CN102707004A - Novel method for researching phase II metabolism of flavone compound by using model organism zebra fish - Google Patents
Novel method for researching phase II metabolism of flavone compound by using model organism zebra fish Download PDFInfo
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Abstract
The invention discloses a novel method for researching the phase II metabolism of a flavone compound by using a model organism zebra fish. In the novel method, the zebra fish is selected as an object of drug metabolism research, experiment grouping design is carried out through an optimized experiment grouping method, drug administration is carried out by adopting an optimized drug administration mode, a metabolite of the flavone compound metabolized by the zebra fish is extracted by adopting an optimized method, the metabolite is analyzed by especially adopting an optimized analysis method, thus, the integral experimental method is high in operability and high in accuracy degree of an experimental result, the real conditions of the phase II metabolism of drugs in vivio and especially the real conditions of the phase II metabolism of the flavone compound in vivio can be objectively reflected, and the defect that a general in-vitro metabolism experiment is difficult to reflect a comprehensive result of in-vivo metabolism and the defects of large used drug quantity and high labor intensity of a general in-vivo metabolism experiment can be overcome. The experimental method has the advantages of high repeatability, small quantity of needed experimented drugs, low cost, low labor intensity, wide application range and high work efficiency by carrying out experiment research in a batched way.
Description
Technical field
The present invention relates to the metabolism research method of a compounds, be specifically related to a kind of new method with the metabolism of model organism zebra fish research chromocor compound II phase.
Background technology
Flavone compound (flavonoids) is the compound with 2-phenyl chromone (flavone) structure.Flavone compound usually is combined into the form existence of glycoside or free state (aglycon) with sugar in plant.All contain flavone compound in most plants; Has important biological; As protect cardiovascular system, antibiotic and antiviral, antitumor activity, anti-inflammatory, anti-oxidant and protect the liver isoreactivity, but the flavone compound bioavilability is low, is difficult to reach onset concentration in vivo.The II of flavones glucuronidation in vivo and sulphation association reaction metabolism mutually is a low major reason of flavones bioavilability.
Drug metabolism is divided into I phase metabolism (oxidation, reduction, hydrolysis etc.) and II metabolism mutually (association reaction) usually; The chromocor compound particularly metabolism of flavone aglycone is main with the metabolism of II phase, and research method also adopts common drug metabolism study method: experimental method and experiment in vitro method in the body.Experimental method in the body: behind the animals administer,, get alimentary canal each several part content and analyze within a certain period of time with sacrifice of animal; And inspection blood, urine, ight soil, bile; Detect the structural change of metabolin, the research drug metabolism and transformation, and infer its metabolic process in alimentary canal.Medicine conversion in vivo can be objectively reacted in the internal metabolism test, but general drug dose is bigger, is unfavorable for the tachymetabolism research of small amount of drug; Component content is humble in some body, even adopt the modern analysis means also to be difficult to satisfy the detection demand of complex system in the body sometimes, also is limited and strengthen dose or means such as further enrichment, purifying sample to result's improvement; Internal metabolism labour intensity is relatively large, often needs multi agent cooperation to accomplish.Experiment in vitro method: external liver cell, liver or intestines microsome and glucuronic acid desmoenzyme, sulfate transferase metabolism.Liver cell metabolism: get LH, liver or enterocyte microsome or glucuronic acid desmoenzyme, sulfate transferase and medicine and hatch altogether, the kind and the content of inspection prototype composition and metabolin thereof.Though the experiment in vitro method has more effectively been simulated the medicine different links of metabolism in vivo, is of value to richness and increases, prepares metabolic product, has broken away from the effect of complete metabolism system to medicine, is difficult to be embodied in the synthesis result of body metabolism; The experiment in vitro conditional request is higher relatively, needs to guarantee the activity of liver cell, microsome and enzyme, and common laboratory is difficult to carry out.Therefore, set up a kind of comprehensive effect that can be embodied in the body metabolism, again can realization condition simple, low labour intensity, the high flux metabolism research method that compound amount is few is significant.
Zebra fish is a kind of fabulous model organism, is to replace the good test model fish as research object such as frog, fruit bat, small white mouse.The expense that zebra fish is fed and keeps is more cheap, and the requisite space place is little, is easy to indoor large-scale breeding.Long 3～the 4cm of adult fish, aquaculture cost is merely the 0.1%-1% that supports mouse.Zebra fish is suitable with the similarity and the mouse of human gene, and on protein level, the homology of its key position almost is 100%, so available zebra fish simulating human disease [P.Goldsmith, Curr Opin Pharmacol, 4,504-12 (2004)].At present, what people were successful sets up many human diseases models with zebra fish, like the disease [S.Sumanas and S.Lin, DDT:TARGETS, 3,89-96 (2004) .] of aspects such as nervous system, the circulation system, the sense of hearing, vision, cancer.A kind of method with zebra fish research drug toxicity is disclosed like Chinese patent 200810019040.5; The formation that patent 200780051025.2 discloses a kind of organ that carries out through stem cell nutrition with and the renovation process of alcohol damaged organ, 200310108710.8 disclose diseases such as utilizing zebra fish gene therapy paraplegia; We disclose 201010258459.3 and have utilized in the zebra fish research red sage root tanshinone IIA, Cryptotanshinone and the Tanshinone I new method of metabolism mutually; The hydroxylation of discovery tanshinone IIA, Cryptotanshinone and Tanshinone I and the I of dehydrogenation reaction be metabolic product mutually; The method is set up system and had the Cytochrome P450 s related enzyme systems of drug metabolism according to zebra fish: the zebra fish P450s gene of being cloned into at present mainly comprises: the member of Cyp1A, Cyp2K6, Cyp3a65 and Cyp3C1, Cyp11a1, Cyp19, Cyp26A1, Cyp26B1 and the several families of Cyp26D1; The homology of they and human corresponding gene is about 40%～73% [P.Collodi, C.L.Miranda, X.Zhao; D.R.Buhler; D.W.Barnes.Xenobiotica, 24 (6), 487-493 (1994)].In recent years research shows that zebra fish still has II phase metabolic enzyme [Y.Morcillo, G.Janer, S.C.M.O ' Hara, D.R.Livingstone, C.Porte.Environmental Toxicology and Chemistry, 23 (4), 990-996 (2004); D.V.Almeida; B.F.Nornberg, L.A.Geracitano, D.M.Barros; J.M.Monserrat; L.F.Marins.Fish Physiol Biochem.36 (3): 347-53 (2010)], this provides foundation for the II phase metabolism that zebra fish is used for medicine, and domestic research in this respect still belongs to blank.
Summary of the invention
Goal of the invention: technical matters to be solved by this invention is the deficiency that overcomes prior art; Provide a kind of easy to operate, cost is low, compound amount is few, labour intensity is low, and the biological zebra fish of quick mode that only needs just can to carry out in common laboratory is studied the new method of medicine such as the metabolism of chromocor compound II phase.
Technical scheme: in order to realize above purpose, the technical scheme that the present invention takes is:
A kind of new method with model organism zebra fish research medicine such as the metabolism of chromocor compound II phase, it may further comprise the steps:
The adult fish of (1) getting zebra fish is put in the container that fills water; Be divided into blank solvent group, blank fish group, blank chromocor compound group and chromocor compound fish group, blank fish group and chromocor compound fish group zebra fish quantity equate, will be tried chromocor compound and be made into desired concn; Join in the WS of chromocor compound fish group; Blank fish group adds the solvent of equivalent, and the blank solvent group only adds the equivalent solvent and do not put the equivalent chromocor compound that zebra fish, blank chromocor compound group add the solvent dissolving and do not put zebra fish, 0h～48h after the zebra fish of chromocor compound fish group is exposed to soup; Get the fish soup in different time points, place in-70 ℃ of refrigerators; Get fish soup 24h or the 48h time point takes out fish at last,, put to death with pure water washing 2～3 times rapidly; Remove fin and fish phosphorus, weigh, place in-70 ℃ of refrigerators; Blank solvent group, blank fish group and blank chromocor compound group are taken a sample with method when 0h, 24h or 48h, and be subsequent use;
(2) get each time point fish soup freeze drying that step (1) obtains or nitrogen, air blow drying; Residue adds an amount of 50%-100% methyl alcohol or acetonitrile dissolving; Cross 0.45 μ m or 0.22 μ m filter membrane or the centrifugal 10～20min of 15000 commentaries on classics/min, get filtrating or supernatant and carry out high-efficient liquid phase chromatogram HPLC analysis or high performance liquid chromatography and mass spectrometry LC-MS analysis; Get 24h or 48h time point zebra fish fish body shreds, weigh, homogenate, homogenate adds methyl alcohol or acetonitrile removes albumen, and 3000～4000 commentaries on classics/min are centrifugal, and 10～20min gets supernatant; Or the organic solvent extraction drug ingedient is directly used in the centrifugal back of homogenate; Centrifuging and taking organic solvent extraction liquid, combining extraction liquid, will except that albumen centrifuged supernatant or extract with nitrogen or air blow drying; Residue adds 50%～100% dissolve with methanol; Cross 0.45 μ m or 0.22 μ m filter membrane or the centrifugal 10～20min of about 15000 commentaries on classics/min,, filtrating or supernatant carry out the HPLC analysis or LC-MS analyzes.
As preferred version; Above-described new method with model organism zebra fish research drug metabolism; Wherein the blank solvent group in the step (1), blank fish group, blank chromocor compound group and chromocor compound fish are organized all parallel 3 groups of establishing, and guarantee the science of experiment.
As preferred version, above-described new method with model organism zebra fish research drug metabolism, the zebra fish of step (1) Chinese traditional medicine fish group is exposed to 0h behind the soup, 2h; 4h, 6h, 8h, 12h; 18h, the 24h time point is got the fish soup, in case of necessity can be with extending to 48h sample time.Adopt continuous time interval point take a sample can abundant clear and definite medicine metabolic way and metabolic characteristic, for the clinical application of instructing medicine and the selection of pharmaceutical dosage form provide scientific basis.
As preferred version, wherein the described solvent of step (1) is the water or the WS that contains 0～1% dimethyl sulfoxide (DMSO) cosolvent.For water-soluble relatively poor medicine, adopt the dimethyl sulfoxide (DMSO) hydrotropy, can effectively improve the water-soluble of medicine, make medicine be dispersed in water-soluble in.
As preferred version, above-described new method with the metabolism of model organism zebra fish research chromocor compound, step (2) is according to the character of being analyzed medicine; Receive the reagent thing according to dissolving of rule of similarity selective solvent or extraction, add 50%-100% methyl alcohol or acetonitrile dissolving like the soup residue, zebra fish fish body shreds; Weigh, homogenate, homogenate adds methyl alcohol or acetonitrile removes albumen; Or the centrifugal back of homogenate is directly with solvent such as ethyl acetate, chloroform or dichloromethane extraction drug ingedient extraction drug ingedient, centrifuging and taking organic solvent layer extract, combining extraction liquid; Nitrogen or air blow drying, residue add the 50%-100% dissolve with methanol.
The present invention has investigated the soup recovered under reduced pressure to dried, direct freeze drying and nitrogen or three kinds of methods of air blow drying; The result shows the loss and the error that the direct freeze-drying of soup or nitrogen, air blow drying can be reduced to greatest extent the sample preparation process; Experimental result is more accurate; Therefore step (2) is preferably with each time point fish soup freeze drying or nitrogen, air blow drying; The dry residue of 50%-100% dissolve with methanol, supernatant carries out high-efficient liquid phase chromatogram HPLC analysis or high performance liquid chromatography and mass spectrometry LC-MS analysis after filtration or the centrifugal treating then.
New method with the metabolism of model organism zebra fish research chromocor compound II phase of the present invention, wherein step (2) adopts Agilent 1100 type series of high efficiency liquid chromatographs or adopts highly sensitive Waters Alliance 2695-ZQ 2000 high performance liquid chromatography-GC-MS to measure in the zebra fish WS situation of change of the content and structure of of chromocor compound and metabolin in the chromocor compound and metabolin and zebra fish in-vivo tissue.
As preferred version, the new method with the metabolism of model organism zebra fish research chromocor compound II phase of the present invention, the reagent thing that receives of screening can be the compound etc. that II phase metabolic response can take place for flavone aglycone, flavonoid glycoside and other.Like 5-flavonol, 7-flavonol or Chrysin.
5-flavonol (5-hydroxyflavone) and 7-flavonol (7-hydroxyflavone) are important monohydroxy chromocor compound; The 5-flavonol has antioxygenic property, photic radiation is had stability, excitatory state Proton-Transfer Reactions, and pharmacological action such as anti-coronary spasm, coronary ischemia crisis; Some 7-bit strips have the flavone compound of hydroxyl to have hemangiectasis, increase the heart coronary flow, increase cerebral blood flow (CBF), anti-platelet aggregation and antiinflammatory action; Chrysin (Chrysin) is 5, and the two flavonols of 7-have pharmacologically active widely, like antitumor, radio therapy sensitization effect, inhibition aromatase activity, anti-inflammatory, anti-oxidant, control cardiovascular and cerebrovascular disease.
Therefore study the metabolism situation of 5-flavonol, 7-flavonol or Chrysin, can effectively understand the internal metabolism situation of flavone compound.
Different with mammals such as mouse, rat, dogs; The volume of zebra fish is less, and oral or drug administration by injection mode is difficulty very, therefore; The present invention will be dissolved in the water that zebra fish lives by the reagent thing in the step (1); Zebra fish can autonomous continuous from solution, absorb receive the reagent thing and carry out metabolism in vivo, the metabolin of medicine also can along with the excreta of zebra fish by continuous being discharged in the water, so just can change the part metabolic information of grasping medicine through the composition of analytical solution herb liquid; Zebra fish adult fish is about 3～4cm; Its body inner blood amount denier is difficult to realize blood sampling, analyzes the feasibility existing problems of blood sample through getting blood; Also can grasp the situation of change of medicine in the fish body and composition in the whole fish analyzed; Like this, can through timing sampling analyze medicine in the zebra fish body and external Changing Pattern come comparatively comprehensively to explore the metabolism situation of zebra fish, this method simple possible to medicine.
Beneficial effect: the new method with the metabolism of model organism zebra fish research flavone compound provided by the invention is compared with prior art and is had the following advantages:
New method with the metabolism of model organism zebra fish research flavone compound II phase provided by the invention is selected the object of zebra fish as drug metabolism study for use, through the experiment group technology of optimizing; Guarantee the accuracy of experimental result, especially to the drug administration mode preferred, medicine is preferred through zebra fish metabolism method for post extraction and analytical approach, whole experimental technique is workable; Can objectively react medicine true metabolism situation in vivo, the experimental result accuracy is high, and it is harsh to overcome general external metabolism experiment condition; And be difficult to be embodied in the shortcoming of body metabolism synthesis result, and can overcome the shortcoming that general mammal internal metabolism experiment institute dosage is big, labour intensity is big, and experimental technique is repeatable strong; It is especially required that to be tried medication amount few; Cost is low, and labour intensity is low, has wide range of applications; And the research that can experimentize in batch, high efficiency.
Description of drawings
The soup that Fig. 1 is a 5-flavonol soup when zebra fish effect 24h and the extraction ion flow graph of fish body total ion current figure and 5-flavonol and its metabolic product.
Fig. 2 is the mass spectrogram of 5-flavonol.
Fig. 3 is the mass spectrogram of 5-flavonol glucuronide conjugate 1.
Fig. 4 is the mass spectrogram of 5-flavonol glucuronide conjugate 2.
Fig. 5 is the mass spectrogram of 5-flavonol sulfuric acid bond.
The soup that Fig. 6 is a 7-flavonol soup when zebra fish effect 24h and the extraction ion flow graph of fish body total ion current figure and 7-flavonol and its metabolic product.
Fig. 7 is the mass spectrogram of 7-flavonol.
Fig. 8 is the mass spectrogram of 7-flavonol glucuronide conjugate.
Fig. 9 is the mass spectrogram of 7-flavonol sulfuric acid bond.
The soup that Figure 10 is the Chrysin soup when zebra fish effect 24h and the extraction ion flow graph of fish body total ion current figure and Chrysin and its metabolic product.
Figure 11 is the mass spectrogram of Chrysin.
Figure 12 is the mass spectrogram of Chrysin glucuronide conjugate 1.
Figure 13 is the mass spectrogram of Chrysin glucuronide conjugate 2.
Figure 14 is the mass spectrogram of Chrysin sulfuric acid bond.
Embodiment
According to following embodiment, can understand the present invention better.Yet, those skilled in the art will readily understand that the described concrete material proportion of embodiment, process conditions and result thereof only are used to explain the present invention, and the present invention that should also can not limit in claims to be described in detail.
The metabolism of embodiment one usefulness model organism zebra fish research 5-flavonol
1. material and instrument
Receive the reagent thing: the 5-flavonol
Compound method: take by weighing and be equivalent to 5-flavonol 1～2mg and be dissolved in the 1ml dimethyl sulfoxide (DMSO), add 250ml Robust pure water, shake up.
Animal: zebra fish is provided by model organism research institute of Nanjing University.
Reagent: acetonitrile, formic acid are chromatographically pure (Germany, Merck company), dimethyl sulfoxide (DMSO) (DMSO, Chemical Reagent Co., Ltd., Sinopharm Group), ultrapure water Robust pure water.
Instrument: Agilent 1100 type series of high efficiency liquid chromatographs comprise G1311A quaternary gradient pump, G1313A automatic sampler, G1316A column oven, G1315B DAD detecting device; Chemstation 6.01 chromatographic work stations (U.S., Agilent company).Waters Alliance 2695-ZQ 2000 liquid chromatograph-mass spectrometers comprise two high-pressure pumps, automatic sampler, column oven, electro-spray ionization interface, 2695 type liquid chromatographs, Masslynx 4.0 chromatographic work stations (U.S., Waters company).METTLER TOLEDO AB135-S analytical balance (Switzerland, METTLERTOLEDO company), KQ3200DE type numerical control supersonic washer (Kunshan Ultrasonic Instruments Co., Ltd.).LABCONCO freeze drier (U.S., LABCONCO company), TGL-16G desk centrifuge (Anting Scientific Instrument Factory, Shanghai).
2. experimental technique
2.1 administration and sampling method:
Get zebra fish, place the brown bottle that contains 30mL solution respectively, be divided into 2 groups, every group of 5 fishes.Wherein 1 group is 0.4%DMSO pure water (blank fish group), and another group is 5-flavonol 0.4%DMSO pure water solution (medicine fish group).Establish blank solvent group (0.4%DMSO pure water) and blank drug group (0.4%DMSO 5-flavonol pure water solution) simultaneously.The medicine fish is organized respectively at 0h behind the zebra fish exposure soup, 2h, and 4h, 6h, 8h, 12h, 18h, every fish taking liquid of 24h time point 0.4ml, each time point soup mixes respectively, places in-70 ℃ of refrigerators.Simultaneously fish is taken out in art sub-sampling time point 24h, wash 3 times rapidly, put to death, remove fin and fish phosphorus, weigh, place (parallel 3 experiments) in-70 ℃ of refrigerators with pure water.The blank solvent group, blank fish group and blank drug group are taken a sample with method during 24h in 0h.
2.2 sample preparation:
Soup is handled: with each 2mL of different time points fish soup, and freeze drying, residue adds the 0.5mL90% dissolve with methanol, crosses 0.22 μ m filter membrane, and filtrating sample introduction 20 μ L carry out HPLC-MS and analyze.
The zebra fish body is handled: mix between the zebra fish parallel-group when getting 24h, shred, take by weighing 1g; Add the homogenate of 5mL physiological saline, the centrifugal 10min of 3500r/min, supernatant add 4 times of amount methyl alcohol vortex mixings and remove albumen; The centrifugal 10min of 3500r/min, supernatant nitrogen dries up, and residue adds the solution that the 1mL90% dissolve with methanol is processed 1g fish/mL; Cross 0.22 μ m filter membrane, filtrating sample introduction 20 μ L carry out HPLC-MS and analyze.
2.3 analysis condition:
Liquid-phase condition: Agilent C
18Post (5 μ m, 150mm * 4.6mm) and C18 pre-column (4.6 * 12.5mm ID, 5 μ m); Column temperature: 32 ℃; Moving phase: A is Robust pure water (with a mass spectrometry time change into 0.05% formic acid water), and B is acetonitrile (with a mass spectrometry time change into 0.05% formic acid acetonitrile), and A ten B=100% adopt gradient elution: 0～5min; 90%B, 5～7min, 90～80%B, 7～20min; 80～75%B, 20～35min, 75～25%B, 35～37min; 25～90%B, 37～40min, 90%B; Flow velocity: 1.0mL/min; Full wavelength scanner detects; Writing time 40min; Sample size is 20 μ L.
Mass spectrum condition: capillary voltage 2.50kV, taper hole voltage 35V, dry gas flow velocity 320L/h, 120 ℃ of ion source temperatures, 310 ℃ of auxiliary temperature degree; The ion detection mode: full scan detects (SCAN), and ion polarity: positive ion (positive) and negative ion (positive) detect simultaneously, extracts ion flow (TIC): [M-H]
-, [M+H]
+[M+Na]
+, ionization mode: pneumatic auxiliary electro-spray ionization (ESI), sweep limit: 100-800m/z.
3. experimental result
Adopt HPLC-MS method positive and negative ion pattern to detect the metabolic product of 5-flavonol after the zebra fish effect simultaneously, wherein positive ion mode is sensitive.As shown in Figure 1,5-flavonol original shape composition during interior 5-flavonol zebra fish soup of discovery 24h or zebra fish body are interior and 5-flavonol glucuronidation, Sulfated II phase metabolic product (concrete experimental result is seen table 1).The existing oral 5-flavonol of rat metabolism research document shows that 5-flavonol metabolic pathway in animal body is mainly glucuronidation, detects 5-flavonol grape alditol bond in the rat body; 5-flavonol grape alditol bond [the CS Shia with consistency that this and zebra fish act on; SY Tsai, SC Kuo, YC Hou and PD Chao.J Agric Food Chem; 2009,57 (1): 83-89; Wei Yingjie, Jiang Jinsheng, Tan Xiaobin; Chen Bin, Liu Wei, Hu Chunping; Ma Shiping, Jia Xiaobin. contemporary Chinese is used pharmacy, 2012; 29 (1): 60-63.], this research also detects 5-flavonol sulfuric acid bond under negative ion mode in addition, shows that positive and negative ion pattern while detection mode can more comprehensively detect metabolic product.Adopt provided by the invention with model organism zebra fish research 5-flavonol metabolism experimental result accurately and reliably, experimental technique is feasible.
The metabolic product of table 15-flavonol after the zebra fish effect identified
The metabolism of embodiment dual-purpose model organism zebra fish research 7-flavonol
1. material and instrument
Receive the reagent thing: 7-flavonol compound
Compound method: take by weighing and be equivalent to 7-flavonol 1～2mg and be dissolved in the 1ml dimethyl sulfoxide (DMSO), add 250ml Robust pure water, shake up.
Animal, reagent, instrument: with embodiment one.
2. experimental technique
2.1 administration and sampling method:
Get zebra fish, place the brown bottle that contains 30mL solution respectively, be divided into 2 groups, every group of 5 fishes.Wherein 1 group is 0.4%DMSO pure water (blank fish group), and another group is 7-flavonol 0.4%DMSO pure water solution (medicine fish group).Establish blank solvent group (0.4%DMSO pure water) and blank drug group (0.4%DMSO 7-flavonol pure water solution) simultaneously.The medicine fish is organized respectively at 0h behind the zebra fish exposure soup, 2h, and 4h, 6h, 8h, 12h, 18h, every fish taking liquid of 24h time point 0.4ml, each time point soup mixes respectively, places in-70 ℃ of refrigerators.Simultaneously fish is taken out in last sampling time point 24h, wash 3 times rapidly, put to death, remove fin and fish phosphorus, weigh, place (parallel 3 experiments) in-70 ℃ of refrigerators with pure water.The blank solvent group, blank fish group and blank drug group are taken a sample with method during 24h in 0h.
2.2 sample preparation:
Soup is handled: with each 2mL of different time points fish soup, and freeze drying, residue adds the 0.5mL90% dissolve with methanol, crosses 0.22 μ m filter membrane, and filtrating sample introduction 20 μ L carry out HPLC-MS and analyze.
The zebra fish body is handled: mix between the zebra fish parallel-group when getting 24h, shred, take by weighing 1g; Add the homogenate of 5mL physiological saline, the centrifugal 10min of 3500r/min, supernatant add 4 times of amount methyl alcohol vortex mixings and remove albumen; The centrifugal 10min of 3500r/min, supernatant nitrogen dries up, and residue adds the solution that the 1mL90% dissolve with methanol is processed 1g fish/mL; Cross 0.22 μ m filter membrane, filtrating sample introduction 20 μ L carry out HPLC-MS and analyze.
2.3 analysis condition: with embodiment one.
3. experimental result
Adopt HPLC-MS method positive and negative ion pattern to detect the metabolic product of 7-flavonol after the zebra fish effect simultaneously, wherein negative ion mode is sensitive.As shown in Figure 6,7-flavonol original shape composition during interior 7-flavonol zebra fish soup of discovery 24h or zebra fish body are interior and 7-flavonol glucuronidation, sulphation II phase metabolic product (concrete experimental result is seen table 2).Existing rat or people's jejunum microsome or the MC 7-flavonol of rat intestine metabolism research document show; 7-flavonol metabolic pathway in animal body is mainly glucuronidation or sulphation; Main metabolites is 7-flavonol grape alditol bond and 7-flavonol sulfuric acid bond, mating type metabolic product [CS Shia, SY Tsai with consistency that this and zebra fish act on; SC Kuo; YC Hou and PD Chao.J Agric Food Chem, 2009,57 (1): 83-89; Wei Yingjie, Jiang Jinsheng, Tan Xiaobin, Chen Bin, Liu Wei, Hu Chunping, Ma Shiping, Jia Xiaobin. Chinese Hospitals pharmaceutical journal, 2012,32 (4): 1-3; ].Adopt provided by the invention with model organism zebra fish research 7-flavonol metabolism experimental result accurately and reliably, experimental technique is feasible.
The metabolic product of table 27-flavonol after the zebra fish effect identified
The metabolism of embodiment three usefulness model organism zebra fish research Chrysin
1. material and instrument
Receive the reagent thing: Chrysin
Compound method: take by weighing and be equivalent to Chrysin 1～2mg and be dissolved in the 1ml dimethyl sulfoxide (DMSO), add 250ml Robust pure water, shake up.
Animal, reagent, instrument: with embodiment one.
2. experimental technique
2.1 administration and sampling method:
Get zebra fish, place the brown bottle that contains 30mL solution respectively, be divided into 2 groups, every group of 5 fishes.Wherein 1 group is 0.4%DMSO pure water (blank fish group), and another group is Chrysin 0.4%DMSO pure water solution (medicine fish group).Establish blank solvent group (0.4%DMSO pure water) and blank drug group (0.4%DMSO Chrysin pure water solution) simultaneously.The medicine fish is organized respectively at 0h behind the zebra fish exposure soup, 2h, and 4h, 6h, 8h, 12h, 18h, every fish taking liquid of 24h time point 0.4ml, each time point soup mixes respectively, places in-70 ℃ of refrigerators.Simultaneously fish is taken out in last sampling time point 24h, wash 3 times rapidly, put to death, remove fin and fish phosphorus, weigh, place (parallel 3 experiments) in-70 ℃ of refrigerators with pure water.The blank solvent group, blank fish group and blank drug group are taken a sample with method during 24h in 0h.
2.2 sample preparation:
Soup is handled: with each 2mL of different time points fish soup, and freeze drying, residue adds the 0.5mL90% dissolve with methanol, crosses 0.22 μ m filter membrane, and filtrating sample introduction 20 μ L carry out HPLC-MS and analyze.
The zebra fish body is handled: mix between the zebra fish parallel-group when getting 24h, shred, take by weighing 1g; Add the homogenate of 5mL physiological saline, the centrifugal 10min of 3500r/min, supernatant add 4 times of amount methyl alcohol vortex mixings and remove albumen; The centrifugal 10min of 3500r/min, supernatant nitrogen dries up, and residue adds the solution that the 1mL90% dissolve with methanol is processed 1g fish/mL; Cross 0.22 μ m filter membrane, filtrating sample introduction 20 μ L carry out HPLC-MS and analyze.
2.3 analysis condition: with embodiment one.
3. experimental result
Adopt HPLC-MS method positive and negative ion pattern to detect the metabolic product of Chrysin after the zebra fish effect simultaneously, wherein negative ion mode is sensitive.Shown in figure 10, Chrysin original shape composition during interior Chrysin zebra fish soup of discovery 24h or zebra fish body are interior and Chrysin glucuronidation, sulphation II phase metabolic product (concrete experimental result is seen table 3).The MC Chrysin metabolism research of existing people, people's jejunum microsome or rat intestine document shows that the metabolic pathway of Chrysin is mainly glucuronidation or sulphation, and main metabolites is Chrysin grape alditol bond and Chrysin sulfuric acid bond; Mating type metabolic product [the T Walle with consistency that this and zebra fish act on; Y Otake, JA Brubaker, UK Walle; PV Halushka.Br J Clin Pharmacol, 2001; 51 (2): 143-6; YC Wong, L Zhang, G Lin and Z Zuo.Int J Pharm, 2009,366 (1-2): 14-20.].Adopt provided by the invention with model organism zebra fish research Chrysin metabolism experimental result accurately and reliably, experimental technique is feasible.
The metabolic product of table 3 Chrysin after the zebra fish effect identified
Show through above experimental result; Provided by the invention strong with II phase metabolism feasibility in the model organism zebra fish research chromocor compound body; Objective and accurate reaction chromocor compound of ability such as 5-flavonol, 7-flavonol or Chrysin II phase metabolism situation in vivo; Can be the metabolism situation of clinical research medicine, the selection of direct clinical medication and pharmaceutical dosage form provides scientific basis.The present invention also provides the method reference for the II phase metabolism with flavonoids.
Zebra fish is suitable with the similarity and the mouse of human gene, and on protein level, the homology of its key position almost is 100%; So the present invention selects the object of zebra fish as the research of medicine internal metabolism for use, through the experiment group technology of optimizing, the medicine dissolution method is optimized; Drug administration mode preferred, especially drug metabolism method for post extraction and analytical approach preferably, can objectively react medicine true metabolism situation in vivo; It is harsh to overcome general external metabolism experiment condition, and is difficult to be embodied in the shortcoming of body metabolism synthesis result, and the experimental result accuracy is high; And experimental technique is repeatable strong; The required medication amount few (be merely in the present invention rat metabolism a thirtieth) of being tried, labour intensity is low, high efficiency.
The above only is a preferred implementation of the present invention; Should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention; Can also make some improvement and retouching, these improvement and retouching also should be regarded as protection scope of the present invention.
Claims (6)
1. the new method with the metabolism of model organism zebra fish research chromocor compound II phase is characterized in that, may further comprise the steps:
The adult fish of (1) getting zebra fish is put in the container that fills water; Be divided into blank solvent group, blank fish group, blank chromocor compound group and chromocor compound fish group, blank fish group and chromocor compound fish group zebra fish quantity equate, will be tried chromocor compound and be made into desired concn; Join in the WS of chromocor compound fish group; Blank fish group adds the solvent of equivalent, and the blank solvent group only adds the equivalent solvent and do not put the equivalent chromocor compound that zebra fish, blank chromocor compound group add the solvent dissolving and do not put zebra fish, 0h～48h after the zebra fish of chromocor compound fish group is exposed to soup; Get the fish soup in different time points, place in-70 ℃ of refrigerators; Get fish soup 24h or the 48h time point takes out fish at last,, put to death with pure water washing 2～3 times rapidly; Remove fin and fish phosphorus, weigh, place in-70 ℃ of refrigerators; Blank solvent group, blank fish group and blank chromocor compound group are taken a sample with method when 0h, 24h or 48h, and be subsequent use;
(2) get each time point fish soup freeze drying that step (1) obtains or nitrogen, air blow drying; Residue adds an amount of 50%-100% methyl alcohol or acetonitrile dissolving; Cross 0.45 μ m or 0.22 μ m filter membrane or the centrifugal 10～20min of 15000 commentaries on classics/min, get filtrating or supernatant and carry out high-efficient liquid phase chromatogram HPLC analysis or high performance liquid chromatography and mass spectrometry LC-MS analysis; Get 24h or 48h time point zebra fish fish body shreds, weigh, homogenate, homogenate adds methyl alcohol or acetonitrile removes albumen, and 3000～4000 commentaries on classics/min are centrifugal, and 10～20min gets supernatant; Or the organic solvent extraction drug ingedient is directly used in the centrifugal back of homogenate; Centrifuging and taking organic solvent extraction liquid; Combining extraction liquid, will except that albumen centrifuged supernatant or extract with nitrogen or air blow drying, residue adds 50%～100% dissolve with methanol; Cross 0.45 μ m or 0.22 μ m filter membrane or the centrifugal 10～20min of about 15000 commentaries on classics/min, filtrating or supernatant carry out the HPLC analysis or HPLC-MS analyzes.
2. the new method with the metabolism of model organism zebra fish research chromocor compound according to claim 1 is characterized in that,
Described high performance liquid chromatography and mass spectrometry HPLC-MS analysis condition are:
Liquid-phase condition: specification is 5 μ m, the Agilent C of 150mm * 4.6mm
18Post and specification are 4.6 * 12.5mm ID, the C18 pre-column of 5 μ m; Column temperature: 32 ℃; Moving phase: A is 0～1.0% formic acid water, and B is 0～1.0% formic acid acetonitrile, and A ten B=100% adopt gradient elution: 0～5min; 90%B, 5～7min, 90～80%B, 7～20min; 80～75%B, 20～35min, 75～25%B, 35～37min; 25～90%B, 37～40min, 90%B; Flow velocity: 1.0mL/min; Full wavelength scanner detects; Writing time 40min; Sample size is 20 μ L;
Mass spectrum condition: capillary voltage 2.50kV, taper hole voltage 35V, dry gas flow velocity 320L/h, 120 ℃ of ion source temperatures, 310 ℃ of auxiliary temperature degree; The ion detection mode: full scan detects, and ion polarity: positive ion and negative ion detect simultaneously, the ionization mode: pneumatic auxiliary electro-spray ionization, sweep limit: 100-800m/z.
3. the new method with the metabolism of model organism zebra fish research chromocor compound according to claim 1 is characterized in that, the zebra fish of chromocor compound fish group is exposed to 0h behind the soup in the step (1); 2h, 4h, 6h; 8h, 12h, 18h; 24h or 48h time point are got the fish soup, when last taking liquid time point 24h or 48h, get the fish body simultaneously.
4. the new method with the metabolism of model organism zebra fish research chromocor compound II phase according to claim 1 is characterized in that the described solvent of step (1) is the water or the WS that contains 0～1% dimethyl sulfoxide (DMSO) cosolvent.
5. the new method with the metabolism of model organism zebra fish research chromocor compound II phase according to claim 1 is characterized in that the described extraction solvent of step (2) is ethyl acetate, chloroform or methylene chloride.
6. the new method with the metabolism of model organism zebra fish research chromocor compound II phase according to claim 1 is characterized in that described chromocor compound is 5-flavonol, 7-flavonol or Chrysin.
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| US20050032070A1 (en) * | 2003-08-05 | 2005-02-10 | Sebastian Raimundo | Polymorphisms in the human gene for CYP2D6 and their use in diagnostic and therapeutic applications |
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