CN106370500A - Extraction separation method of fat-soluble metabolite in faeces - Google Patents
Extraction separation method of fat-soluble metabolite in faeces Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/34—Purifying; Cleaning
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- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/42—Selective adsorption, e.g. chromatography characterised by the development mode, e.g. by displacement or by elution
- B01D15/424—Elution mode
- B01D15/426—Specific type of solvent
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
- G01N1/405—Concentrating samples by adsorption or absorption
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Abstract
The invention discloses an extraction separation method of a fat-soluble metabolite in faeces. The extraction separation method comprises the following steps: extracting the metabolite in the faeces by an organic solvent, carrying out gradient chromatography by a petroleum ether/ethyl acetate elution system I and a dichloromethane/methyl alcohol elution system II, and separating metabolite monomers through high performance liquid chromatography. According to the extraction separation method, large-polarity and small-polarity impurities in a sample are removed through normal-phase silica gel column chromatography of gradient elution, the method plays a good role in enrichment and separation for the metabolite, the metabolite monomers can be obtained through extraction and separation, and the disadvantage that isomerides of the metabolite are difficultly identified by HPLC-MS is overcome; and moreover, while subsequent structure determination is guaranteed to be completed, an extracted and separated metabolite standard sample can be obtained.
Description
Technical field
A kind of it relates to extraction separation method of metabolite, in particular it relates to lipid soluble metabolites in feces
Extraction separation method.
Background technology
Nutritional labeling in food or medicine etc. enter internal, through the digested suction of the organs such as oral cavity, esophagus, stomach, small intestinal
Receive, nutrient enters blood circulation from intestinal absorption, THPV enters liver.Liver is to endogenouss and exogenous material
These nutrients or medicine are resolved into other products by metabolism by the major organs of metabolism.Product after metabolism may
There is the biological activity more higher than parent, so research interior metabolism product is all significant to medicine and food service industry.
At present, the method that the research of metabolite relies primarily on hplc-ms.But, hplc-ms cannot isolation identification have
The metabolite of same molecular quality, this is totally unfavorable for the research of metabolite.
Content of the invention
In order to overcome drawbacks described above, present disclose provides a kind of extraction separation method of lipid soluble metabolites, the method
Extract separation efficiency high, the advantage being obtained in that metabolite monomer.
Present disclose provides in a kind of feces lipid soluble metabolites extraction separation method, methods described specifically includes that
A animal wastes are carried out reflux, extract, in organic solvent by (), obtain primary extract；B () is by described primary extract with washing
De- system i carries out Gradient column chromatography, then carries out gradient chromatography using elution system ii, collects the primary separation product of gained；
Described elution system i contains petroleum ether and ethyl acetate, and described elution system ii contains dichloromethane and methanol, described chromatographic column
For purification on normal-phase silica gel filled column；C () carries out isolation using high performance liquid chromatography (hplc) to described primary separation product,
Elution system is acetonitrile/water mixed liquor.
By technique scheme, instant invention overcomes cannot distinguish between same molecular amount using hplc-ms in existing research
Metabolite shortcoming, the extraction separation efficiency of the present invention is high simultaneously, process is simple, is ensureing to complete follow-up structure determination
Meanwhile, the detached metabolite composition of extraction can also be obtained.
Other feature and advantage of the disclosure will be described in detail in subsequent specific embodiment part.
Specific embodiment
Specific embodiment of this disclosure is described in detail below.It should be appreciated that it is described herein concrete
Embodiment is merely to illustrate and explains the disclosure, is not limited to the disclosure.
Extraction ratio described in the disclosure is to extract target product content in the target product yield/feces obtaining.
The invention provides in a kind of feces lipid soluble metabolites extraction separation method, methods described step includes:
A animal wastes are carried out reflux, extract, in organic solvent by (), obtain primary extract；B () is by described primary extract with washing
De- system i carries out Gradient column chromatography, then carries out gradient chromatography using elution system ii, collects the primary separation product of gained；
Described elution system i contains petroleum ether and ethyl acetate, and described elution system ii contains dichloromethane and methanol, described chromatographic column
For purification on normal-phase silica gel filled column；C () carries out isolation using high performance liquid chromatography (hplc) to described primary separation product,
Elution system is acetonitrile/water mixed liquor.
By technique scheme, instant invention overcomes cannot distinguish between same molecular amount using hplc-ms in existing research
Metabolite shortcoming, the extraction separation efficiency of the present invention is high simultaneously, process is simple, is ensureing to complete follow-up structure determination
Meanwhile, the detached metabolite composition of extraction can also be obtained.
Further, in order to improve the extraction efficiency of the present invention, described step (a) can carry out twice, and described backflow carries
The time taking can be 4-24 hour.
Further, in order to improve the content of metabolite, described animal wastes can need to study metabolism product for feeding
The feces that after the composition of thing, animal produces.
Further, in step (a), described feces are 1:(10-1000 with the weight/volume of organic solvent).
Further, in step (a), described organic solvent is selected from ethyl acetate, normal hexane and n-butyl alcohol at least
One kind, the higher extraction efficiency that above organic solvent can obtain.
Further, in order to remove the impurity of the big polarity in sample and little polarity, in step (b), described elution system i
In the eluent petrochina ether that uses successively be 9:1,8:2,7:3 with the volume ratio of ethyl acetate；Described elution system ii is successively
Use eluent in dichloromethane and methanol volume ratio be 100:1,90:1,80:1,50:1,30:1,20:1,15:1,10:
1st, 8:1,5:1；The applied sample amount of described primary extract is (3-10) with chromatographic column bed volume ratio: 1；Each gradient elution described
The volume of liquid is (1-3) with chromatographic column bed volume ratio: 1；Described purification on normal-phase silica gel filled column is 100-400 mesh；Described eluent
Flow velocity be 2ml/min-20ml/min, eluting temperature be 4-40 DEG C.
Further, in order to obtain more preferable enrichment and separating effect, chromatographic column described in step (b) is 200 mesh -300
Mesh purification on normal-phase silica gel filled column.
Further, the formic acid that can also add 1% in elution system described in step (c) makes the separation of metabolite
Effect is more preferable.
Further, described animal wastes are the rat feces of feeding 5- hydroxyl Nobiletin；The method includes: (1) is given
Described feeding rats with the addition of the food of described 5- hydroxyl Nobiletin, and collects rat feces, and described feces are crushed；(2)
Using organic solvent, reflux, extract, is carried out to described feces, obtain primary extraction product；(3) described primary extraction product is adopted
Purification on normal-phase silica gel filler column chromatography, through petrol ether/ethyl acetate elution system i, methylene chloride/methanol elution system ii gradient elution
Afterwards, obtain primary separation product；(4) described primary separation product is carried out high performance liquid chromatography through acetonitrile/water elution system to divide
From obtaining metabolite monomer.
Further, in the extraction separation method of the above-mentioned metabolite to 5- hydroxyl Nobiletin, in order to carry further
Rise and extract separation efficiency, the eluent petrochina ether that can use successively in elution system i described in step (2) and ethyl acetate
Volume ratio be 9:1,8:2,7:3；The body of dichloromethane and methanol in the eluent that described elution system ii can use successively
Long-pending ratio is 100:1,90:1,80:1,50:1,30:1,20:1,15:1,10:1,8:1,5:1；The flow velocity of described eluent can be
2ml/min-20ml/min, eluting temperature can be 4-40 DEG C.
With reference to specific embodiment, the present invention is further elaborated, but the present invention does not therefore suffer from any restriction.
Methods described is conventional method if no special instructions.Described raw material all can obtain from open commercial sources if no special instructions
?.
In embodiment 1 polymethoxyflavone (5- hydroxyl Nobiletin) feed rat feces, the extraction of metabolite separates and grinds
Study carefully
This case study object is the f344 rat of feeding 5- hydroxyl Nobiletin.Raised in metabolic cage, and given
Give ain-76a Mus standard chow (adding gross weight 0.1%5- hydroxyl Nobiletin).Daily timing carries out supplementing simultaneously to food pot
Rat feces are collected in one week.
Be metabolite in rat 5- hydroxyl Nobiletin feces is carried out deep excavate with exploitation, present invention employs as
Lower extraction separation method:
(1) 4.2g rat fecal specimens are pulverized, using organic solvent 1000ml reflux, extract, 24 hours, concentrating under reduced pressure steamed
Dry solvent；Feces residue adopts identical organic solvent reflux, extract, again, obtains primary and carry after merging with first time extract
Take product.Primary extraction product concentrates after being evaporated and weighs.
Three kinds of organic solvent-normal hexanes, ethyl acetate, n-butyl alcohol reflux, extract, metabolite respectively is employed in this step,
Using hplc liquid phase analysis, reflux, extract, metabolite extraction ratio twice is detected and be respectively as follows: ethyl acetate 93%；N-butyl alcohol
92%；Normal hexane 74%.
(2) purification on normal-phase silica gel filler column chromatography, chromatographic column bed volume and loading are adopted to ethyl acetate primary extraction product
Amount volume ratio is 6:1；It is respectively adopted the purification on normal-phase silica gel filled column that mesh number is 100-200,200-300,300-400；Using oil
Ether/ethyl acetate elution system i and methylene chloride/methanol elution system ii carry out gradient elution；In elution system i, make successively
Eluent petrochina ether is respectively 9:1,8:2,7:3 with the volume ratio of ethyl acetate；In elution system ii, use successively
In eluent the volume ratio of dichloromethane and methanol be respectively 100:1,95:1,90:1,80:1,50:1,30:1,20:1,15:1,
10:1,8:1,5:1, the flow velocity of eluent is 10ml/min；Eluting temperature is room temperature；Each gradient elution agent volume and chromatographic column
The ratio of bed volume is 2:1；Collect the eluent of each eluting ratio, tlc detects, weighs.Post layer is detected by positive lamellae
The separating effect of analysis.
Result proves: compound eluent system petrol ether/ethyl acetate 9:1,8:2 can remove oils and fatss, volatility abnormal flavour by eluting
The common 660mg of impurity of the little polarity such as composition, accounts for the 60% of primary extraction product weight；Methylene chloride/methanol 8:1 and 5:1 can
Remove the big common 120mg of polar impurity of saccharide, protein etc. in sample, account for the 11% of primary extraction product weight；Dichloromethane/
The composition weight that methanol system 100:1 gradient elution obtains is 143mg, accounts for the 13% of primary extraction product weight.Use
The purification on normal-phase silica gel filled column of 200-300 mesh, metabolite is enriched about 7.7 times, and gradient separations have to metabolite divides well
Level effect.
(3) efficient liquid phase preparation is carried out to the component of each metabolite, using acetonitrile/water elution system, elution requirement
Different according to the polarity difference of each metabolite, add 1% formic acid to adjust ph value thus improving separation efficiency.Add 1%
Formic acid after, the sample concentrating under reduced pressure for preparing is evaporated 3 main metabolite monomers of acquisition, respectively 4 ', 5 '-dihydroxy
Base Nobiletin 28mg, purity reaches 98%；3 ', 5 '-dihydroxy Nobiletin 7mg, purity reaches 99%；3 ', 4 ', 5 '-three
Hydroxyl Nobiletin 15mg, purity reaches 98%.
The extraction Separation Research of metabolite in embodiment 2 dihydrochalcone (phlorhizin) feeding mice feces
This case study object is the aj mice of feeding table phlorhizin.Raised in metabolic cage, and given ain-76a
Mus standard chow (adds gross weight 0.1% phlorhizin).Daily timing is supplemented to food pot and was collected rat in one week
The common 3.6g of all feces.To phlorhizin interior metabolism product in stool in mice adopt the present invention extraction separation method:
(1) 3.6g rat fecal specimens are pulverized, be respectively adopted normal hexane, ethyl acetate, three kinds of organic solvents of n-butyl alcohol
Each 1000ml reflux, extract, 24 hours, concentrating under reduced pressure solvent evaporated；Feces residue is carried using the backflow of identical organic solvent again
Take, after merging with first time extract, obtain primary extraction product.Concentrate to be evaporated to weigh and obtain the weight of primary extraction product.
Using hplc liquid phase analysis, detect that the extraction ratio of organic solvent reflux, extract, primary metabolite twice is respectively as follows:
Ethyl acetate 90%；N-butyl alcohol 88%；Normal hexane 59%.
(2) purification on normal-phase silica gel filler column chromatography, chromatographic column bed volume and sample are adopted to ethyl acetate primary extraction product
The ratio of applied sample amount volume is 8:1；Attempting mesh number respectively is 100-200 mesh, 200-300 mesh, the purification on normal-phase silica gel filler of 300-400 mesh
Post；Gradient elution is carried out using petrol ether/ethyl acetate elution system i and methylene chloride/methanol elution system ii；Elution system
In i, the eluent petrochina ether and the volume ratio of ethyl acetate that use successively are respectively 9:1,8:2,7:3；In elution system ii,
In the eluent using successively the volume ratio of dichloromethane and methanol be respectively 100:1,95:1,90:1,80:1,50:1,30:1,
20:1、15:1、10:1、8:1、5:1；The flow velocity of eluent is 8ml/min；Eluting temperature is room temperature；Each gradient elution agent body
The long-pending ratio with chromatographic column bed volume collects the eluent of each eluting ratio for 2:1, and tlc detects, weighs.By positive lamellae
The separating effect of detection column chromatography.
Result proves: compound eluent system petrol ether/ethyl acetate 9:1,8:2 can remove oils and fatss, volatility abnormal flavour by eluting
The common 647mg of impurity of the little polarity such as composition, accounts for the 65% of primary extraction product weight；Methylene chloride/methanol 8:1 and 5:1 can
Remove the big common 101mg of polar impurity of saccharide, protein etc. in sample, account for the 10% of primary extraction product weight；Dichloromethane/
The composition weight that methanol system 100:1 gradient elution obtains is 148mg, accounts for the 15% of primary extraction product weight.Use
The purification on normal-phase silica gel filled column of 200-300 mesh, metabolite is enriched about 6.7 times, and gradient separations have to metabolite divides well
Level effect.
(3) efficient liquid phase preparation is carried out to the component of each metabolite, using acetonitrile/water elution system, elution requirement
Different according to the polarity difference of each metabolite, add 1% formic acid to adjust ph value thus improving separation efficiency.Add 1%
Formic acid after, the sample for preparing is evaporated 1 main metabolite monomer of acquisition through concentrating under reduced pressure, and phloretin 68mg is pure
Degree reaches 98%.
By embodiment 1,2 as can be seen that instant invention overcomes cannot distinguish between identical point using hplc-ms in existing research
The shortcoming of the metabolite of son amount, the extraction separation efficiency height of the present invention, process is simple simultaneously, ensureing that completing subsequent structural surveys
While determining, also can obtain the detached high-purity metabolite composition of extraction, and the present invention extracts fat-soluble generation from feces
Thank and there is during product good separating effect.Present invention organic solvent used in reflux, extract, process is normal hexane, acetic acid
When ethyl ester, n-butyl alcohol, effect preferably, and preferably uses ethyl acetate and can make extraction rate reached to more than 90%.
The preferred implementation of the disclosure described in detail above, but, the disclosure is not limited in above-mentioned embodiment
Detail, in the range of the technology design of the disclosure, multiple simple variant can be carried out with technical scheme of this disclosure, this
A little simple variant belong to the protection domain of the disclosure.
It is further to note that each particular technique feature described in above-mentioned specific embodiment, in not lance
In the case of shield, can be combined by any suitable means, in order to avoid unnecessary repetition, the disclosure to various can
The compound mode of energy no longer separately illustrates.
Additionally, combination in any can also be carried out between the various different embodiment of the disclosure, as long as it is without prejudice to this
Disclosed thought, it equally should be considered as disclosure disclosure of that.
Claims (9)
1. in a kind of feces the extraction separation method of lipid soluble metabolites it is characterised in that methods described comprises the steps:
A animal wastes are carried out reflux, extract, in organic solvent by (), obtain primary extract；
B described primary extract is carried out Gradient column chromatography with elution system i by (), then carry out gradient layer using elution system ii
Analysis, collects the primary separation product of gained；Described elution system i contains petroleum ether and ethyl acetate, and described elution system ii contains
There are dichloromethane and methanol, described chromatographic column is purification on normal-phase silica gel filled column；
C () carries out isolation using high performance liquid chromatography (hplc) to described primary separation product, elution system is second
Nitrile/water mixed liquid.
2. method according to claim 1, wherein, described step (a) carries out twice, and the time of described reflux, extract, is 4-
24 hours.
3. method according to claim 1, wherein, in step (a), described animal wastes need to study generation for feeding animals
The feces producing after thanking to the composition of product.
4. method according to claim 1, wherein, in step (a), the weight/volume of described feces and organic solvent
For 1:(10-1000).
5. method according to claim 1, wherein, in step (a), described organic solvent be selected from ethyl acetate, just oneself
At least one in alkane and n-butyl alcohol.
6. method according to claim 1, wherein, in step (b), the eluent that uses successively in described elution system i
Petrochina ether is 9:1,8:2,7:3 with the volume ratio of ethyl acetate；Dichloro in the eluent that described elution system ii uses successively
The volume ratio of methane and methanol is 100:1,90:1,80:1,50:1,30:1,20:1,15:1,10:1,8:1,5:1；Described primary
Extracting the applied sample amount of product and chromatographic column bed volume ratio is (3-10): 1；The volume of each gradient eluent described and chromatographic column
The ratio of bed volume is (1-3): 1；Described purification on normal-phase silica gel filled column is 100 mesh -400 mesh；The flow velocity of described eluent is 2ml/
Min-20ml/min, eluting temperature is 4-40 DEG C.
7. method according to claim 6, wherein, the applied sample amount of the first extract described in step (b) and chromatographic column post
Bed volume is than for 6:1；The volume of each gradient eluent described is 2:1 with the ratio of chromatographic column bed volume.
8. method according to claim 1, wherein, described animal wastes are the big Oletum Ratti norvegici of feeding 5- hydroxyl Nobiletin
Just；The method includes:
(1) with the addition of the food of described 5- hydroxyl Nobiletin to described feeding rats, and collect rat feces, by described feces
Broken；
(2) using organic solvent, reflux, extract, is carried out to described feces, obtain primary extraction product；
(3) by described primary extraction product adopt purification on normal-phase silica gel filler column chromatography, through petrol ether/ethyl acetate elution system i, two
After chloromethanes/methanol-eluted fractions system ii gradient elution, obtain primary separation product；
(4) described primary separation product is obtained metabolite monomer through high performance liquid chromatography separation, described elution system contains
Acetonitrile/water mixed liquor.
9. method according to claim 8, wherein, in the eluent using successively in elution system i described in step (2)
Petroleum ether is 9:1,8:2,7:3 with the volume ratio of ethyl acetate；Dichloromethane in the eluent that described elution system ii uses successively
The volume ratio of alkane and methanol is 100:1,90:1,80:1,50:1,30:1,20:1,15:1,10:1,8:1,5:1；Described eluent
Flow velocity be 2ml/min-20ml/min, eluting temperature be 4-40 DEG C.
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Cited By (1)
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