CN102608241A - Traditional Chinese medicine (TCM) active ingredient fingerprint quality control method established on basis of Caco-2 cell model - Google Patents
Traditional Chinese medicine (TCM) active ingredient fingerprint quality control method established on basis of Caco-2 cell model Download PDFInfo
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- CN102608241A CN102608241A CN2011103527179A CN201110352717A CN102608241A CN 102608241 A CN102608241 A CN 102608241A CN 2011103527179 A CN2011103527179 A CN 2011103527179A CN 201110352717 A CN201110352717 A CN 201110352717A CN 102608241 A CN102608241 A CN 102608241A
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Abstract
The invention relates to a traditional Chinese medicine (TCM) active ingredient fingerprint quality control standard establishing method with strong operability, reliable experiment results and good repeatability on the basis of a Caco-2 cell model. The technical points of the method are that a medicament is prepared to serve as a sample; the Caco-2 monolayer cell model is established; research on transport through cell membranes of the medicament is performed; HPLC (High Performance Liquid Chromatography) detection is performed on chemical compositions of the medicament after the transport through cell membranes; similarities and differences of active ingredients of rhizoma coptidis and fructus evodiae with different proportions are defined through comparison, and differences and similarities of index ingredients of the rhizoma coptidis and the fructus evodiae before and after supersession are defined through comparison; and fingerprint quality control standards on the active ingredients of the rhizoma coptidis and the fructus evodiae are established. The method belongs to the technical field of TCM analysis.
Description
Technical field
The present invention relates to a kind of method for building up of traditional Chinese medicine ingredients collection of illustrative plates quality control standard, specifically, a kind of active ingredient of Chinese herbs finger-print quality control method of setting up based on the Caco-2 cell model; Belong to the Chinese medicine analysis technical field.
Background technology
The drug matching rule is one of elite of traditional Chinese medical theory.Ancients' cloud " medication is as using military forces ", and in the compatibility of Chinese medicine was used, the right use of medicine more is original and different, and was quite characteristic.Medicine is to being the minimum compatibility unit of Chinese medicine; Be form and core basic, the most the most frequently used in the compound compatibility; Also being the key link that medicine rises to prescription, is the bridge between simple and prescription, has both strengthened the effect of medicine; Enlarged the therapeutic domain of medicine, laid a good foundation for the compatibility of Chinese medicinal formulae again.
Two simple compatibilities of medicine centering, or strengthen efficacy of drugs, or lower poisonous side effect of medicine.Behind the simple compatibility, its chemical constitution what has change and with the relation of simple, promptly medicine is the core of compound chemical to chemistry, is worth further investigation.
Middle pharmaceutically active ingredient is through metabolism; Its chemical constitution quality and quantity all changes; With the index property composition of the chemical substance in the effective constituent, can not accurately reflect medicine pharmacodynamics effect and toxicity size, and can be used as the index that traditional Chinese medicine quality detects as the active ingredient of Chinese herbs that medicine is really brought into play the material base of drug effect as traditional Chinese medicine quality; Through chemical constitution comparative study before and after the metabolism of Chinese medicine; Inquire into the active ingredient of Chinese herbs metabolic rule, the material base of clear and definite herbal pharmacology effect, foundation is that the traditional Chinese medicine quality examination criteria of index is significant with the active component.
Summary of the invention
The present invention relates to a kind of method for building up of the active ingredient of Chinese herbs finger-print quality control standard based on the Caco-2 cell model, this method is workable, reliable experiment result, can repeat.
Technical scheme of the present invention is such: a kind of method for building up of the active ingredient of Chinese herbs finger-print quality control standard based on the Caco-2 cell model, this method comprises the steps: successively
1) preparation medicine test sample
Take by weighing the coptis and evodia rutaecarpa in 1: 1,2: 1,6: 1 by mass ratio, add in the round-bottomed flask, add 8 times of water gagings of sample gross mass for the first time, soak 30min; Second and third time adds 4 times of water gagings of medicine gross mass respectively, the timing of boiling back, and each 1h merges three times decocting liquid, is concentrated into driedly, puts that drying under reduced pressure becomes powder in the vacuum drying chamber, and it is subsequent use to put 4 ℃ of refrigerators preservations;
2) set up Caco2 cell monolayer model
Cultivate based on the cultivation of going down to posterity in the culture flask with the high sugared DMEM that contains 10% hyclone; On Nunc plug-in type culture plate, planting density is 5 * 10 with the cell inoculation that grows fine
4Individual cell mL
-1The nutrient culture media that adds 0.5mL and 1.5mL respectively at placenta percreta A face and mucous layer B face is put into 37 ℃ of incubators and is hatched; Before 14d change liquid every other day, change liquid later every day 1 time, cultivate about 21d, mensuration is striden the film resistance value, when times blank well TEER value of TEER＞1.5～2, explaining that cell reaches converges, is differentiated to form monolayer, is used for testing;
3) transmembrane transport of medicine research
The Caco-2 cell is after forming closely complete monolayer on the Nunc plug-in type culture plate, and each hole is swung with HBSS liquid and washed cell 3 times; Every then hole adds 37 ℃ of HBSS liquid of 2mL, places and continues to hatch 1h on the air shaking table, removes the attachment of cell surface, discards HBSS liquid; Medicine is diluted to the maximum cell non-toxic concn with HBSS liquid.Medicine from the transhipment test operation of villous surface side to basal surface side is: add the 0.5mL medicine in the AP side, the B face adds 2.0mLHBSS liquid; Placing 37 ℃ of rotating speeds is 50rmin
-1The air shaking table on, respectively at behind the medicine carrying 0,30,60,90,120,180min collects acceptable solution 0.5mL from the B side, and adds equivalent HBSS liquid immediately; The transhipment of medicine from the BL side to the AP side then adds the BL side with medicine, and the AP side adds blank HBSS liquid, and other step is with the transhipment test operation of AP side to BL side; Investigation receives in the test solution composition at the absorption expression of cellular layer, and calculates the composition that can express and the absorption rate of characteristic;
4) chemical constitution behind the transmembrane transport being carried out HPLC detects
5) set up the finger-print quality control standard of Chinese medicine coptis evodia rutaecarpa effective constituent.
From the globality of chromatogram, relatively the chromatogram of all mensuration and record confirms that at last 19 peaks of the coptis, evodia rutaecarpa are characteristic peak, forms its finger-print.
The method for building up of above-mentioned traditional Chinese medicine ingredients collection of illustrative plates quality control standard based on the Caco-2 cell model is characterized in that described miillpore filter is the miillpore filter of 0.45 μ m.
The method for building up of above-mentioned traditional Chinese medicine ingredients collection of illustrative plates quality control standard based on the Caco-2 cell model is characterized in that described condition of culture is 37 ℃, 5%CO2.
The method for building up of above-mentioned traditional Chinese medicine ingredients collection of illustrative plates quality control standard based on the Caco-2 cell model is characterized in that diameter is 25mm on the described Nunc plug-in type culture plate, micro-pore diameter: 0.4 μ m.
The method for building up of above-mentioned traditional Chinese medicine ingredients collection of illustrative plates quality control standard based on the Caco-2 cell model is characterized in that described chromatographic condition: coptis alkaloid chromatographic condition: chromatographic column: Agilent Extend C18 post (4.6mm * 150mm, 5 μ m); Moving phase: acetonitrile (A)-0.3% phosphoric acid 0.2% triethylamine (B); Flow velocity: 0.7ml/min; Detect wavelength: 268nm; Column temperature: 20 ℃; Working time: 65min; Back working time: 10min.
The chromatographic condition of rutaecarpin: chromatographic column: Agilent Extend C18 post (4.6mm * 150mm, 5 μ m); Moving phase: acetonitrile (A)-2% tetrahydrofuran 0.2% acetate (B)=47: 53; Flow velocity: 1ml/min; Detect wavelength: 225nm; Column temperature: 30 ℃; Working time: 15min, sample size: 50 μ L.
The present invention has following beneficial effect:
1, method provided by the present invention is workable, reliable experiment result, can repeat.
2, method provided by the present invention has been set up the finger-print quality control standard of the different proportionings of coptis evodia rutaecarpa; Expectation has fundamentally solved because the Chinese medicine compound prescription complicated component causes existing traditional Chinese medicine quality standard can not accurately reflect the defective of Chinese medicine curative effect, for modernization of Chinese medicine development provides new experimental technique.
3, research contents of the present invention will provide demonstration research for the traditional Chinese medicine quality establishment of standard, and the technology platform of being set up will be served whole traditional Chinese medicine quality research industry, promote its development, have the wide application prospect of sending out.
Description of drawings
Fig. 1 is the coptis, evodia rutaecarpa compatibility finger-print feature spectrogram;
Fig. 2 is I～X finger-print chromatogram;
Fig. 3 is Jatrorrhizine chloride, palmatin hydrochloride and hydrochloric acid barberry reference substance chromatogram;
Fig. 4 is Jatrorrhizine chloride in the coptis-evodia rutaecarpa (1: 1), palmatin hydrochloride and Berberine hydrochloride chromatogram;
Fig. 5 is Jatrorrhizine chloride in the coptis-evodia rutaecarpa (2: 1), palmatin hydrochloride and Berberine hydrochloride chromatogram;
Fig. 6 is Jatrorrhizine chloride in the coptis-evodia rutaecarpa (6: 1), palmatin hydrochloride and Berberine hydrochloride chromatogram;
Fig. 7 is a rutaecarpin reference substance chromatogram;
Fig. 8 is a rutaecarpin chromatogram in the coptis-evodia rutaecarpa (1: 1);
Fig. 9 is a rutaecarpin chromatogram in the coptis-evodia rutaecarpa (2: 1);
Figure 10 is a rutaecarpin chromatogram in the coptis-evodia rutaecarpa (6: 1).
Embodiment
Below in conjunction with embodiment the present invention is done further detailed description, but do not constitute any restriction of the present invention.
1. prepare medicine
Getting each sample, a certain amount of (coptis: evodia rutaecarpa=1: 1 o'clock is each 100g; The coptis: evodia rutaecarpa=2: 1 o'clock is 200g and 100g; The coptis: evodia rutaecarpa=6: 1 o'clock is 600g and 100g), the accurate title, decide, and adds in the round-bottomed flask, adds 8 times of water gagings of sample quality for the first time, soaks 30min; Second and third time adds 4 times of water gagings, the timing of boiling back, and each 1h merges three times decocting liquid, is concentrated into driedly, puts that drying under reduced pressure becomes powder in the vacuum drying chamber, and it is subsequent use to put 4 ℃ of refrigerators preservations.
2. set up Caco2 cell monolayer model
High sugared DMEM with containing 10% hyclone cultivates based on 25cm
2Go down to posterity in the culture flask cultivation (37 ℃, 5%CO
2).(diameter: 25mm, micro-pore diameter: 0.4 μ m), planting density is 5 * 10 on 6 hole Nunc plug-in type culture plates with the cell inoculation that grows fine
4Individual cell mL
-1The nutrient culture media that adds 0.5mL and 1.5mL respectively at placenta percreta (Apical side) A face and mucous layer (basolateral side) B face is put into 37 ℃ of incubators and is hatched.Before 14d (day) change liquid every other day, change liquid later every day 1 time, cultivate about 21d, mensuration is striden film resistance value (TEER), when times blank well TEER value of TEER＞1.5～2, explaining that cell reaches converges, is differentiated to form the Caco-2 monolayer, can be used for testing.
3. the transmembrane transport of medicine research
The Caco-2 cell is after forming closely complete monolayer on the Nunc plug-in type culture plate, and each hole is swung with 37 ℃ of HBSS (balanced salt solution) liquid and washed cell 3 times.Every then hole adds 37 ℃ of HBSS liquid of 2mL, places and continues to hatch 1h on the air shaking table, removes the attachment of cell surface, discards HBSS liquid.The medicinal powder of preparation is diluted to maximum cell non-toxic concn (1.2mg/mL) with HBSS liquid in getting 1., and is subsequent use with the membrane filtration in 0.45um aperture.Medicine from the transhipment test operation of villous surface (AP) side to basal surface (BL) side is: the medicine B face that adds each proportioning of 0.5mL in the AP side adds 2.0mL HBSS liquid; Placing 37 ℃ of rotating speeds is 50rmin
-1The air shaking table on, respectively at behind the medicine carrying 0,30,60,90,120,180min collects acceptable solution 0.5mL from the B side, and adds equivalent HBSS liquid immediately.The transhipment of medicine from the BL side to the AP side then adds the BL side with medicine, and the AP side adds blank HBSS liquid, and other step is with the transhipment test operation of AP side to BL side.Investigation receives in the test solution composition at the absorption expression of cellular layer, and calculates the composition that can express and the absorption rate (apparent penetrating coefficient Papp) of characteristic, computing formula: Papp=Δ Q (/ Δ tAC
0).In the formula, Δ Q is the amount that medicine sees through in the Δ t time; A is the monolayer area, is 4.9cm in this test
2C
0Be the medicine initial concentration; The unit of Papp is with cms
-1Expression.Concrete outcome sees the following form.
Can know by table 1; The principal ingredient of the different proportionings of coptis evodia rutaecarpa absorbs and the transport velocity that effluxes has notable difference at the Caco-2 cell; After carrying out in twos relatively, find the obvious increase (P＜0.05～0.01) of Papp than 1: 1 of 4 kinds of alkaloids (jamaicin, palmatine, jateorrhizine and rutaecarpin) of 2: 1 and 6: 1; Wherein 6: 1 Papp is maximum (P＜0.05～0.01).And the main chemical compositions P of each proportioning
AP-BL/ P
BL-APAll greater than 1.
The different proportioning alkaloid osmotic coefficient investigating results
of table 1 coptis evodia rutaecarpa
Tab?1?Apparent?permeability?coefficient(Papp)of?Alkaloids?of?Coptis?and?Evodia?across?the?Caco-2cell?monolaye(rx±s，n＝3)
Annotate: compare * * P＜0.01 for 1: 1 group with yellow Wu; Compare ##P＜0.01 for 2: 1 groups with yellow Wu.
4. the chemical constitution behind the transmembrane transport being carried out HPLC detects
Accurate respectively reference substance solution (providing the title of reference substance) the 5 μ L that draw, acceptable solution 50 μ L measure by following chromatographic condition.
Coptis alkaloid chromatographic condition: chromatographic column: Agilent Extend C18 post (4.6mm * 150mm, 5 μ m); Moving phase: acetonitrile (A)-0.3% phosphoric acid 0.2% triethylamine (B); Flow velocity: 0.7ml/min; Detect wavelength: 268nm; Column temperature: 20 ℃; Working time: 65min; Back working time: 10min.
The chromatographic condition of rutaecarpin: chromatographic column: Agilent Extend C18 post (4.6mm * 150mm, 5 μ m); Moving phase: acetonitrile (A)-2% tetrahydrofuran 0.2% acetate (B)=47: 53; Flow velocity: 1ml/min; Detect wavelength: 225nm; Column temperature: 30 ℃; Working time: 15min, sample size: 50 μ L.
5. the similarities and differences of active principle after the different proportionings of coptis evodia rutaecarpa relatively
Consult Fig. 3 to Figure 10; Through statistical study; The principal ingredient of finding the different proportionings of coptis evodia rutaecarpa absorbs and the transport velocity that effluxes has notable difference at the Caco-2 cell; After carrying out in twos relatively, find the obvious increase of Papp than 1: 1 of 4 kinds of alkaloids (jamaicin, palmatine, jateorrhizine and rutaecarpin) of 2: 1 and 6: 1; Wherein 6: 1 Papp is maximum.And the main chemical compositions P of each proportioning
AP-BL/ P
BL-APAll greater than 1.
6. set up the finger-print quality control standard of Chinese medicine coptis evodia rutaecarpa effective constituent
From the globality of chromatogram, through the chromatogram of all mensuration relatively and record, confirm that at last 19 peaks of the coptis, evodia rutaecarpa are characteristic peak, form its finger-print.Compare for the ease of identification and analysis, whole chromatogram is divided into I, two districts of II.The position of fingerprint peaks: first district comprises 1
#～12
#Peak (retention time scope 5～32min); Second district comprises 13
#～19
#Peak (retention time scope 32～55min).1: 1 (283.5mg/mL) chromatogram is representative to close decocting liquid with the coptis, evodia rutaecarpa, demarcates the peak number and the retention time of characteristic fingerprint pattern, and each characteristic peak-to-peak number and retention time are seen table 2, representative finger-print See Figure 1.
Precision is drawn each 10 μ L of reference substance solution, and each 3 μ L of need testing solution are sample introduction respectively, through contrasting with finger-print retention time and ultraviolet spectrum, has confirmed 15
#The peak is a jateorrhizine, 18
#The peak is a palmatine, 19
#The peak is a Berberine hydrochloride.Because Berberine hydrochloride is one of important effective constituent in the coptis, and the peak area number percent of this peak in finger-print is big and stable, and degree of separation is good, therefore select its be in the finger-print with reference to the peak.Through single decocting liquid and the check analysis that closes the decocting liquid finger-print, confirmed the contribution of medicinal material of respectively distinguishing the flavor of basically in addition to compatibility decoction finger-print.
Wherein total peak 1
#Peak, 11
#Peak, 13
#Peak, 14
#Peak, 15
#Peak, 16
#Peak, 18
#Peak, 19
#The peak comes from the coptis, and 2
#Peak, 3
#Peak, 4
#Peak, 5
#Peak, 6
#Peak, 7
#Peak, 10
#Peak, 12
#Peak, 17
#The peak comes from evodia rutaecarpa.The coptis, evodia rutaecarpa medicine are seen Fig. 2 to the characteristic fingerprint pattern of different compatibilities.
Table 2 characteristic peak-to-peak number and retention time
Claims (5)
1. the method for building up based on the traditional Chinese medicine ingredients collection of illustrative plates quality control standard of Caco-2 cell model is characterized in that this method comprises the steps: successively
1) preparation medicine test sample
Take by weighing the coptis and evodia rutaecarpa in 1: 1,2: 1,6: 1 by mass ratio, add in the round-bottomed flask, add 8 times of water gagings of sample gross mass for the first time, soak 30min; Second and third time adds 4 times of water gagings of medicine gross mass respectively, the timing of boiling back, and each 1h merges three times decocting liquid, is concentrated into driedly, puts that drying under reduced pressure becomes powder in the vacuum drying chamber, and it is subsequent use to put 4 ℃ of refrigerators preservations;
2) set up Caco2 cell monolayer model
Cultivate based on the cultivation of going down to posterity in the culture flask with the high sugared DMEM that contains 10% hyclone; On Nunc plug-in type culture plate, planting density is 5 * 10 with the cell inoculation that grows fine
4Individual cell mL
-1The nutrient culture media that adds 0.5mL and 1.5mL respectively at placenta percreta A face and mucous layer B face is put into 37 ℃ of incubators and is hatched; Before 14d change liquid every other day, change liquid later every day 1 time, cultivate about 21d, mensuration is striden the film resistance value, when times blank well TEER value of TEER＞1.5～2, explaining that cell reaches converges, is differentiated to form monolayer, is used for testing;
3) transmembrane transport of medicine research
The Caco-2 cell is after forming closely complete monolayer on the Nunc plug-in type culture plate, and each hole is swung with HBSS liquid and washed cell 3 times; Every then hole adds 37 ℃ of HBSS liquid of 2mL, places and continues to hatch 1h on the air shaking table, removes the attachment of cell surface, discards HBSS liquid; Medicine is diluted to the maximum cell non-toxic concn with HBSS liquid.Medicine from the transhipment test operation of villous surface side to basal surface side is: add the 0.5mL medicine in the AP side, the B face adds 2.0mLHBSS liquid; Placing 37 ℃ of rotating speeds is 50rmin
-1The air shaking table on, respectively at behind the medicine carrying 0,30,60,90,120,180min collects acceptable solution 0.5mL from the B side, and adds equivalent HBSS liquid immediately; The transhipment of medicine from the BL side to the AP side then adds the BL side with medicine, and the AP side adds blank HBSS liquid, and other step is with the transhipment test operation of AP side to BL side; Investigation receives in the test solution composition at the absorption expression of cellular layer, and calculates the composition that can express and the absorption rate of characteristic;
4) chemical constitution behind the transmembrane transport being carried out HPLC detects
5) set up the finger-print quality control standard of Chinese medicine coptis evodia rutaecarpa effective constituent.
2. require the method for building up of described traditional Chinese medicine ingredients collection of illustrative plates quality control standard based on the Caco-2 cell model according to right 1, it is characterized in that described condition of culture is 37 ℃, 5%CO2.
3. require the method for building up of described traditional Chinese medicine ingredients collection of illustrative plates quality control standard based on the Caco-2 cell model according to right 1, it is characterized in that described Nunc plug-in type culture plate 6 hole Nunc plug-in type culture plates.
4. require the method for building up of described traditional Chinese medicine ingredients collection of illustrative plates quality control standard based on the Caco-2 cell model according to right 1, it is characterized in that diameter is 25mm on the described Nunc plug-in type culture plate, micro-pore diameter: 0.4 μ m.
5. require the method for building up of described active ingredient of Chinese herbs finger-print quality control standard based on the Caco-2 cell model according to right 1; It is characterized in that; Described chromatographic condition: coptis alkaloid chromatographic condition: chromatographic column: Agilent Extend C18 post; Specification is: 4.6mm * 150mm, 5 μ m; Moving phase: acetonitrile (A)-0.3% phosphoric acid 0.2% triethylamine (B); Flow velocity: 0.7ml/min; Detect wavelength: 268nm; Column temperature: 20 ℃; Working time: 65min; Back working time: 10min.The chromatographic condition of rutaecarpin: chromatographic column: Agilent Extend C18 post, specification: 4.6mm * 150mm, 5 μ m; Moving phase: acetonitrile (A)-2% tetrahydrofuran 0.2% acetate (B)=47: 53; Flow velocity: 1ml/min; Detect wavelength: 225nm; Column temperature: 30 ℃; Working time: 15min, sample size: 50 μ L.
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Application publication date: 20120725 |