CN102879539A - New method for using model organism zebrafish to research metabolism of glucoside compound asperosaponin VI - Google Patents
New method for using model organism zebrafish to research metabolism of glucoside compound asperosaponin VI Download PDFInfo
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Abstract
The invention discloses a new method for using a model organism zebrafish to research deglycosylated metabolism of glucoside compound asperosaponin VI. The new method comprises the following steps of selecting a zebrafish as a subject of a drug metabolism research, carrying out an experimental grouping design by an optimized experimental grouping method, dosing through an optimized medicine dose way, and extracting asperosaponin VI metabolin metabolized by the zebrafish through an optimized method, especially analyzing the metabolin by the optimized analysis method, so that the entire experimental method is strong in operability and is capable of objectively responding a true deglycosylated metabolism condition of a medicine in vivo, particularly the glucoside compound asperosaponin VI. In addition, the hydroxylated metabolism of the glucoside compound asperosaponin VI is high in accuracy of the experimental results, is capable of overcoming the defect that general in-vitro metabolic experiments are difficult to embody in aggregate results in vitro and the defects that the general in-vivo metabolic experiments are large in dosage and high in labor intensity, and the experimental method is strong in repeatability, required tested drug dose is little, cost is low, the labor intensity is low, a range of application is wide, and the method can be experimentized and researched in batches and working efficiency is high.
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 model organism zebra fish research glycoside compounds asperosaponin VI metabolism.
Background technology
Glucoside compound often refers to the glycosides compound that aglycon and sugar are combined into; such as saponin(e, flavonoid glycoside etc.; it is important natural products; be distributed widely in the plant; have important biologically active, as protection cardiovascular system, antibiotic and antiviral, antitumor activity, anti-inflammatory, anti-oxidant, protect the liver, the nourishing disney and strengthening bone isoreactivity, glucoside compound in vivo usually in body the gastrointestinal bacterial flora effect slough glycosyl; change into time glucosides or even aglycon, improve activity and improve simultaneously bioavilability.
Drug metabolism is divided into I phase metabolism (oxidation, reduction, hydrolysis etc.) and mutually metabolism of II (association reaction) usually, glycoside compounds main metabolic pathway be the hydrolysis reaction of desugar base, this type of reaction is mediated by gastrointestinal bacterial flora usually.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, within a certain period of time with sacrifice of animal, get alimentary canal each several part content and analyze, and check blood, urine, ight soil, bile, detect the structural change of metabolin, the metabolic conversion of drugs, 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 larger, is unfavorable for the tachymetabolism research of a small amount of medicine; Component content is humble in some body, even adopt the modern analysis means sometimes also to be difficult to satisfy the detection demand of complex system in the body, also is limited and strengthen dose or the means such as further enrichment, purifying sample to result's improvement; Internal metabolism labour intensity is relatively large, often needs multi agent cooperation to finish.The experiment in vitro method: gastrointestinal bacterial flora metabolism ight soil temperature incubates method and the stripped alimentary canal content temperature method of incubating is the method for drugs desugar base hydrolysis metabolism in alimentary canal.The metabolism under the effect of alimentary canal flora of many traditional Chinese medicine ingredients, particularly in the enteron aisle glycosidic bond hydrolytic enzyme of bacterium or people's ight soil suspending liquid and medicine under anaerobic temperature incubate, checking kind and the content of prototype composition and metabolin thereof, is that the interior bacterium of research intestines is to the effective ways of drug metabolism.Although the experiment in vitro method has more effectively been simulated the in vivo different links of metabolism of medicine, is of value to richness and increases, prepares metabolic product, has broken away from complete metabolism system to the effect of medicine, is difficult to be embodied in the synthesis result of body metabolism; The experiment in vitro conditional request is relatively high, needs to guarantee the activity of intestines bacterium, 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 frog, fruit bat, small white mouse etc. as the good test model fish of research object.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 are only for supporting the 0.1%-1% of mouse.Zebra fish is suitable with 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, such as the disease [S.Sumanas and S.Lin, DDT:TARGETS, 3,89-96 (2004) .] of the aspects such as nervous system, the circulation system, the sense of hearing, vision, cancer.A kind of method with zebra fish drugs toxicity is disclosed such as Chinese patent 200810019040.5; Patent 200780051025.2 disclose a kind of organ that is undertaken by stem cell nutrition formation and 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 zebra fish research tanshinone in salvia miltiorrhiza bunge IIA, Cryptotanshinone and Tanshinone I be the new method of metabolism mutually, find tanshinone IIA, the hydroxylation of Cryptotanshinone and Tanshinone I and the I of dehydrogenation reaction be metabolic product mutually, the method is set up system and had the Cytochrome P450s related enzyme systems of drug metabolism according to zebra fish: the zebra fish P450s gene of being cloned at present mainly comprises: Cyp1A, Cyp2K6, Cyp3a65 and Cyp3C1, Cyp11a1, Cyp19, Cyp26A1, the member of 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)].Research in recent years 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)], we disclose the mutually metabolism of II that utilizes zebra fish research chromocor compound Chrysin, 5-flavonol and 7-flavonol 201210187191.8, find their glucuronide conjugate and the II of sulfates metabolic product mutually; Zebra fish also has gut flora [Lv Aijun in addition, Yang Zhenghang, Liu Huan, Hu Xiucai, Zhang Yanhua, Cheng Chao. Chinese agronomy circular, 2010,26 (24): 412-415], can this class flora make glucoside compound occur and the similar desugar base of intestinal flora of mammals hydrolysis reaction, and it is blank that domestic research in this respect still belongs to.
Summary of the invention
Goal of the invention: technical matters to be solved by this invention is to overcome the deficiencies in the prior art, provide a kind of easy to operate, cost is low, compound amount is few, labour intensity is low, and only needs the quick mode biology zebra fish drugs that just can carry out in common laboratory such as the new method of glycoside compounds asperosaponin VI desugar base metabolism.
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 drugs such as the metabolism of glycoside compounds asperosaponin VI desugar base, 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 the blank solvent group, blank fish group, blank glycoside compounds group and glycoside compounds fish group, blank fish group and glycoside compounds fish group zebra fish quantity equate, tested glycoside compounds is made into desired concn, join in the aqueous solution of glycoside compounds fish group, blank fish group adds the solvent of equivalent, the blank solvent group only adds the equivalent solvent and does not put zebra fish, the equivalent glycoside compounds that blank glycoside compounds group adds the solvent dissolving is not put zebra fish, 0h～48h after the zebra fish of glycoside compounds fish group is exposed to liquid, get the fish liquid in different time points, place in-70 ℃ of refrigerators; Get fish liquid 24h or the 48h time point takes out fish at last, with rapidly washing 2～3 times of pure water, put to death, remove fin and fish phosphorus, weigh, place in-70 ℃ of refrigerators, blank solvent group, blank fish group and blank glycoside compounds group are taken a sample with method when 0h, 24h or 48h, and be for subsequent use;
(2) get each time point fish liquid freeze drying that step (1) obtains or with 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 15000 and turn/the centrifugal 10～20min of min, get filtrate or supernatant and carry out high-efficient liquid phase chromatogram HPLC analysis or High performance liquid chromatography 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, 3000～4000 turn/min is centrifugal, and 10～20min gets supernatant, and supernatant adds the dissolving of 50%～100% methyl alcohol with nitrogen or air blow drying, residue, cross 0.45 μ m or 0.22 μ m filter membrane or about 15000 and turn/the centrifugal 10～20min of min, filtrate or supernatant carry out the HPLC analysis or HPLC-MS analyzes.
As preferred version, above-described new method with the metabolism of model organism zebra fish research glycoside compounds, wherein the blank solvent group in the step (1), blank fish group, blank glycoside compounds group and glycoside compounds fish are organized all parallel 3 groups, sciences of assurance experiment of establishing.
As preferred version, above-described new method with the metabolism of model organism zebra fish research glycoside compounds, the zebra fish of glycoside compounds fish group is exposed to 0h behind the liquid in the step (1), 3h, 6h, 9h, 12h, 18h, the 24h time point is got the fish liquid, can be extended to 48h sample time in case of necessity.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 water or the aqueous solution that contains 0～1% dimethyl sulfoxide (DMSO) cosolvent.For water-soluble relatively poor medicine, adopt the dimethyl sulfoxide (DMSO) hydrotropy, can the Effective Raise medicine water-soluble, make medicine be dispersed in water-soluble in.
As preferred version, above-described new method with the metabolism of model organism zebra fish research glycoside compounds, step (2) is dissolved tested medicine according to the character of analyzed medicine according to the rule of similarity selective solvent, add 50%-100% methyl alcohol or acetonitrile dissolving such as the liquid residue, zebra fish fish body shreds, and weighs homogenate, homogenate adds methyl alcohol or acetonitrile removes albumen, the centrifuging and taking supernatant, nitrogen or air blow drying, residue adds 50%-100% methyl alcohol or acetonitrile dissolving.
The present invention has investigated the liquid recovered under reduced pressure to dried, direct freeze drying and nitrogen or three kinds of methods of air blow drying, the result shows loss and the error that the direct freeze-drying of liquid 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 liquid freeze drying or nitrogen, air blow drying, 50%-100% methyl alcohol or acetonitrile dissolve dry residue, then filter or centrifugal treating, supernatant carries out the high-efficient liquid phase chromatogram HPLC analysis or High performance liquid chromatography mass spectrometry LC-MS analyzes.
New method with model organism zebra fish research glycoside compounds asperosaponin VI metabolism of the present invention, wherein step (2) adopts Agilent 1100 type series of high efficiency liquid chromatographs or adopts highly sensitive Waters Alliance 2695-ZQ2000 HPLC-MS instrument to measure in the zebra fish aqueous solution situation of change of the content and structure of of glycoside compounds and metabolin in the glycoside compounds and metabolin and zebra fish in-vivo tissue.
As preferred version, the new method with model organism zebra fish research glycoside compounds asperosaponin VI metabolism of the present invention, the tested medicine of screening can be the compound etc. that desugar base hydrolysis metabolic response can occur for flavonoid glycoside, saponin(e and other.Such as asperosaponin VI.
Teasel root is the dry root of Dipsacaceae plant teasel (Dipsacus asper Wall.ex Henry), tool filling liver kidney, strengthening the bones and muscles, the effect of continuous folding wound.Modern study shows that the teasel root main active is saponin component, and wherein asperosaponin VI is main active trisaccharide saponin(e, has the promotion union, prevents and treats osteoporosis, promotes Gegenbaur's cell to generate, and anticancer isoreactivity.
Therefore study the metabolism situation of asperosaponin VI, can effectively understand the internal metabolism situation of glucoside compound.
Different from mammals such as mouse, rat, dogs, the small volume of zebra fish, oral or the drug administration by injection mode is very difficult, therefore, in the step (1) the present invention with tested medicine dissolving in the water that zebra fish lives, zebra fish can also carry out metabolism by the autonomous continuous tested medicine that absorbs in vivo from solution, the metabolin of medicine also can along with the excreta of zebra fish by continuous being discharged in the water, so just can grasp by the variation of analytical solution herb liquid composition the part metabolic information of medicine; Zebra fish adult fish is about 3～4cm, its body inner blood amount denier, be difficult to realize blood sampling, analyze the feasibility existing problems of blood sample by 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 by timing sampling analyze medicine in the zebra fish body and external Changing Pattern come comparatively comprehensively to explore zebra fish to the metabolism situation of medicine, this method simple possible.
Beneficial effect: the new method with the metabolism of model organism zebra fish research glucoside compound provided by the invention is compared with existing technology and has the following advantages:
New method with the metabolism of model organism zebra fish research glucoside compound provided by the invention, select zebra fish as the object of drug metabolism study, by the experiment group technology of optimizing, guarantee the accuracy of experimental result, especially preferred to the drug administration mode, 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, it is harsh to overcome general external intestinal flora metabolism experiment condition, and be difficult to be embodied in the shortcoming of body metabolism synthesis result, and it is large to overcome general mammal internal metabolism experiment institute dosage, the shortcoming that labour intensity is large, and experimental technique is repeatable strong, and especially required tested medication amount is few, and cost is low, labour intensity is low, have wide range of applications, and can carry out in batch experimental study, high efficiency.
Description of drawings
Fig. 1 is liquid and the fish body total ion current figure of asperosaponin VI liquid when zebra fish effect 24h
Fig. 2 is the asperosaponin VI of asperosaponin VI liquid when zebra fish effect 24h and the extraction ion flow graph of its metabolic product.
Fig. 3 is the positive ion mode mass spectrogram of asperosaponin VI.
Fig. 4 is the negative ion mode mass spectrogram of asperosaponin VI.
Fig. 5 is asperosaponin VI takes off 1 molecule aralino product B M1 after the zebra fish metabolism mass spectrogram.
Fig. 6 is asperosaponin VI takes off 1 molecule glucose based products BM2 after the zebra fish metabolism mass spectrogram.
Fig. 7 is asperosaponin VI takes off 1 molecule aralino and 1 molecule glucose based products BM3 after the zebra fish metabolism mass spectrogram.
Fig. 8 is asperosaponin VI takes off 2 molecule glucose based products BM4 after the zebra fish metabolism mass spectrogram.
Fig. 9 is asperosaponin VI takes off 1 molecule aralino and 2 molecule glucose based products BM5 after the zebra fish metabolism mass spectrogram.
Figure 10 is the mass spectrogram of asperosaponin VI hydroxylation product BM6 after the zebra fish metabolism.
Figure 11 is the total ion current figure of rat bile behind the Oral Administration in Rats asperosaponin VI, blood, urine and ight soil.
Figure 12 is the extraction ion flow graph of the asperosaponin VI in the rat body and its metabolic product behind the Oral Administration in Rats asperosaponin VI.
Figure 13 is asperosaponin VI takes off 1 molecule arabinose based products RM1 behind metabolism of rat mass spectrogram.
Figure 14 is asperosaponin VI takes off 1 molecule glucose based products RM2 behind metabolism of rat mass spectrogram.
Figure 15 is asperosaponin VI takes off 1 molecule aralino and 1 molecule glucose based products RM3 behind metabolism of rat mass spectrogram.
Figure 16 is asperosaponin VI takes off 2 molecule glucose based products RM4 behind metabolism of rat mass spectrogram.
Figure 17 is asperosaponin VI takes off 1 molecule aralino and 2 molecule glucose based products RM5 behind metabolism of rat mass spectrogram.
Figure 18 is the mass spectrogram of asperosaponin VI hydroxylation product RM6 behind metabolism of rat.
Embodiment
According to following embodiment, the present invention may be better understood.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 for explanation the present invention, and should also can not limit the present invention described in detail in claims.
The metabolism of embodiment one usefulness model organism zebra fish research asperosaponin VI
1. material and instrument
Tested medicine: asperosaponin VI (Asperosaponin VI)
Compound method: take by weighing and be equivalent to asperosaponin VI3～4mg 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, Electrospray Ionization Interface, 2695 type liquid chromatographs, Masslynx 4.0 chromatographic work stations (U.S., Waters company).METTLER TOLEDO AB 135-S analytical balance (Switzerland, METTLER TOLEDO company), KQ3200DE type numerical control ultrasonic cleaner (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 respectively the brown bottle that contains 30mL solution, 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 the 0.4%DMSO pure water solution of asperosaponin VI (medicine fish group).Establish simultaneously blank solvent group (0.4%DMSO pure water) and blank medicine group (0.4%DMSO asperosaponin VI pure water solution).The medicine fish is organized respectively at 0h behind the zebra fish exposure liquid, 3h, and 6h, 9h, 12h, 18h, every fish taking liquid of 24h time point 0.4ml, each time point liquid mixes respectively, places in-70 ℃ of refrigerators.Simultaneously fish is taken out in last sampling time point 24h, wash 3 times rapidly with pure water, put to death, remove fin and fish phosphorus, weigh, place (parallel 3 experiments) in-70 ℃ of refrigerators.The blank solvent group, blank fish group and blank medicine group are taken a sample with method during 24h in 0h.
2.2 sample preparation:
Liquid is processed: with each 2mL of different time points fish liquid, and freeze drying, residue adds the dissolving of 0.5mL90% methyl alcohol, crosses 0.22 μ m filter membrane, and filtrate sample introduction 20 μ L carry out HPLC-MS and analyze.
The zebra fish body is processed: mix between parallel group of zebra fish when getting 24h, shred, take by weighing 1g, add the homogenate of 5mL physiological saline, the centrifugal 10min of 3500r/min, supernatant adds 4 times of amount methyl alcohol vortex mixings except albumen, the centrifugal 10min of 3500r/min, supernatant nitrogen dries up, and residue adds the solution that 1g fish/mL is made in the dissolving of 1mL90% methyl alcohol, cross 0.22 μ m filter membrane, filtrate 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: 25 ℃; Mobile phase: A is Robust pure water (with mass spectrometry time change into 0.05% formic acid water), and B is acetonitrile (with mass spectrometry time change into 0.05% formic acid acetonitrile), A ten B=100%, adopt gradient elution: 0～12min, 25～27%B, 12～30min, 27～50%B, 30～40min, 50～80%B, 40～45min, 80～90%B, 45～50min, 90%B; Flow velocity: 1.0mL/min; Full wavelength scanner detects; Writing time 50min; 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, 350 ℃ of assisted gas temperature; The ion detection mode: full scan detects (SCAN), and ion polarity: positive ion (positive) and negative ion (positive) detect simultaneously, extracts ion current (TIC): [M-H]
-, [M+HCOO]
-, [M+H]
+[M+Na]
+, ionization mode: Aeroassisted electro-spray ionization (ESI), sweep limit: 100-1200m/z.
3. experimental result
Adopt HPLC-MS method positive and negative ion pattern to detect simultaneously the metabolic product of asperosaponin VI after the zebra fish effect, wherein negative ion mode is sensitive.Such as Fig. 1-shown in Figure 9, find that the main metabolic path of asperosaponin VI after the zebra fish effect is the desugar radical reaction, the asperosaponin VI original shape composition in detecting in asperosaponin VI zebra fish liquid in the 24h or the zebra fish body and 5 desugar base metabolic products (BM1～BM5) (concrete experimental result sees Table 1) of asperosaponin VI.There are some researches show, behind the Oral Administration in Rats asperosaponin VI, in rat ight soil, detect 5 desugar base metabolic products, asperosaponin VI desugar based products [the LI Kai with consistency of this and zebra fish effect, YANG Xiao-Lin, ZHANG Chun-Feng and YANG Zhong-Lin.Chin J Nat Med, 2009,7 (6): 440-443.], in addition, this research also detects 1 the hydroxylated I phase metabolic product (BM6) of asperosaponin VI after the zebra fish effect, and hydroxylating by the mediation of Cytochrome P450s related enzyme systems, shows that zebra fish can react in the comprehensive metabolism result of body animal model usually.Adopt provided by the invention with model organism zebra fish research asperosaponin VI metabolism result comprehensively, accurately and reliably, experimental technique is feasible.
The metabolite identification of table 1 asperosaponin VI after the zebra fish effect
The metabolism of embodiment dual-purpose rat studies asperosaponin VI
Existing asperosaponin VI's is still not comprehensive in body mammal metabolism research, except document [LI Kai, YANG Xiao-Lin, ZHANG Chun-Feng and YANG Zhong-Lin.Chin J Nat Med, 2009,7 (6): 440-443.] behind the report Oral Administration in Rats asperosaponin VI outside the metabolic product in the ight soil, there is not yet the Methanogenesis in blood plasma, bile, the urine, for multianalysis, comparison zebra fish and the rat Difference of Metabolism to asperosaponin VI, we to asperosaponin VI the metabolism in the rat body carried out systematic study.
1. material and instrument
Tested medicine: asperosaponin VI compound
Prepare for reagent: it is an amount of to take by weighing asperosaponin VI, adds 0.8%CMC-Na and is made into 10mgmL
-1Solution, for rat oral gavage.
Heparin is purchased from Jiangsu Wanbang Biological Pharmaceutical Co., Ltd., physiological saline (sodium chloride injection, Nanjing Xiaoying Medicine Group Co.,Ltd, lot number 2010070203).Acetonitrile (chromatographically pure, U.S. TEDIA company), ultrapure water (Milli-Q), the Robust pure water.All the other reagent are pure for analyzing.
Animal: SD rat, body weight 200～300g.Shanghai Slac Experimental Animal Co., Ltd..
Instrument: Waters Alliance 2695-ZQ 2000 liquid chromatograph-mass spectrometers (U.S. Waters company) comprise two high-pressure pumps, automatic sampler, column oven, Electrospray Ionization Interface, 2695 type liquid chromatographs, Masslynx 4.0 chromatographic work stations.TGL-16G type supercentrifuge (Anting Scientific Instrument Factory, Shanghai); TDZ5B-WS low speed self-poise centrifuge (Shanghai Lu Xiang instrument hydro-extractor instrument company limited); XW-80A vortex mixer (Qingpu Shanghai Luxi device factory of instrument plant); Balance (1,/10 ten thousand, METTLER AB135-S type, plum Teller 2 holder benefit Instr Ltd.), OrganomationN-EVAPTM 112 Nitrogen evaporators (Organomation Associates, Jnc.USA)
2. experimental technique
2.1 medication and sample collection
The rat urine sample: get 6 of SD rats,
, body weight 200～300g is divided into 2 groups, every group of 3 rats, blank group and administration group.Fasting be can't help water 12 hours before the experiment.Administration group single gavage 100mgkg
-1The 0.8%CMC-Na solution of asperosaponin VI, blank group gives equal-volume 0.8%CMC-Na aqueous solution.Collect respectively twenty-four-hour urine liquid.It is for subsequent use to put-70 ℃ of Refrigerator stores.
Rat ight soil: above-mentioned SD rat.Collect twenty-four-hour urine liquid simultaneously, collect respectively rat ight soil in 24 hours.It is for subsequent use to put-70 ℃ of Refrigerator stores.
Rat plasma: above-mentioned SD rat is being collected urine sample and ight soil simultaneously, and respectively at 0.5h, 1h, 2h, 4h, 6h after the administration, eye socket is got blood, puts in the heparin test tube, fully mixing.3000rmin
-1Get supernatant behind the centrifugal 10min, each time point blood plasma merges respectively, puts in-70 ℃ of refrigerators to save backup.
Rat bile: above-mentioned SD rat is raised the convalescence more than 10 days after getting blood.Fasting be can't help water 12 hours before the administration, used Ethylurethanm 1gkg
-1Intramuscular anesthesia, the operation of belly cystic duct cannula.Respectively gastric infusion and 0.8%CMC-Na aqueous solution when treating that rat is clear-headed, each medicine is collected respectively 12h bile, puts-70 ℃ of refrigerator and cooled and freezes and save backup.
2.2 sample preparation:
Rat urine sample, blood plasma, bile are processed: get respectively each time period of rat and mix urine 7mL, blood plasma 1.5mL or bile 3mL, respectively add 4 times of methyl alcohol, vortex mixing 3min, 3000rmin
-1Get supernatant behind the centrifugal 15min, N
2Lower 40 ℃ dry up, and residue adds respectively 2.0m L, 0.8mL or the dissolving of 1.5mL70% methyl alcohol, 13000rmin
-1Get supernatant behind the centrifugal 15min, sample introduction 20 μ L analyze.The blank urine of rat, blood plasma or bile are processed with method.
Rat ight soil is processed: each time period ight soil mixes, and grinds with mortar, takes by weighing 1.6g, adds 20mL 70% methyl alcohol and soaks and ultrasonic 30min 3000rmin
-1Get supernatant behind the centrifugal 15min, N
2Lower 40 ℃ dry up, and residue adds the dissolving of 1mL70% methyl alcohol, 15000rmin
-1Get supernatant behind the centrifugal 15min, sample introduction 20 μ L analyze.The blank excrement of rat is processed with method.
2.3 analysis condition: with embodiment one.
3. experimental result
Employing HPLC-MS method positive and negative ion pattern detects the metabolic product in blood plasma, urine, bile and the ight soil of asperosaponin VI after the rat effect simultaneously, and wherein negative ion mode is sensitive.Such as Figure 11-shown in Figure 180, in blood plasma, urine, bile and ight soil, all detect the original shape composition, the desugar radical reaction is the main metabolic path of asperosaponin VI, detect 5 kinds of asperosaponin VI desugar base metabolic products (RM1～RM5), consistent [LI Kai with the metabolic product of reported in literature, YANG Xiao-Lin, ZHANG Chun-Feng and YANG Zhong-Lin.Chin J Nat Med, 2009,7 (6): 440-443.], these 5 kinds of desugar based products with embodiment one are consistent.In addition, in rat bile, find first 1 hydroxylation product of asperosaponin VI, point out this hydroxylating mainly by hepatomicrosome cytochrome P450s monooxygenase mediation, this with embodiment one in zebra fish to the hydroxylating tool similarity (specifically experimental result sees Table 2) of asperosaponin VI.Metabolism of rat research comprehensively, the reaction of system comprehensive metabolism result in the body of asperosaponin VI, and with consistency to the metabolism of asperosaponin VI with zebra fish.
The metabolite identification of table 2 asperosaponin VI after the rat effect
Show by above experimental result, provided by the invention strong with model organism zebra fish research glycoside compounds desugar base hydrolysis metabolism feasibility, the desugar base hydrolysis metabolism situation by the gut flora mediation of the objective and accurate reaction glycoside compounds of energy such as asperosaponin VI, in addition also can be objective the hydroxylating (this type of reaction is open in patent 201010258459.3) of reacting cells cytochrome p 450 s related enzyme systems mediation, illustrate that zebra fish can react medicine in the synthesis result of body metabolism comprehensively, be the metabolism situation of clinical research medicine, instruct the selection of clinical application and pharmaceutical dosage form that the overall scientific foundation is provided.The present invention also is the desugar base hydrolysis metabolism supplying method reference of glucoside compound.
Zebra fish is suitable with similarity and the mouse of human gene, and on protein level, the homology of its key position almost is 100%, zebra fish has the related enzyme systems of drug metabolism, also has gut flora, the present invention selects zebra fish as the object of medicine internal metabolism research, by the experiment group technology of optimizing, the medicine dissolving method is optimized, drug administration mode preferred, and especially drug metabolism method for post extraction and analytical approach is preferred, can objectively react medicine true metabolism situation in vivo, it is harsh to overcome general In vitro 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, required tested medication amount few (only be about in the present invention metabolism of rat a thirtieth), labour intensity is low, high efficiency.
The above only is 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 improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (5)
1. the new method with the metabolism of model organism zebra fish research glycoside compounds asperosaponin VI desugar base 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 the blank solvent group, blank fish group, blank glycoside compounds group and glycoside compounds fish group, blank fish group and glycoside compounds fish group zebra fish quantity equate, tested glycoside compounds is made into desired concn, join in the aqueous solution of glycoside compounds fish group, blank fish group adds the solvent of equivalent, the blank solvent group only adds the equivalent solvent and does not put zebra fish, the equivalent glycoside compounds that blank glycoside compounds group adds the solvent dissolving is not put zebra fish, 0h～48h after the zebra fish of glycoside compounds fish group is exposed to liquid, get the fish liquid in different time points, place in-70 ℃ of refrigerators; Get fish liquid 24h or the 48h time point takes out fish at last, with rapidly washing 2～3 times of pure water, put to death, remove fin and fish phosphorus, weigh, place in-70 ℃ of refrigerators, blank solvent group, blank fish group and blank glycoside compounds group are taken a sample with method when 0h, 24h or 48h, and be for subsequent use;
(2) get each time point fish liquid freeze drying that step (1) obtains or with 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 15000 and turn/the centrifugal 10～20min of min, get filtrate or supernatant and carry out high-efficient liquid phase chromatogram HPLC analysis or High performance liquid chromatography mass spectrometry LC-MS analysis; Get 24h or 48h time point zebra fish fish body shreds, weigh, homogenate, homogenate adds methyl alcohol or acetonitrile except albumen, 3000～4000 turn/min is centrifugal, and 10～20min gets supernatant; Supernatant adds the dissolving of 50%～100% methyl alcohol with nitrogen or air blow drying, residue, crosses 0.45 μ m or 0.22 μ m filter membrane or about 15000 and turns/the centrifugal 10～20min of min, and filtrate or supernatant carry out the HPLC analysis or HPLC-MS analyzes.
2. the new method with the metabolism of model organism zebra fish research glycoside compounds according to claim 1 is characterized in that,
Described High performance liquid chromatography mass spectrometry HPLC-MS analysis condition is:
Liquid-phase condition: specification is 5 μ m, the Agilent C of 150mm * 4.6mm
18Post and specification are 4.6 * 12.5mm, the C18 pre-column of ID5 μ m; Column temperature: 25 ℃; Mobile phase: A is 0～0.1% formic acid water, and B is 0～0.1% formic acid acetonitrile, and A ten B=100% adopt gradient elution: 0～12min, 25～27%B, 12～30min, 27～50%B, 30～40min, 50～80%B, 40～45min, 80～90%B, 45～50min, 90%B; Flow velocity: 1.0mL/min; Full wavelength scanner detects; Writing time 50min; 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, 350 ℃ of assisted gas temperature; The ion detection mode: full scan detects, and ion polarity: positive ion and negative ion detect simultaneously, the ionization mode: Aeroassisted electro-spray ionization, sweep limit: 100-1200m/z.
3. the new method with the metabolism of model organism zebra fish research glycoside compounds according to claim 1, it is characterized in that, the zebra fish of glycoside compounds fish group is exposed to 0h behind the liquid in the step (1), 3h, 6h, 9h, 12h, 18h, 24h or 48h time point are got the fish liquid, get simultaneously the fish body when last taking liquid time point 24h or 48h.
4. the new method with model organism zebra fish research glycoside compounds asperosaponin VI metabolism according to claim 1 is characterized in that the described solvent of step (1) is water or the aqueous solution that contains 0～1% dimethyl sulfoxide (DMSO) cosolvent.
5. the new method with the metabolism of model organism zebra fish research glycoside compounds according to claim 1 is characterized in that described glycoside compounds is asperosaponin VI.
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