CN102645364A - Renal toxicity causing toxic effect portion of rhizoma alismatis and method for preparing toxic effect portion - Google Patents
Renal toxicity causing toxic effect portion of rhizoma alismatis and method for preparing toxic effect portion Download PDFInfo
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Abstract
The invention provides a renal toxicity causing toxic effect portion of rhizoma alismatis, a method for preparing the toxic effect portion and a testing method for determining the toxic effect portion, which solve the problem of bottleneck of modern, international and sustainable development of traditional Chinese medicines due to unclear renal toxicity causing material bases and portions of rhizoma alismatis. The toxic effect portion is mainly characterized in that the renal toxicity causing toxic effect portion of the rhizoma alismatis is chloroform extract of rhizoma alismatis water decoction. The method for preparing the toxic effect portion includes the steps: preparing the extract of the rhizoma alismatis water decoction; and preparing the chloroform extract. The testing method for the renal toxicity adopts a pathological section and metabonomics. As the renal toxicity causing toxic effect portion of the rhizoma alismatis is determined as the chloroform extract of the rhizoma alismatis water decoction, evidence can be provided for clinical safety application of the rhizoma alismatis.
Description
Technical field
The present invention relates to field of medicaments, be specifically related to rhizoma alismatis and cause renal toxicity effect toxic effect position and preparation method thereof.
Background technology
Rhizoma alismatis is an Alismataceae plant rhizoma alismatis
Alisma orientalis(Sam.) Juzep
.Dry tuber.Head is stated from Shennong's Herbal, is clinical medicine commonly used, and " Chinese pharmacopoeia is all recorded to go through version.Only record the Chinese patent drug that contains rhizoma alismatis in the current edition pharmacopeia kind surplus Maiwei Dihuang Wan, seven flavor Duqi Wans, three health-care capsules, mountain chrysanthemum hypertension pill, wuling powder, Liuwei Dihuang Wan, the tablets for treating hyperlipemia etc. 20 is just arranged; Remove as clearing damp and promoting diuresis property Chinese medicine, also be used as in herbal cuisine, the slimming health product, this makes them be able to taken for a long time.But all the time, people only pay close attention to its therapeutic efficiency, have but ignored its long-term use or the excessive toxic action that uses the back that kidney is caused.The toxicity research of Chinese medicine is subject to development in science and technology level and scientific research thinking, and mostly ancient traditional Chinese medicine is general and perception based on clinical practice to the description of Chinese materia medica tocixity, like large, medium and small poison and upper, middle and lower article (Shennong's Herbal); The thinking of modern Chinese herbal medicine toxicity research is then for a long time always with reference to chemicals toxicity assessment method; Research means mainly concentrates in acute, subacute and the long term toxicity test, and index choice is main with the change of observation organ techtology and the detection of some biochemical indicator.Though this has disclosed the symptom and the degree of toxicity for us, can't reach from integral viewpoint for the description of phenomenon and characteristic and to illustrate Changing Pattern, and then disclose the purpose of mechanism of poisoning by the caused endogenous material of toxicity basis.A kind of in learning as numerous groups, metabolism group is through investigating the subject that the variation of its metabolic product after living things system irriate or the disturbance comes postgraduate's objects system.Research object is made the as a whole viewpoint of studying to metabolism group and the whole sight of the traditional Chinese medicine and pharmacy mode of thinking is perfectly in harmony, and the comprehensive system strategy of using metabolism group discloses the rule of Chinese materia medica tocixity, with the modernization that effectively promotes traditional Chinese medicine.
Summary of the invention
For solving the problems of the technologies described above, the invention provides the experimental technique that rhizoma alismatis causes renal toxicity effect toxic effect position and preparation method thereof, confirms the toxic effect position.
The used rhizoma alismatis of the present invention is from Liaoning Province's medical material company limited.
Technical scheme of the present invention is:
1, rhizoma alismatis to cause renal toxicity effect toxic effect position be the chloroform extract of rhizoma alismatis decocting liquid.
2, the preparation method of the chloroform extract of rhizoma alismatis decocting liquid is: at first rhizoma alismatis is added the water of 8 ~ 12 times of amounts of its weight, the room temperature cold soaking spends the night, and heated and boiled 1 ~ 3 time is extracted 0.5 ~ 2 h at every turn, filters, and merging filtrate, concentrating under reduced pressure obtain rhizoma alismatis decocting liquid extract; Secondly, will concentrate back decocting liquid extract, with chloroform equal-volume extraction 2 ~ 4 times, combining extraction liquid, through 50 ℃ of Rotary Evaporators, solvent evaporated obtains chloroform extract.
3, the toxic effect confirmation method of the chloroform extract of rhizoma alismatis decocting liquid:
(1) pathological section method:
The chloroform extract of obtaining, thin up is to being equivalent to medicinal material concentration 2 g/mL, as the chloroform extract test liquid;
Get caulis aristologhiae manshuriensis and pulverize, adds 4 times of distilled water, soak 30 min after, be heated to boiling, decoct 60 min, inclining soup, filters, and decocts repeatedly 2 times, merges soup, filtration is concentrated into and is equivalent to medicinal material concentration 0.5 g/ml as positive control solution; At random animal is equally divided into 3 groups by body weight: the normal control group, rhizoma alismatis chloroform extract group and caulis aristologhiae manshuriensis positive controls, irritated stomach once, continuous irrigation stomach 120 days every day after rat put into metabolic cage and adapt to 7 days; Rat is put to death after 120 days in administration, kidney is weighed, and gets renal tissues in rats and use 10% formaldehyde fixed, and FFPE is also made slice, processes 3 μ m section, through haematine-Yi red colouring, under optical microscope, observes morphological change.
(2) metabolism group method
The chloroform extract of obtaining, thin up is to being equivalent to medicinal material concentration 2 g/mL, as the chloroform extract test liquid;
Get caulis aristologhiae manshuriensis and pulverize, adds 4 times of distilled water, soak 30 min after, be heated to boiling, decoct 60 min, inclining soup, filters, and decocts repeatedly 2 times, merges soup, filtration is concentrated into and is equivalent to medicinal material concentration 0.5 g/ml as positive control solution; At random animal is equally divided into 3 groups by body weight: the normal control group, rhizoma alismatis chloroform extract group and caulis aristologhiae manshuriensis positive controls, irritated stomach once, continuous irrigation stomach 120 days every day after rat put into metabolic cage and adapt to 7 days;
1. the collection of biological sample
Urine collecting:Adopt the metabolic cage method, collected 24 h urines in 120 days to centrifuge tube, behind centrifugal 3 min of urine sample 13000 rpm, get supernatant and place-20 ℃ of refrigerators preservations subsequent use in administration;
Serum is collected:In administration eye socket blood sampling in 120 days, place test tube, placed 30 minutes, take out the blood cake, centrifugal 5 min of solution 13000 rpm, separation obtains serum and places-20 ℃ of refrigerators preservations subsequent use;
2. the pre-service of metabolism group biological sample
The urine sample pre-service:Get urine sample 200 μ L, place 1.5 mL EP pipe, add 200 μ L methyl alcohol, vortex 3 min, centrifugal 5 min under the 13000 rpm conditions get supernatant 5 μ L and carry out the UPLC-MS analysis;
The blood serum sample pre-service:Get 200 μ L serum and add in the 1.5 mL EP pipe, add 400 μ L acetonitrile precipitation albumen, in 4 ℃ low temperature environment, centrifugal 10 min of 15000 rpm, supernatant are transferred in another EP pipe, keep 30 ℃ of constant temperature, at N
2Dry up under the air-flow, with 100 μ L Yi Jing – water (15: 85,
V/v) redissolve, ultrasonic 3 min, vortex 1 min behind centrifugal 5 min of 13000 rpm, gets supernatant 5 μ L and carries out the UPLC-MS analysis;
3. the foundation of UPLC-MS analytical approach
Chromatographic conditionChromatographic column: ACQUITY UPLC
TMBEH C
18Post 6; Column temperature: 35 ℃; Moving phase: 0.1% formic acid acetonitrile (A)-0.1% formic acid water (B), gradient elution;
The mass spectrum conditionIon gun ESI positive ion full scan pattern, capillary voltage 3.2 kV, taper hole voltage 30 V, 120 ℃ of ion source temperatures, 350 ℃ of desolventizing temperature, desolventizing gas N
2With taper hole gas N
2Flow velocity is set to 600 and 50 L/h respectively.Adopt full surface sweeping scan mode, sweep limit is 100 ~ 900 amu, and when the scanning of ion multi-stage ms was extracted in utilization, argon gas was as collision gas.It is mass calibration that NaCsI is used at the experiment previous crops;
The conclusive evidence of UPLC-MS analytical approachThe precision and the study on the stability of urine sample and blood sample analysis method all meet the requirements;
4. data processing
Utilization Markerlynx software carries out chromatographic peak identification and peak match, and adopts PCA (PCA) that the metabolin group of each group rat plasma and urine sample is analyzed.
Specify the present invention below in conjunction with accompanying drawing.
Description of drawings
Fig. 1 is that rat oral gavage gives rhizoma alismatis chloroform extract kidney histopathology slice map after 120 days among the embodiment nine-3, wherein,
(a) be the pathology slice map of normal blank group glomerulus.Glomerulus demonstrates tangible normal configuration.
(b) be the pathology slice map of normal blank group renal tubule.Renal tubule demonstrates tangible normal configuration.
(c) be the pathology slice map of rhizoma alismatis chloroform extract group glomerulus.The glomerulus atrophy is lobulated, the capsular space expansion, and capillary is accidental, and sees many capsules red blood cell.
(d)-(f) be the pathology slice map of rhizoma alismatis chloroform extract group renal tubule.Visible a small amount of albumen oozes out in the renal tubule, and visible a small amount of inflammatory cell.The renal cells enlargement, tube chamber dwindles, and powder occurs and dyes structureless protein cast.
(g) be the pathology slice map of positive control caulis aristologhiae manshuriensis group glomerulus.Glomerulus is not seen obvious pathology with respect to the blank group.
(h)-(i) be the pathology slice map of positive controls caulis aristologhiae manshuriensis group renal tubule.Protein cast appears in renal tubule, the renal cells enlargement, and tube chamber dwindles.
Fig. 2 is that rat oral gavage gives rhizoma alismatis chloroform extract urine sample chromatogram after 120 days among the embodiment nine-4, provides with the form of BPI (base peak intensity) figure.Results of statistical analysis shows that model group and blank group can obviously distinguish.
Fig. 3 is that rat oral gavage gives rhizoma alismatis chloroform extract typical blood sample chromatogram after 120 days among the embodiment nine-4, provides with the form of BPI (base peak intensity) figure.Results of statistical analysis shows that model group and blank group can obviously distinguish.
Embodiment
One, the preparation method of the chloroform extract of the decocting liquid of 8 times of water additions of rhizoma alismatis is:
At first rhizoma alismatis is added the water of 8 times of amounts of its weight, the room temperature cold soaking spends the night, and heated and boiled 2 times is extracted 1 h at every turn, filters, and merging filtrate, concentrating under reduced pressure obtain rhizoma alismatis decocting liquid extract; Secondly, will concentrate back decocting liquid extract, with chloroform equal-volume extraction 3 times, combining extraction liquid, through 50 ℃ of Rotary Evaporators, solvent evaporated obtains chloroform extract.
Two, the preparation method of the chloroform extract of the decocting liquid of 12 times of water additions of rhizoma alismatis is:
At first rhizoma alismatis is added the water of 12 times of amounts of its weight, the room temperature cold soaking spends the night, and heated and boiled 2 times is extracted 1 h at every turn, filters, and merging filtrate, concentrating under reduced pressure obtain rhizoma alismatis decocting liquid extract; Secondly, will concentrate back decocting liquid extract, with chloroform equal-volume extraction 3 times, combining extraction liquid, through 50 ℃ of Rotary Evaporators, solvent evaporated obtains chloroform extract.
Three, rhizoma alismatis is boiled the preparation method of chloroform extract of 1 time decocting liquid and is:
At first rhizoma alismatis is added the water of 10 times of amounts of its weight, the room temperature cold soaking spends the night, and heated and boiled 1 time is extracted 1 h at every turn, filters, and merging filtrate, concentrating under reduced pressure obtain rhizoma alismatis decocting liquid extract; Secondly, will concentrate back decocting liquid extract, with chloroform equal-volume extraction 3 times, combining extraction liquid, through 50 ℃ of Rotary Evaporators, solvent evaporated obtains chloroform extract.
Four, rhizoma alismatis is boiled the preparation method of chloroform extract of 3 times decocting liquid and is:
At first rhizoma alismatis is added the water of 10 times of amounts of its weight, the room temperature cold soaking spends the night, and heated and boiled 3 times is extracted 1 h at every turn, filters, and merging filtrate, concentrating under reduced pressure obtain rhizoma alismatis decocting liquid extract; Secondly, will concentrate back decocting liquid extract, with chloroform equal-volume extraction 3 times, combining extraction liquid, through 50 ℃ of Rotary Evaporators, solvent evaporated obtains chloroform extract.
Five, rhizoma alismatis is extracted the preparation method of chloroform extract of the decocting liquid of 0.5 h at every turn and is:
At first rhizoma alismatis is added the water of 10 times of amounts of its weight, the room temperature cold soaking spends the night, and heated and boiled 2 times is extracted 0.5 h at every turn, filters, and merging filtrate, concentrating under reduced pressure obtain rhizoma alismatis decocting liquid extract; Secondly, will concentrate back decocting liquid extract, with chloroform equal-volume extraction 3 times, combining extraction liquid, through 50 ℃ of Rotary Evaporators, solvent evaporated obtains chloroform extract.
Six, rhizoma alismatis is extracted the preparation method of chloroform extract of the decocting liquid of 2 h at every turn and is:
At first rhizoma alismatis is added the water of 10 times of amounts of its weight, the room temperature cold soaking spends the night, and heated and boiled 2 times is extracted 2 h at every turn, filters, and merging filtrate, concentrating under reduced pressure obtain rhizoma alismatis decocting liquid extract; Secondly, will concentrate back decocting liquid extract, with chloroform equal-volume extraction 3 times, combining extraction liquid, through 50 ℃ of Rotary Evaporators, solvent evaporated obtains chloroform extract.
Seven, the preparation method of the extract of 2 extractions of chloroform of rhizoma alismatis decocting liquid is:
At first rhizoma alismatis is added the water of 10 times of amounts of its weight, the room temperature cold soaking spends the night, and heated and boiled 2 times is extracted 1 h at every turn, filters, and merging filtrate, concentrating under reduced pressure obtain rhizoma alismatis decocting liquid extract; Secondly, will concentrate back decocting liquid extract, with chloroform equal-volume extraction 2 times, combining extraction liquid, through 50 ℃ of Rotary Evaporators, solvent evaporated obtains chloroform extract.
Eight, the preparation method of the extract of 4 extractions of chloroform of rhizoma alismatis decocting liquid is:
At first rhizoma alismatis is added the water of 10 times of amounts of its weight, the room temperature cold soaking spends the night, and heated and boiled 2 times is extracted 1 h at every turn, filters, and merging filtrate, concentrating under reduced pressure obtain rhizoma alismatis decocting liquid extract; Secondly, will concentrate back decocting liquid extract, with chloroform equal-volume extraction 4 times, combining extraction liquid, through 50 ℃ of Rotary Evaporators, solvent evaporated obtains chloroform extract.
Nine, rhizoma alismatis causes renal toxicity effect chloroform extract and causes renal toxicity effect test method
1. the preparation of test liquid
The chloroform extract of obtaining, thin up is to being equivalent to medicinal material concentration 2 g/mL, as the chloroform extract test liquid.
Get caulis aristologhiae manshuriensis and pulverize, adds 4 times of distilled water, soak 30 min after, be heated to boiling, decoct 60 min, inclining soup, filtration decocts 2 times repeatedly.The merging soup filters, and is concentrated into to be equivalent to medicinal material concentration 0.5 g/ml as positive control solution.Get distilled water as the normal control group.
2. animal divides into groups and medication
At random animal is equally divided into 3 groups by body weight: normal control group, rhizoma alismatis chloroform extract group (40 g/kg/day calculate by drinking raw sheet) and caulis aristologhiae manshuriensis positive controls (15 g/kg/day).Irritated stomach once, continuous irrigation stomach 120 days every day after rat put into metabolic cage and adapt to 7 days.
3. pathological section
Rat is put to death after 120 days in administration; Kidney is weighed; And get renal tissues in rats and use 10% formaldehyde fixed, FFPE is also made slice (every section comprises the scope from the cortex renis to the kidney medulla), processes 3 μ m section; Through haematine-Yi red (HE) dyeing, under optical microscope, observe morphological change.
4. the method for metabolism group
(1) collection of biological sample
Urine collecting:Adopt the metabolic cage method, collected 24 h urines in 120 days to centrifuge tube, behind centrifugal 3 min of urine sample 13000 rpm, get supernatant and place-20 ℃ of refrigerators preservations subsequent use in administration.
Serum is collected:In administration eye socket blood sampling in 120 days, place test tube, placed 30 minutes, take out the blood cake, centrifugal 5 min of solution 13000 rpm, separation obtains serum and places-20 ℃ of refrigerators preservations subsequent use.
(2) pre-service of metabolism group biological sample
The urine sample pre-service:Get urine sample 200 μ L, place 1.5 mL EP pipe, add 200 μ L methyl alcohol, vortex 3 min, centrifugal 5 min under the 13000 rpm conditions get supernatant 5 μ L and carry out the UPLC-MS analysis.
The blood serum sample pre-service:Get 200 μ L serum and add in the 1.5 mL EP pipe, add 400 μ L acetonitrile precipitation albumen, in 4 ℃ low temperature environment, centrifugal 10 min of 15000 rpm, supernatant are transferred in another EP pipe, keep 30 ℃ of constant temperature, at N
2Dry up under the air-flow, with 100 μ L Yi Jing – water (15: 85,
V/v) redissolve, ultrasonic 3 min, vortex 1 min behind centrifugal 5 min of 13000 rpm, gets supernatant 5 μ L and carries out the UPLC-MS analysis.
(3) foundation of UPLC-MS analytical approach
Chromatographic conditionChromatographic column: ACQUITY UPLC
TMBEH C
18Post (50 mm * 2.1 mm, 1.7 μ m); Column temperature: 35 ℃; Moving phase: 0.1% formic acid acetonitrile (A)-0.1% formic acid water (B), gradient elution
The mass spectrum conditionIon gun ESI positive ion full scan pattern, capillary voltage 3.2 kV, taper hole voltage 30 V, 120 ℃ of ion source temperatures, 350 ℃ of desolventizing temperature, desolventizing gas N
2With taper hole gas N
2Flow velocity is set to 600 and 50 L/h respectively.Adopt full surface sweeping scan mode, sweep limit is 100 ~ 900 amu, and when the scanning of ion multi-stage ms was extracted in utilization, argon gas was as collision gas.It is mass calibration that NaCsI is used at the experiment previous crops.
The conclusive evidence of analytical approachThe precision and the study on the stability of urine sample and blood sample analysis method all meet the requirements.
(4) data processing
This research application Markerlynx software carries out chromatographic peak identification and peak match, and adopts PCA (PCA) that the metabolin group of each group rat plasma and urine sample is analyzed.
5. experimental result
(1) pathological section, as shown in Figure 1, rats in normal control group kidney pathological section demonstrates tangible normal configuration; The nephridial tissue section of rhizoma alismatis chloroform extract group embodies the obvious impairment state: the glomerulus atrophy is lobulated, the capsular space expansion, and capillary is accidental, and sees many capsules red blood cell.Visible a small amount of albumen oozes out in the renal tubule, and visible a small amount of inflammatory cell.The renal cells enlargement, tube chamber dwindles, and powder occurs and dyes structureless protein cast.The result shows that long-term filling stomach gives the imitative extract of rhizoma alismatis decocting liquid chlorine and can cause the kidney of rats damage.Caulis aristologhiae manshuriensis group rat is as positive controls, irritates stomach and gives after 120 days glomerulus and do not see obvious pathology, and protein cast appears in renal tubule, the renal cells enlargement, and tube chamber dwindles, and is consistent with bibliographical information, proved the reliability of test findings.
(2) rat urine sample metabolism group (referring to Fig. 2)
The principal component analysis (PCA) result
Typical urine sample chromatogram collection of illustrative plates after 120 days has been carried out statistical analysis.From urine sample score figure, can find out the chloroform extract group obviously away from the normal control group, the result is consistent with the nephridial tissue pathological section.The chloroform extraction position of having proved rhizoma alismatis decocting liquid from the metabolism group angle has renal toxicity.
The evaluation of label
Through the one-level to label, second order ms figure resolves, and its structure is made preliminary judgement; Then through the retrieval of KEGG (http://www.genome.jp/kegg/) and METLIN Relational databases such as (http://metlin.scripps.edu/) has been carried out Preliminary Identification to the label identity; At last through with the retention time of standard items, one-level, second order ms figure contrasts, and has finally confirmed tag structure.Figure is a foundation with the principal component analysis (PCA) factor loading, through standard control, and database retrieval, literature search, identify following label (table 1):
Table 1Chloroform layer group urine sample label
(3) rat blood sample metabolism group is referring to Fig. 3.
The principal component analysis (PCA) result
Typical blood sample chromatogram collection of illustrative plates after 120 days has been carried out statistical analysis.Display model group and blank group can significantly be distinguished on shot chart as a result.
The evaluation of label
Method identifies that with the urine sample label figure is a foundation with the principal component analysis (PCA) factor loading, through standard control, and database retrieval, literature search, identify following label (table 2):
Table 2Chloroform layer group plasma sample label
Claims (8)
- Rhizoma alismatis to cause renal toxicity effect toxic effect position be the chloroform extract of rhizoma alismatis decocting liquid.
- 2. the method for preparing the chloroform extract of the described rhizoma alismatis decocting of claim 1 liquid; It is characterized in that: at first rhizoma alismatis is added the water of 8 ~ 12 times of amounts of its weight, the room temperature cold soaking spends the night, heated and boiled 1 ~ 3 time; Each 0.5 ~ 2 h that extracts; Filter, merging filtrate, concentrating under reduced pressure obtain rhizoma alismatis decocting liquid extract; Secondly, will concentrate back decocting liquid extract, with chloroform equal-volume extraction 2 ~ 4 times, combining extraction liquid, through 50 ℃ of Rotary Evaporators, solvent evaporated obtains chloroform extract.
- 3. like the said preparation method of claim 2, the addition that it is characterized in that water is 10 times of rhizoma alismatis weight.
- 4. like the said preparation method of claim 2, it is characterized in that the heated and boiled number of times is 2 times.
- 5. like the said preparation method of claim 2, it is characterized in that extraction time is 1h.
- 6. like the said preparation method of claim 2, it is characterized in that extraction times is 3 times.
- 7. confirm that it is the test method of the chloroform extract of rhizoma alismatis decocting liquid that the described rhizoma alismatis of claim 1 causes renal toxicity effect toxic effect position, is characterized in that: adopt the method for pathological section, specifically be(1) chloroform extract of the described rhizoma alismatis decocting of preparation claim 1 liquid;(2) preparation of test liquidThe chloroform extract of obtaining, thin up is to being equivalent to medicinal material concentration 2 g/mL, as the chloroform extract test liquid;Get caulis aristologhiae manshuriensis and pulverize, adds 4 times of distilled water, soak 30 min after, be heated to boiling, decoct 60 min, inclining soup, filters, and decocts repeatedly 2 times, merges soup, filtration is concentrated into and is equivalent to medicinal material concentration 0.5 g/ml as positive control solution;(3) animal divides into groups and medicationAt random animal is equally divided into 3 groups by body weight: the normal control group, rhizoma alismatis chloroform extract group and caulis aristologhiae manshuriensis positive controls, irritated stomach once, continuous irrigation stomach 120 days every day after rat put into metabolic cage and adapt to 7 days;(4) pathological sectionRat is put to death after 120 days in administration, kidney is weighed, and gets renal tissues in rats and use 10% formaldehyde fixed, and FFPE is also made slice, processes 3 μ m section, through haematine-Yi red colouring, under optical microscope, observes morphological change.
- 8. confirm that it is the test method of the chloroform extract of rhizoma alismatis decocting liquid that the described rhizoma alismatis of claim 1 causes renal toxicity effect toxic effect position, is characterized in that: adopt the method for the method of metabolism group, specifically be(1) chloroform extract of the described rhizoma alismatis decocting of preparation claim 1 liquid;(2) preparation of test liquidThe chloroform extract of obtaining, thin up is to being equivalent to medicinal material concentration 2 g/mL, as the chloroform extract test liquid;Get caulis aristologhiae manshuriensis and pulverize, adds 4 times of distilled water, soak 30 min after, be heated to boiling, decoct 60 min, inclining soup, filters, and decocts repeatedly 2 times, merges soup, filtration is concentrated into and is equivalent to medicinal material concentration 0.5 g/ml as positive control solution;(3) animal divides into groups and medicationAt random animal is equally divided into 3 groups by body weight: the normal control group, rhizoma alismatis chloroform extract group and caulis aristologhiae manshuriensis positive controls, irritated stomach once, continuous irrigation stomach 120 days every day after rat put into metabolic cage and adapt to 7 days;(4) collection of biological sampleUrine collecting:Adopt the metabolic cage method, collected 24 h urines in 120 days to centrifuge tube, behind centrifugal 3 min of urine sample 13000 rpm, get supernatant and place-20 ℃ of refrigerators preservations subsequent use in administration;Serum is collected:In administration eye socket blood sampling in 120 days, place test tube, placed 30 minutes, take out the blood cake, centrifugal 5 min of solution 13000 rpm, separation obtains serum and places-20 ℃ of refrigerators preservations subsequent use;(5) pre-service of metabolism group biological sampleThe urine sample pre-service:Get urine sample 200 μ L, place 1.5 mL EP pipe, add 200 μ L methyl alcohol, vortex 3 min, centrifugal 5 min under the 13000 rpm conditions get supernatant 5 μ L and carry out the UPLC-MS analysis;The blood serum sample pre-service:Get 200 μ L serum and add in the 1.5 mL EP pipe, add 400 μ L acetonitrile precipitation albumen, in 4 ℃ low temperature environment, centrifugal 10 min of 15000 rpm, supernatant are transferred in another EP pipe, keep 30 ℃ of constant temperature, at N 2Dry up under the air-flow, with 100 μ L Yi Jing – water (15: 85, V/v) redissolve, ultrasonic 3 min, vortex 1 min behind centrifugal 5 min of 13000 rpm, gets supernatant 5 μ L and carries out the UPLC-MS analysis;(6) foundation of UPLC-MS analytical approachChromatographic conditionChromatographic column: ACQUITY UPLC TMBEH C 18Post 6; Column temperature: 35 ℃; Moving phase: 0.1% formic acid acetonitrile (A)-0.1% formic acid water (B), gradient elution;The mass spectrum conditionIon gun ESI positive ion full scan pattern, capillary voltage 3.2 kV, taper hole voltage 30 V, 120 ℃ of ion source temperatures, 350 ℃ of desolventizing temperature, desolventizing gas N 2With taper hole gas N 2Flow velocity is set to 600 and 50 L/h respectively; Adopt full surface sweeping scan mode, sweep limit is 100 ~ 900 amu, and when the scanning of ion multi-stage ms was extracted in utilization, argon gas was as collision gas; It is mass calibration that NaCsI is used at the experiment previous crops;The conclusive evidence of UPLC-MS analytical approachThe precision and the study on the stability of urine sample and blood sample analysis method all meet the requirements;(7) data processingThis research application Markerlynx software carries out chromatographic peak identification and peak match, and adopts PCA (PCA) that the metabolin group of each group rat plasma and urine sample is analyzed.
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