted extraction twice, after centrifugation, collecting supernate twice, mixing the supernate, adding sterilized deionized water into the mixed supernate, and after conducting centrifugation, still standing and drying, obtaining a mixed solution; 4 after conducting vacuum drying to the mixed solution, conducting derivatization through a methyl amine hydrochloride oxygen-pyridine solution and N-methyl-N-(trimethylsilane)-trifluoroacetamide two-step method, and obtaining a zebra fish metabolite derivatization sample. The method for extracting the zebra fish metabolite has the advantages of being simple, accurate and easy to carry out, the method is used for the experiment that a zebra fish is exposed to low-concentration nanometer materials, and the sensitivity of zebra fish toxicity detection can be improved.
Description
Technical field
The present invention relates to extracting method and the application thereof of Environmental Toxicological and biomedicine, particularly a kind of zebra fish metabolin.
Background technology
Metabolite analysis is that a kind of important biosome is sought peace the analytical approach of toxicological parameters, by carrying out biological cylinder metabolism-ure qualitative or determining a series of change that quantitative analysis can occur in vivo and the node changed, contribute to the analysis of the situation such as judgement and environmental pollution of bio-toxicity.
Biological subject used in the present invention is zebra fish.Zebra fish have small, be easy to cultivation breeding and low cost and other advantages, the use in fields such as ecotoxicological is very extensive.And there is the similarity of height in zebra fish, therefore can be used as good model animal in heredity, physiology and pharmacological reaction with the mankind.
Summary of the invention
The object of the invention is for above-mentioned technical Analysis, a kind of extracting method and application thereof of zebra fish metabolin are provided, this extracting method technique is simple, easy to implement, for the toxicity test that nano material exposes zebra fish low concentration, sensitiveer and easy relative to other detection method of toxicity obtains more how available data.
Technical scheme of the present invention:
An extracting method for zebra fish metabolin, step is as follows:
1) by the zebra fish sample clean rear correct amount of normal saline flushing, be then immersed in liquid nitrogen rapidly and save backup;
2) above-mentioned zebra fish sample is placed in the mortar filling liquid nitrogen to grind at-80 DEG C of temperature and become homogeneous slurry to zebra fish tissue in 5 minutes;
3) above-mentioned slurry shape zebra fish sample is added in the extract of-20 DEG C of precoolings, microwave abstracting 30 minutes at 40 DEG C of temperature, microwave abstracting power is (sample pot number+2) × 100W, collected after centrifugation first time supernatant, then in solid residue, add the extract of above-mentioned-20 DEG C of identical amount again, continue microwave abstracting 30 minutes at 40 DEG C of temperature, collected after centrifugation second time supernatant;
4) will mix with second time supernatant by supernatant for the first time, sterilizing deionized water is added in mixed liquor, under 4000rpm centrifugal 5 minutes, leave standstill solution layering after 5 minutes, upper solution is methanol/water phase, and lower floor's solution is chloroform phase, is dried up by chloroform nitrogen after two-phase laminated flow, then by methanol/water phase transfer to dry chloroform mutually in, obtain mixed solution;
5) above-mentioned mixed solution is put into freeze drier with after-30 DEG C of--50 DEG C of vacuum drying, two-step approach is adopted to carry out derivatization: first to add methoxy semicarbazide hydrochloride-pyridine solution that concentration is 20mg/mL, vortex 1 minute after sealing, after by its centrifugal 1 minute at 3,000 rpm, at 30 DEG C, temperature is bathed 90 minutes again, then silylating reagent N-methyl-N-(trimethyl silane)-trifluoroacetamide (MSTFA) is added, temperature bath 30 minutes at 37 DEG C, obtains zebra fish metabolin derivatization sample and transfers in the internal lining pipe of applicable GC-MS analysis preserving.
Described step 3) extract is the mixed liquor of methyl alcohol, chloroform and water, in mixed liquor, the volume ratio of methyl alcohol, chloroform and water is 2.5:1:1, and wherein methyl alcohol, chloroform and water are chromatographically pure; The amount ratio of zebra fish sample and extract is 0.3-0.5g:15mL.
Described step 4) in the amount ratio of zebra fish sample and sterilizing deionized water be 0.3-0.5g:500 μ L.
Described step 5) in the amount ratio of zebra fish sample and methoxy semicarbazide hydrochloride-pyridine solution be 0.3-0.5g:50 μ L, the amount ratio of zebra fish sample and silylating reagent is 0.3-0.5g:80 μ L.
An application for the extracting method of described zebra fish metabolin, for zebra fish nanometer toxicity detection.
Advantage of the present invention is: the extraction of this zebra fish metabolin and analytical approach are simply, accurately, easy to implement, is exposed to the experiment of low concentration nano material, the sensitivity of zebra fish toxicity detection can be made to improve for zebra fish.
Accompanying drawing explanation
Fig. 1 is the active comparison diagram of total number born of the zebra fish adult fish of same batch of contamination.
Fig. 2 is the mda content comparison diagram of the zebra fish adult fish of same batch of contamination.
Embodiment
Embodiment 1:
An extracting method for zebra fish metabolin, step is as follows:
1) by the zebra fish sample clean rear correct amount of normal saline flushing, be then immersed in liquid nitrogen rapidly and save backup;
2) above-mentioned zebra fish sample is placed in the mortar filling liquid nitrogen to grind at-80 DEG C of temperature and become homogeneous slurry to zebra fish tissue in 5 minutes;
3) above-mentioned slurry shape zebra fish sample is added in the extract of-20 DEG C of precoolings, extract extract is methyl alcohol, the mixed liquor of chloroform and water, methyl alcohol in mixed liquor, the volume ratio of chloroform and water is 2.5:1:1, wherein methyl alcohol, chloroform and water are chromatographically pure, the amount ratio of zebra fish sample and extract is 0.3g:15mL, microwave abstracting 30 minutes at 40 DEG C of temperature, microwave abstracting power is (sample pot number+2) × 100W, collected after centrifugation first time supernatant, then in solid residue, add the extraction mixed liquor of above-mentioned-20 DEG C of identical amount again, continue microwave abstracting 30 minutes at 40 DEG C of temperature, collected after centrifugation second time supernatant,
4) will mix with second time supernatant by supernatant for the first time, sterilizing deionized water is added in mixed liquor, the amount ratio of zebra fish sample and sterilizing deionized water is 0.3g:500 μ L, under 4000rpm centrifugal 5 minutes, leave standstill solution layering after 5 minutes, upper solution was methanol/water phase, lower floor's solution is chloroform phase, after two-phase laminated flow, chloroform nitrogen is dried up, then by methanol/water phase transfer to dry chloroform mutually in, obtain mixed solution;
5) above-mentioned mixed solution is put into freeze drier with after-30 DEG C of--50 DEG C of vacuum drying, two-step approach is adopted to carry out derivatization: first to add methoxy semicarbazide hydrochloride-pyridine solution that concentration is 20mg/mL, the amount ratio of zebra fish sample and methoxy semicarbazide hydrochloride-pyridine solution is 0.3g:50 μ L, vortex 1 minute after sealing, after by its centrifugal 1 minute at 3,000 rpm, at 30 DEG C, temperature is bathed 90 minutes again, then silylating reagent N-methyl-N-(trimethyl silane)-trifluoroacetamide (MSTFA) is added, the amount ratio of zebra fish sample and silylating reagent is 0.3g:80 μ L, temperature bath 30 minutes at 37 DEG C, obtain zebra fish metabolin derivatization sample and transfer in the internal lining pipe of applicable GC-MS analysis preserving.
Above zebra fish sample is set to first group of sample.
Contrast experiment:
1) second group of sample
The extracting method of this group zebra fish metabolin, step is substantially identical with first group of sample, and difference is: by step 3) in microwave abstracting change into ultrasonic extraction, ultrasonic power is 100W.
2) the 3rd group of sample
The extracting method of this group zebra fish metabolin, step is substantially identical with first group of sample, and difference is: by step 3) in extraction mixed liquor change into chromatographically pure ethanol.
3) the 4th group of sample
The extracting method of this group zebra fish metabolin, step is substantially identical with first group of sample, and difference is:
By step 3) in extraction mixed liquor change into chromatographically pure ethanol, simultaneously microwave abstracting changes into ultrasonic extraction, and ultrasonic power is 100W.
The analysis of above four groups of samples:
Analytical instrument is Agilent gas chromatography tandem mass spectrum.Optimum configurations is as follows:
Gas chromatographic sample introduction parameter is: adopt automatic sampler sample introduction, sample size 1 μ L, injector temperature 230 DEG C, zebra fish adult fish split sampling ratio are 1:10, and juvenile fish is not shunted, carrier gas is helium, flow velocity 2mL/min.
Gas chromatograph parameters is: MDN-35 capillary chromatographic column (30m), temperature program(me) are constant temperature 2min at 80 DEG C, then with the ramp of 15 DEG C/min to 325 DEG C, constant temperature 6min, transmission line temperature are 250 DEG C.
Mass spectrometry parameters is: ion source temperature is 250 DEG C, mass scan range is 70-600m/z, acquisition rate 20 scannings per second, mass spectrum electron bombardment ionization source filament opening time postpone after 170s at chromatographic solvent, the loss of detector voltage 1700-1850V, mass spectrum is set to 0, filament bias current is 70V, instrument hands-off tuning.
The optimum configurations of the spectrogram adjustment of data processing stations:
Spectrogram deconvolution parameter is: the business software Chroma TOF that Leco Corporation carries, baseline are eliminated (Baseline Offset) and arranged 1 (0.5-1), level and smooth (Smoothing) data point 5 (3-7) of spectrogram, peak width (Peak Width) 3s (3s-4s), signal to noise ratio (S/N ratio) S/N (Signal-Tonoise Ratio) 10 (2-15).
Obtain analysis result by the analysis contrast of Nist.08 database, table 1 is that four kinds of Different Extraction Method extract information slip.
Table 1
Table 2
Table 3
Table 4
Table 5
As can be seen from table 1-table 5: the zebra fish metabolin using the method extracting mixed liquor-microwave abstracting to extract on average has 64 kinds, it comprises amino acid, carbohydrate and carbohydrate derivative, oil substances and Small molecular acid and part alkane, alkene alcohols etc., Species distributing is more even, the substance classes that its excess-three kind method is extracted is on average within 60 kinds, and deviation is larger; Amino acid, part lipid, carbohydrate and Small molecular acid difference in kind that four kinds of methods detect is little, but using method one complete detection can go out three kinds of carbohydrates: glucose, mannose and fructose, and all the other methods but and unstable; This method can detect the existence of alkane and alkene in addition, and its excess-three kind method does not detect; In addition as the important substance lactic acid of biological metabolism, only have first method energy stable detection to analyze, all the other methods occur that the chance of lactic acid is random or response is not high.
The application of the extracting method of described zebra fish metabolin, for zebra fish nanometer toxicity detection, method is as follows: select healthy adult zebra fish, it is evenly divided into four groups, wherein three components does not add in the graphene oxide of 0.01mg/L, carbon nano-tube, quantum dot graphene oxide solution three kinds of nano materials and contaminates, another group puts into the salt solution not adding any nano material, changes water feeding every day, brings up 21d for 27 DEG C; Then operate according to the five steps of the extracting method of zebra fish metabolin respectively, will zebra fish metabolin derivatization sample be obtained and transfer in the internal lining pipe of applicable GC-MS analysis preserving.Obtain four groups of samples are analyzed according to method as hereinbefore.Table 2 is that three kinds of different nano material contaminations and control group metabolin after extracting extract situation (unit: peak area/0.1g organizes).
Table 6
Table 7
Table 8
Table 9
Table 10
As can be seen from Table 4: being changed significantly of creatinine in the data using this extracting method to obtain, can be used for the situation assessing fish renal function; In table 1, the marked change of ornithine also can indicate the change of zebra fish internal metabolism situation; But this species diversity is not fairly obvious in the result of the enzyme such as total number born and MDA index measurement alive.
Fig. 1 is the active comparison diagram of total number born of the zebra fish adult fish of same batch of contamination, shows: in figure compared with the zebra fish of control group, through the not significant change of total number born activity of the zebra fish of three kinds of nano material process.。
Fig. 2 is the mda content comparison diagram of the zebra fish adult fish of same batch of contamination, and show in figure: compared with the zebra fish of control group, the mda content through the zebra fish of three kinds of nano material process does not have marked change.
Claims (5)
1. an extracting method for zebra fish metabolin, is characterized in that step is as follows:
1) by the zebra fish sample clean rear correct amount of normal saline flushing, be then immersed in liquid nitrogen rapidly and save backup;
2) above-mentioned zebra fish sample is placed in the mortar filling liquid nitrogen to grind at-80 DEG C of temperature and become homogeneous slurry to zebra fish tissue in 5 minutes;
3) above-mentioned slurry shape zebra fish sample is added in the extract of-20 DEG C of precoolings, microwave abstracting 30 minutes at 40 DEG C of temperature, microwave abstracting power is (sample pot number+2) × 100W, collected after centrifugation first time supernatant, then in solid residue, add the extract of above-mentioned-20 DEG C of identical amount again, continue microwave abstracting 30 minutes at 40 DEG C of temperature, collected after centrifugation second time supernatant;
4) will mix with second time supernatant by supernatant for the first time, sterilizing deionized water is added in mixed liquor, under 4000rpm centrifugal 5 minutes, leave standstill solution layering after 5 minutes, upper solution is methanol/water phase, and lower floor's solution is chloroform phase, is dried up by chloroform nitrogen after two-phase laminated flow, then by methanol/water phase transfer to dry chloroform mutually in, obtain mixed solution;
5) above-mentioned mixed solution is put into freeze drier with after-30 DEG C of--50 DEG C of vacuum drying, two-step approach is adopted to carry out derivatization: first to add methoxy semicarbazide hydrochloride-pyridine solution that concentration is 20mg/mL, vortex 1 minute after sealing, after by its centrifugal 1 minute at 3,000 rpm, at 30 DEG C, temperature is bathed 90 minutes again, then silylating reagent N-methyl-N-(trimethyl silane)-trifluoroacetamide (MSTFA) is added, temperature bath 30 minutes at 37 DEG C, obtains zebra fish metabolin derivatization sample and transfers in the internal lining pipe of applicable GC-MS analysis preserving.
2. the extracting method of zebra fish metabolin according to claim 1, it is characterized in that: described step 3) extract is the mixed liquor of methyl alcohol, chloroform and water, in mixed liquor, the volume ratio of methyl alcohol, chloroform and water is 2.5:1:1, and wherein methyl alcohol, chloroform and water are chromatographically pure; The amount ratio of zebra fish sample and extract is 0.3-0.5g:15mL.
3. the extracting method of zebra fish metabolin according to claim 1, is characterized in that: described step 4) in the amount ratio of zebra fish sample and sterilizing deionized water be 0.3-0.5g:500 μ L.
4. the extracting method of zebra fish metabolin according to claim 1, it is characterized in that: described step 5) in the amount ratio of zebra fish sample and methoxy semicarbazide hydrochloride-pyridine solution be 0.3-0.5g:50 μ L, the amount ratio of zebra fish sample and silylating reagent is 0.3-0.5g:80 μ L.
5. an application for the extracting method of zebra fish metabolin as claimed in claim 1, for zebra fish nanometer toxicity detection.
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| CN106568931A (en) * | 2016-10-31 | 2017-04-19 | 首都医科大学 | Method for detecting cardiotoxicity of nano-material |
| CN115015450A (en) * | 2022-05-26 | 2022-09-06 | 贵州省烟草科学研究院 | Method for analyzing metabolites in soil through microwave derivatization-quasi-target gas chromatography-mass spectrometry |
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