CN1282872C - Process for detecting active ingredients of traditional Chinese medicine - Google Patents
Process for detecting active ingredients of traditional Chinese medicine Download PDFInfo
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- CN1282872C CN1282872C CN 200410041024 CN200410041024A CN1282872C CN 1282872 C CN1282872 C CN 1282872C CN 200410041024 CN200410041024 CN 200410041024 CN 200410041024 A CN200410041024 A CN 200410041024A CN 1282872 C CN1282872 C CN 1282872C
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Abstract
The present invention relates to a method for measuring effective components in traditional Chinese medicine by the interaction of biomembranes and effective components in traditional Chinese medicine. The method comprises: extract of traditional Chinese medicine and biomembranes are added in a dialysis bag for dialysis, blank control is carried out simultaneously, liquid dialyzed by the extract of traditional Chinese medicine and liquid dialyzed by blank control liquid are respectively detected in high efficiency chromatography, the changes of fingerprint spectrums of the two kinds of liquid are compared, peaks with changed peak areas are components with interaction with biomembranes, and structural information with interaction components with membranes is obtained by applying technologies of LC-MS, NMR, etc. The method has the advantages of quickness, accuracy, corresponding simplicity and cost lower. The efficiency of screening effective components from traditional Chinese medicine is obviously improved, and the present invention can be used as a supplementary method for the quality control of traditional Chinese medicine.
Description
Technical field
The detection method of pharmaceutically active ingredient in the invention belongs to is specially the method that the interaction that utilizes effective constituent in biological membrane and the Chinese medicine detects middle pharmaceutically active ingredient.
Background technology
Phytochemical method extraction active component compound group or monomeric compound wherein normally adopted in the research of traditional active ingredient of Chinese herbs, determines through pharmacological evaluation again.The traditional Chinese medicine ingredients complexity, Chinese medicine just contains tens even hundreds of compounds simply, and a compound of being made up of plurality of Chinese, wherein chemical constitution is just more, pass through decoction process again after, wherein chemical constitution may produce new variations such as chemical combination or decomposition again.In numerous chemical constitutions, and effective constituent often content is humble, so the operation of its compartment analysis is loaded down with trivial details, workload is big, the cycle is long, success ratio is low, and can not illustrate the Chinese medicine multicomponent fully, the effect characteristics of many target spots.
Method and the technology of seeking effective medicine rapid screening are the focuses that domestic and international drugmaker pays close attention to always.Modern scientific research shows that the permeability of medicine cell membrane plays a crucial role for its activity.Because most medicines must at first enter cell and could show its activity, and the innerlich anwenden thing also must just can work through the cell membrane of target cell.In brief, distribution and other physiology course in the activity of medicine, toxicity, the body is all relevant with the allocation situation of medicine on film.So the permeability of investigating the cell membrane of compound is one of primary committed step of pharmaceutically active screening and evaluation.
The model of the permeability of detection of drugs cell membrane commonly used has: organic solvent-water system such as octanol-water, ODS (18 alkyl silica gel) is the individual layer intestinal villi cell model of the reversed-phase liquid chromatography system and the ARTIFICIAL CULTURE of stationary phase, referring to: simulation biological membrane as chromatographic such as Mao Xiqin are used to predict the small intestine absorption analysis chemistry of medicine, 2001 (10): 1135-1139.Preceding two kinds of patterns all can only the hydrophobic effect of aids drug on film, and the polar head that can't analogue membrane phosphatide and the interaction of medicine, then a kind ofly then seems loaded down with trivial details, time-consuming.
Biological membrane as chromatographic be used to investigate the medicine cell membrane permeability and with film on a kind of newer method of protein combination situation, this method is to be carrier with silica gel or gel, on it on coating or the bonding manual simulation's cell membrane or competent cell film preparation form the chromatographic stationary phase, different and different medicines is separated according to the interactional power of medicine and cell membrane.This method also can be used for screening with biological membrane from Chinese medicine interactional chemical constitution, but there is many shortcoming in actual use in it.For example: carriers such as l, biological membrane and silica gel combine its biological property of back and can be subjected to certain influence.2, this method has strict restriction to chromatogram flow phase, promptly must be with physiological buffer as moving phase, otherwise meeting disrupting biofilm, make the different moving phases of chromatogram utilization like this and realize that the characteristics of the separation of compound of different nature are difficult to performance, compound is difficult to realize being separated from each other each other on biological chromatography.3, the biological membrane on the chromatographic column all have at any time by moving phase elute gradually may, therefore be difficult to realize and MS, the NMR coupling, thereby the structural information of compound can not be provided.4, the preparation procedure complexity of biological chromatography, strictness, cost is higher.With the simulation biological membrane as chromatographic method of coating process preparation because increased silica gel medium and the biomembranous cohesive process of simulation, so more complicated.Referring to: biological membrane as chromatographic and the applied analysis chemistry 2002 (2) in active constituents of medicine is analyzed: 231-236 thereof such as Mao Xiqin; Mao Xiqin etc., the preparation of lecithin coating biological membrane as chromatographic stationary phase and the investigation of stability thereof, chromatogram, 2001 (5): 433-435; He Lang dashes and waits enzymatic activity and the chromatographic characterization Science Bulletin thereof that is fixed on silica gel surface detail after birth, 1999 (6): 633-637.The collection of illustrative plates that above-mentioned all articles provide is all bad to the separating effect of medicine, and all moving phase is to use damping fluid, can not with the LC-MS coupling, and the chromatographic column life-span have only about a week.Silica gel medium price is higher, and the biomembranous preparation method of common simulation is very classical, and is simple.At Mao Xiqin etc., three kinds of chromatogram mode coupling application in the active ingredient of Chinese herbs preliminary screening, analytical chemistry 2003,31 (8): in the document of 992-995., there is Ligusticum wallichii simulating on the biological membrane as chromatographic post and the separating spectrum on the reversed-phase column, the separation efficiency of apparent biological membrane as chromatographic is undesirable, not as reverse-phase chromatographic column; And the biological chromatography post that is prepared from real cell membrane, its separation efficiency is just poorer.The analysis and the comparison of shorthorned epimedium roots such as Zhao Xiaojuan and leaf active component, analytical chemistry, 2002 (2): 195-197; Effective constituent Chinese Pharmaceutical Journal in the cellular membrane chromatography screening study Radix Caulophylli such as Gao Kun, 2003 (1): 14-16.
Summary of the invention
The problem that will solve of the present invention is: study a kind of quick, accurately, embody the method that interactional chemical constitution is arranged with biological membrane in the synergistic detection Chinese medicine of Chinese medicine multicomponent, avoid as much as possible because of the be affected accuracy of the screening that has influence on effective constituent of biomembranous biological property, improve the separating effect of effective constituent, and in screening effective constituent, obtain its structural information, may have active lead compound or compound group so that from Chinese medicine, seek fast.This method is simple relatively simultaneously, cost is lower, and can be used as the means of supplementing out economy of traditional Chinese medicine quality control.
For addressing the above problem, the invention provides following technical scheme.
Pharmaceutically active ingredient detection method in a kind of comprises the steps: to get Chinese medicine to be measured, and after solvent extraction, it is standby to make Chinese medicine extract; Chinese medicine extract and biological membrane joined in the bag filter dialyse; Detect the liquid that Chinese medicine extract was dialysed with the high efficiency chromatography method.In the described detection method: Chinese medicine to be measured is single medicinal material or Chinese medicine compound prescription; The solvent that extracts Chinese medicine can be water and/or organic solvent; To containing the high extract of organic solvent or organic solvent content, concentrated removal all or behind certain amount of organic solvent and/or the dilute with water, it is standby to make Chinese medicine extract.
The solvent that extracts Chinese medicine in this detection method is selected from water, C
1-C
5Lower alcohol, ethyl acetate, ether, chloroform, sherwood oil, or their potpourri; Add solubilizer in the Chinese medicine extract.Described C
1-C
5Lower alcohol be methyl alcohol, ethanol, normal butyl alcohol; The solvent that extracts Chinese medicine is neutral, acid or alkaline; Can select for use in mineral acid, inorganic base, organic acid or the organic base and acid or alkaline Chinese medicine extract; Solubilizer is selected from tween, dimethyl sulfoxide (DMSO), polyglycol, Qu Latong or their potpourri; The volume of pressing Chinese medicine extract adds the solubilizer of 0.1-5% volume.
Method for optimizing in the above-mentioned detection method is: Chinese medicine extract is water or the Chinese medicine extract that contains the preparation of 1-30% alcohol extract; Biological membrane is prepared from for the cell membrane of simulation biological membrane, human body or animal tissue cell, the potpourri of wherein simulating biological membrane and be with lecithin or lecithin and natural phospholipid; Cell membrane comprises endothelial cell membrane, erythrocyte membrane, myocardial cell membrane, smooth muscle cell film.
Comprise in the aforementioned detection method and place the damping fluid under the simulation physiological condition to dialyse Chinese medicine extract and biomembranous bag filter are housed, to not contain biomembranous Chinese medicine extract simultaneously as blank, pack into and dialyse in the bag filter, temperature and time is inversely proportional to during dialysis, temperature is low more, it is long more that the film both sides reach the needed time of balance, after treating film two lateral balances, detect the liquid of dialysing with the high efficiency chromatography method respectively, compare both finger-prints, the peak that peak area changes promptly is with biological membrane interactional composition to be arranged.Biological membrane is a liposome; The damping fluid of the simulation physiological condition of dialysis usefulness is phosphate or acetate buffer; When the dialysis temperature was 4-25 ℃, dialysis time was 12-24 hour.Phosphate buffer pH2.0-7.4 in the method more preferably, acetate buffer pH2.0-7.4; After detecting the liquid that Chinese medicine extract dialyse with the high efficiency chromatography method, with liquid phase-mass spectrum and/or nuclear magnetic resonance coupling, acquisition and film have the structural information of interaction composition.
The invention provides a kind of quick, accurately, embody the method that interactional chemical constitution is arranged with biological membrane in the synergistic detection Chinese medicine of Chinese medicine multicomponent, described detection comprises screening and measures, avoided because of the be affected accuracy of the screening that has influence on effective constituent of biomembranous biological property, can with LC-MS (liquid phase-mass spectrum, coupling Hereinafter the same), improve the separating effect of effective constituent, and in screening effective constituent, obtain its structural information, can from Chinese medicine, seek the lead compound or the compound group that may have activity fast.The inventive method is simple relatively simultaneously, cost is lower, and the efficient of screening effective constituent significantly improves from Chinese medicine, and can be used as the means of supplementing out economy of traditional Chinese medicine quality control.Traditional Chinese medicine fingerprint is a gordian technique that is used for traditional Chinese medicine quality control now, but the outstanding problem that existing finger-print exists is exactly the pharmacology information that this Chinese medicine can not be provided.For example, the red line collection of illustrative plates of the danggui buxue decoction of representing among Fig. 2 (position up) promptly is the finger-print of danggui buxue decoction, and it only can provide the chemical information of Chinese crude drug ingredient.Use the inventive method and make finger-print again with after danggui buxue decoction and the effect of simulation biological membrane, as the blue line chart of danggui buxue decoction among Fig. 2 (position below), obvious variation has taken place in the peak area that has 12 peaks as can be seen.Medicine and biomembranous interaction are the prerequisites that medicine produces pharmacologically active, and when carrying out traditional Chinese medicine quality control with finger-print, these vicissitudinous peaks should be the objects of emphasis control, because they just may be the active substance bases of this medicine.The inventive method will dialyse technology and liquid chromatography coupling, the result is easier than biological chromatography method, and conclusion is more reliable.Fig. 1 of the present invention is that danggui buxue decoction is at the separating spectrum on simulation biological membrane as chromatographic post under the testing conditions of 280nm, Fig. 2 is that the danggui buxue decoction under the same detection wavelength combines back dialysis and the relatively change of collection of illustrative plates of blank with the simulation biological membrane, and label is represented the peak that peak area changes.As seen the peak that changes of peak area is more than the peak of simulation biological chromatography post.
Below further describing pharmaceutically active ingredient screen method among the present invention:
The selection of dialysis membrane.Described dialysis membrane is meant that having certain molecule freely sees through less than its material by molecular weight by the molecular weight that can allow of amount in solution, and molecular weight also is called semi-permeable diaphragm greater than its impervious film of material by molecular weight.Can it be made certain shape according to purposes, just be bag filter as the bag shape.Commercially available dialysis membrane has tens of kinds of specifications at present, the criteria for classifying of its model is to be foundation with its molecule by amount, is that 12000 dialysis membrane allows molecular weight freely to see through less than 12000 molecule as molecule by amount, and molecular weight then can not see through greater than 12000 molecule.The experimental dialysis membrane that needs the selection different molecular by amount, complicated component in the Chinese medicine, it has composition may be various compounds, as protein, polysaccharide, the peptide class, and small-molecule substance etc., if our research object is the macromolecular substances in the Chinese medicine, then need to select for use molecule by the bigger semi-permeable diaphragm of amount, can freely see through with the protein that guarantees to dissociate, as only needing to study small-molecule substance wherein, then can select molecule by the smaller dialysis membrane of amount, only need to guarantee that small-molecule substance can freely see through dialysis membrane just, this dialysis membrane can effectively guarantee in the Chinese medicine can not to see through dialysis membrane with molecule after biological membrane combines, and does not have can freely seeing through of combination.
Biomembranous preparation.Biological membrane can be the simulation biological membrane, and described simulation biological membrane is to use the potpourri of the natural phospholipid that refers to lecithin or lecithin and composition biological cell membrane to be prepared from; Can be the cell membrane that comes from human body or animal tissue cell also, as endothelial cell membrane, erythrocyte membrane etc.The biological membrane of simulation is one of focus of the foreign study of research at present, because it is similar to genuine membrane structure, but making and preservation are simpler than genuine cell membrane, make things convenient for manyly, therefore investigated lead compound and the interactional situation of cell membrane usually bringing in early days of new drug research, to instruct further screening active ingredients work.Liposome is a kind of easier simulation biological membrane, biomembranous basic structure is the Lipid Bilayer Structure that is same as liposome, but real biological membrane also contains other material such as cholesterol, protein etc., approach genuine biomembranous simulation biological membrane so more, its preparation is also just complicated more.Along with updating of simulation biological membrane technology of preparing, its difference with genuine cell membrane will be more and more littler.Also not having commercial biological membrane at present, generally is according to the preparation of experiment needs oneself.But simulate biomembranous preparation method's list of references: flat its can wait the modern medicinal agents first published, Chinese Medicine science and technology publishing house, 597-606; But the preparation list of references of cell membrane: Xu Shuyun etc., pharmacological experimental methodology, People's Health Publisher's second edition: 522-527.
The extraction of Chinese medicine.Described Chinese medicine comprises single medicinal material and the compound of being made up of number flavor Chinese medicine.At the physicochemical property of the contained effective constituent of different Chinese medicines, can choose suitable solvent wantonly and extract.But the extract that will contact with dialysis membrane with the biological membrane effect must be can disrupting biofilm and dialysis membrane.Generally be advisable with the alcohol of water or low concentration, for example under the ethanol condition below 30%, the simulation biological membrane has certain stability, the biomembranous Lipid Bilayer Structure of meeting crash simulation of high concentration.That is to say, can use any solvent when the Chinese medicine for the treatment of sieve extracts.Be under the situation of pure liquid of water liquid or low concentration at extract, can directly extract and biological membrane effect be dialysed.If extract Chinese medicine with the alcohol of high concentration or organic solvent, acidity or the basic solvent of other possibility disrupting biofilm structure, to carry out certain processing to this extract, as adopt to concentrate and remove all or the method for certain amount of organic solvent and/or dilute with water, the bulking agent that perhaps after removing organic solvent, adds a spot of not disrupting biofilm structure, tweens as 1% etc. perhaps neutralize.In principle, content that it concentrate to be removed the amount of the degree of organic solvent and/or dilute with water and/or adds bulking agent is appropriateness with not disrupting biofilm and semi-permeable diaphragm structure, and in practical operation, this appropriateness is grasp easily by simple test.For example: the extract to be measured that takes a morsel contacts with biological membrane, observes biomembranous variation, shows then that if any melt and dissolved phenomenon this extract need further remove organic solvent and/or dilute with water.As long as the concentration of extract reaches the limit that can detect in the extract, concentration is to experiment effect and do not make significant difference.
Dialysis.With after biological membrane mixes, dialysis process is dialysed routinely, the laggard circumstances in which people get things ready for a trip analysis of spectrum of film two lateral balances with Chinese medicine extract.Simultaneously, with not making a parallel blank with the membrane-bound Chinese medicine extract of biology.Can dialyse with the damping fluid of the different pH value under the simulation physiological condition, medicine is different with biological membrane-bound situation under different PH conditions, it is fixed that this can come according to the experiment needs, the pH value of the different parts of human body also be different as pH in the stomach for acid, be alkaline in the intestines.Generally reach balance during 4 ℃ of low temperature dialysis and need 24 hours, only need 12 hours and reach balance during the dialysis of 25 ℃ of room temperatures, rising along with temperature, reaching balance time will shorten, but when temperature is too high, the extract of Chinese medicine particularly water extract is easy to go bad, and the temperature that therefore needs adjustment to suit is saved time with assurance and guaranteed the stable of traditional Chinese medicine ingredients.Generally be advisable in 24 hours with 4 ℃ of balances.
Chromatogram detects.Detect liquid that Chinese medicine extract dialysed respectively and as the extract of blank with the high efficiency chromatography method, relatively, analyze the change of both collection of illustrative plates.Wherein the peak that significantly reduces of peak area promptly is to have interactional chemical constitution with biological membrane, and the relative percentage of the change of its peak area shows the power of itself and biological membrane effect.If specimen is the extract of whole Chinese medicine or compound, say that so what obtain is exactly composition and biomembranous interaction situation in the whole Chinese medicine, if sample is simplification compound or a class or a few compounds, what obtain so is exactly simplification compound or a class or a few compounds and biomembranous interaction situation.As for simplification compound, a compounds, a few compounds, single medicinal material extract, the herbal mixture extract interacts with biological membrane between them for what difference, also can further investigate, as: the forulic acid monomer, forulic acid in the angelica extract, what difference forulic acid in the danggui buxue decoction extract has with the biological membrane effect respectively, we can by relatively they with the biological membrane effect after whether the number percent of change of peak area consistent finds out whether other composition influential to it and biomembranous interaction in the Chinese medicine.Owing to contain many compositions in the Chinese medicine, because high performance liquid chromatogram has very high separation efficiency, Chinese medicine extract many peaks can occur by high performance liquid chromatogram, and generally speaking, under a kind of elution requirement, the several components of a composition or structural similarity is just represented at a peak.Simultaneously, if the composition that can not separate under certain elution requirement, can change a kind of elution requirement will be separately.By screening technique of the present invention, just can be fast, find easily: in whole peaks, the peak and the biological membrane that have have interaction, the not interaction that has, and the interaction that has is strong, a little less than the interaction that has.The technical problem to be solved in the present invention will be sought from Chinese medicine exactly with biological membrane interactional peak, is the prerequisite that medicine plays a role because take place to interact with film.For not having interactional peak, be invalid components very likely just with biological membrane.
Description of drawings
Fig. 1, be danggui buxue decoction at the separating spectrum on simulation biological membrane post under the testing conditions of 280nm, there is tangible reservation at rarely seen 5 peaks on simulation biological membrane as chromatographic post, and the peak is very wide.
Fig. 2, to be danggui buxue decoction with the simulation biological membrane combine back dialysis and blank be collection of illustrative plates relatively, as seen there is the peak area at 12 peaks that significant change is arranged, the peak that these peak areas change is with biological membrane interactional composition to be arranged, and wherein No. 12 peak is through being defined as Ligustilide with standard control.
Fig. 3 is that Radix Angelicae Sinensis combines the back dialysis with the simulation biological membrane and as seen blank comparison collection of illustrative plates has the peak area at 6 peaks that significant change is arranged.Wherein No. 6 peak is through being defined as Ligustilide with standard control.
Fig. 4 is that the Radix Astragali combines relatively collection of illustrative plates of back dialysis and blank with the simulation biological membrane, as seen has the peak area at 8 peaks that significant change is arranged.
Fig. 5 is that danggui buxue decoction combines the back with endothelial cell membrane and blank compares collection of illustrative plates, as seen has the peak area at 7 peaks that significant change is arranged.
Embodiment
Effective constituent in the embodiment 1 screening Radix Astragali
The Radix Astragali is the qi-invigorating herb of classics, studies show that, wherein contains polysaccharide, saponin(e, alkaloid, volatile oil, multiple composition such as flavones.If composition is wherein extracted the screening of carrying out active component one by one, individual beyond doubt very complicated process adopts screening technique of the present invention therefrom to seek it fast and biological membrane has interactional possible active component.
The extraction of the Radix Astragali: the Radix Astragali 20g that gets pulverizing, the ethanol 200ml of adding 90% soaks 1h, water-bath refluxing extraction 1h, extract is removed solvent with Rotary Evaporators under 45 degree conditions, it is that 7.4 phosphate buffer fully dissolves that remainder adds 20ml pH, the centrifugal 10min of 4000rpm/min, common filter paper filtering, put preserve in the negative 20 degree refrigerators standby.
The preparation of simulation biological membrane (liposome): the injection of employing classics (can wait the modern medicinal agents first published referring to flat its, Chinese Medicine science and technology publishing house, 597-606), biological membrane (liposome) is simulated in preparation.Liposome is a kind of easier simulation biological membrane, and biomembranous basic structure is the Lipid Bilayer Structure that is same as liposome.
Take by weighing lecithin (East China Normal University chemical plant) 0.5g, use the 4ml anhydrous alcohol solution, in the syringe with its 10ml that packs into, other gets a small beaker, interior dress 10ml pH value is 7.4 phosphate buffer, it is inserted in the water-bath of band magnetic agitation, put a stirrer in the small beaker, with about water-bath adjustment to 60 degree, open magnetic agitation, with the fixing syringe of iron stand, the lecithin soln in the syringe is freely splashed in the small beaker, continuing to be stirred to after dripping off does not have the alcohol flavor and promptly gets liposome solutions.
Dialysis: getting the molecule that is about 7cm is 1 of 12000 bag filter (the magnificent bioengineering in Beijing company limited) by amount, and an end is tightened with fine rule.Wherein add 1ml and go up the liposome solutions and the lml angelica extract of method preparation; Insert that dialysis 15h reaches balance inside and outside making bag under the 20 degree room temperatures, other gets 1 bag filter and does parallel blank, promptly replaces liposome with the 1ml phosphate buffer, and all the other operations are identical.Get invisible spectro solution lml, filter the back and analyze with high performance liquid chromatogram.
Testing conditions: C
18Chromatographic column, Angilent series of high efficiency liquid chromatographic system, DAD UV-detector, moving phase: methyl alcohol: water gradient elution.Detect wavelength: 280nm, the peak that peak area changes is represented at the peak that is marked with arabic numeral, promptly with the simulation biological membrane composition that combines is arranged.
Result: Fig. 4 is that the Radix Astragali combines relatively collection of illustrative plates of back dialysis and blank with the simulation biological membrane, as seen has the peak area at 8 peaks that significant change is arranged.
The effective constituent of embodiment 2 screening Radix Angelicae Sinensis
The extraction of Radix Angelicae Sinensis: the Radix Angelicae Sinensis 5g that gets pulverizing, add 100ml water logging bubble lh, lh is extracted in 100 degree oil baths, extract is removed solvent with Rotary Evaporators under 50 degree conditions, it is that 7.0 acetate buffer solution fully dissolves that remainder adds 5ml pH, the centrifugal 10min of 4000rpm/min, common filter paper filtering, put preserve in the negative 20 degree refrigerators standby.
The preparation of simulation biological membrane (liposome): take by weighing lecithin (East China Normal University chemical plant) 0.5g, use the 5ml anhydrous alcohol solution, in the syringe with its 10ml that packs into, other gets a small beaker, interior dress 10ml pH value is 7.0 acetate buffer solutions, it is inserted in the water-bath of band magnetic agitation, put a stirrer in the small beaker, with about water-bath adjustment to 60 degree, open magnetic agitation, with the fixing syringe of iron stand, the lecithin soln in the syringe is freely splashed in the small beaker, continuing to be stirred to after dripping off does not have the alcohol flavor and promptly gets liposome solutions.
Dialysis: getting the molecule that is about 7cm is 1 of 12000 bag filter (the magnificent bioengineering in Beijing company limited) by amount, an end is tightened the back with fine rule add liposome solutions and the 1ml angelica extract that lml goes up the method preparation, then the other end is tightened with fine rule; The refrigerators dialysis 24h that put into 4 degree reach balance inside and outside making bag, and other gets 1 bag filter and does parallel blank, promptly replaces liposome with the 1ml acetate buffer solution, and all the other operations are identical.Get invisible spectro solution 1ml, filter the back and analyze with high performance liquid chromatogram.
Testing conditions: with example 1
Result: Fig. 3 is that Radix Angelicae Sinensis combines relatively collection of illustrative plates of back dialysis and blank with the simulation biological membrane, as seen has the peak area at 6 peaks that significant change is arranged, and wherein No. 6 peak is through being defined as Ligustilide with standard control.
Danggui buxue decoction and simulation biological membrane have the screening of interactional composition
The extraction of danggui buxue decoction: get and pulverize Radix Angelicae Sinensis (commercially available) 5g, the Radix Astragali (commercially available) 25g, the 75% alcohol immersion 1h that adds 10 times of amounts, refluxing extraction 1h, extract is removed solvent with Rotary Evaporators under 50 degree conditions, it is that 7.0 phosphate buffer 30ml fully dissolves the centrifugal 10min of 4000rpm/min that remainder adds 30ml pH, common filter paper filtering, put preserve in the negative 20 degree refrigerators standby.
Simulate biomembranous preparation: take by weighing lecithin (East China Normal University chemical plant) 0.6g and cholesterol 0.3g, use the 6ml anhydrous alcohol solution, in the syringe with its 10ml that packs into, other gets a small beaker, interior dress 10ml pH value is 7.0 phosphate buffers, it is inserted in the water-bath of band magnetic agitation, put a stirrer in the small beaker, with about water-bath adjustment to 60 degree, open magnetic agitation, with the fixing syringe of iron stand, the lecithin soln in the syringe is freely splashed in the small beaker, continuing to be stirred to after dripping off does not have the alcohol flavor and promptly gets liposome solutions.
Dialysis: getting the molecule that is about 7cm is 1 of 12000 bag filter (the magnificent bioengineering in Beijing company limited) by amount, and an end is tightened with fine rule.Add 1ml and go up the liposome solutions and the 1ml danggui buxue decoction of method preparation; The other end is tightened.Put into a pH that 8ml is housed and be the test tube of 7.0 phosphate buffer, the refrigerators dialysis 24h that put into 4 degree reach balance inside and outside making bag, and other gets 1 bag filter and does parallel blank, promptly replaces liposome with the lml phosphate buffer, and all the other operations are identical.Get invisible spectro solution lml, filter the back and analyze with high performance liquid chromatogram.
Testing conditions: with embodiment 1
Result: Fig. 2 is that Radix Angelicae Sinensis combines relatively collection of illustrative plates of back dialysis and blank with the simulation biological membrane, as seen has the peak area at 12 peaks that significant change is arranged, and wherein No. 12 peak is through being defined as Ligustilide with standard control.：
Embodiment 4
The screening of interactional composition is arranged with the simulation biological membrane in the extract of the different extraction processes of danggui buxue decoction
1. get and pulverize Radix Angelicae Sinensis 5g, Radix Astragali 25g, the water logging bubble 1h that adds 10 times of amounts, 1h is extracted in 100 degree oil baths, extract is removed solvent with Rotary Evaporators under 50 degree conditions, it is that 7.0 phosphate buffer fully dissolves the centrifugal 10min of 4000rpm/min that remainder adds 15ml pH, common filter paper filtering, put preserve in the negative 20 degree refrigerators standby.
Simulate biomembranous preparation: with embodiment 3
Dialysis: with embodiment 3
Testing conditions: with embodiment 3
The result: the basically identical of the number at peak and embodiment three, but the peak area at part peak reduces to some extent, promptly the content carried of water reduces to some extent.But the peak that peak area reduces in embodiment three collection of illustrative plates equally in this example peak area reduced.
2. get and pulverize Radix Angelicae Sinensis (commercially available) 5g, the Radix Astragali (commercially available) 25g, the 75% alcohol immersion 1h that adds 10 times of amounts, refluxing extraction 1h, extract is removed solvent with Rotary Evaporators under 50 degree conditions, it is that 7.0 phosphate buffer 30ml fully dissolves the centrifugal 10min of 4000rpm/min that remainder adds pH that 30ml contains 0.5% tween, common filter paper filtering, put preserve in the negative 20 degree refrigerators standby.
Simulate biomembranous preparation: with embodiment 3
Dialysis: getting the molecule that is about 7cm is 1 of 12000 bag filter (the magnificent bioengineering in Beijing company limited) by amount, and an end is tightened with fine rule.Add lml and go up the liposome solutions and the 1ml danggui buxue decoction of method preparation; The other end is tightened, put into one the test tube that pH that 8ml contains 0.5% tween is 7.0 phosphate buffer is housed, the refrigerators dialysis 24h that put into 4 degree reach balance inside and outside making bag, and other gets 1 bag filter and does parallel blank, promptly replace liposome with the 1ml phosphate buffer, all the other operations are identical.Get invisible spectro solution 1ml, filter the back and analyze with high performance liquid chromatogram.
Testing conditions: with embodiment 3
The result: the basically identical of the number at peak and embodiment three, but the peak area at part peak increases to some extent, significantly increase as the peak area of No. 12 peak ligustilides among the embodiment 3, promptly tween has solubilization to some composition.But the peak that peak area reduces in embodiment three collection of illustrative plates equally in this example peak area reduced.
The screening that danggui buxue decoction and interior epithelium have interactional effective constituent
The extraction of danggui buxue decoction: with embodiment 3
The preparation of endothelial cell membrane: get after healthy puerpera's caesarean section 12 hours with interior umbilical cord, be immersed in the aseptic umbilical cord of 4 degree immediately and gather in the liquid.Cultivate after being improved by the Jaffe method.Collagenase digesting with 0.1% separates endothelial cell, with 2 * 10
4/ ml cell inoculation is cultivated with the M199 nutrient solution that contains 20% calf serum in 96 well culture plates.Be passaged to cell growth stable after, change in the culture flask of 4 500ml and cultivate, merge 4 bottles of cells, reference literature prepares endothelial cell membrane: Xu Shuyun etc., pharmacological experimental methodology, People's Health Publisher's second edition: 524-525.
Dialysis: getting the molecule that is about 7cm is 1 of 12000 bag filter (the magnificent bioengineering in Beijing company limited) by amount, and an end is tightened with fine rule.Add 1ml and go up the endothelial cell coating solution and the 1ml danggui buxue decoction of method preparation; The other end is tightened.Put into a pH that 8ml is housed and be the test tube of 7.0 phosphate buffer, the refrigerators dialysis 24h that put into 4 degree reach balance inside and outside making bag, other gets 1 bag filter and does parallel blank, promptly replaces the endothelial cell coating solution with the 1ml phosphate buffer, and all the other operations are identical.Get invisible spectro solution 1ml, filter the back and analyze with high performance liquid chromatogram.
Testing conditions: C
18Chromatographic column, Angilent series of high efficiency liquid chromatographic system, DAD UV-detector, moving phase: methyl alcohol: water gradient elution.Detect wavelength: 313nm, the peak that peak area changes is represented at the peak that is marked with arabic numeral, promptly with endothelial cell membrane the composition that combines is arranged.
Result: Fig. 5 is that danggui buxue decoction combines the back dialysis with endothelial cell membrane and blank compares collection of illustrative plates, as seen has the peak area at 7 peaks that significant change is arranged.
The interpretation of result of screening experiment (referring to Fig. 1-5):
Fig. 1 is a danggui buxue decoction at the separating spectrum on simulation biological membrane post under the testing conditions of 280nm, and there is tangible reservation at rarely seen 5 peaks on simulation biological membrane as chromatographic post, and the peak is very wide.Fig. 2 is that danggui buxue decoction combines relatively collection of illustrative plates of back dialysis and blank with the simulation biological membrane, as seen there is the peak area at 12 peaks that significant change is arranged, the peak that these peak areas change is with biological membrane interactional composition to be arranged, and wherein No. 12 peak is through being defined as the effective constituent Ligustilide with standard control.Fig. 3 is that Radix Angelicae Sinensis combines relatively collection of illustrative plates of back dialysis and blank with the simulation biological membrane, as seen has the peak area at 6 peaks that significant change is arranged, and wherein No. 6 peak is through being defined as Ligustilide with standard control.Fig. 4 is that the Radix Astragali combines relatively collection of illustrative plates of back dialysis and blank with the simulation biological membrane, as seen has the peak area at 8 peaks that significant change is arranged.Fig. 5 is that danggui buxue decoction combines the back dialysis with endothelial cell membrane and blank compares collection of illustrative plates, as seen has the peak area at 7 peaks that significant change is arranged.Ligustilide is the principal ingredient in the Chinese angelica volatile oil, accounts for 45% of Chinese angelica volatile oil, and Chinese angelica volatile oil has dual regulation to the uterus.
Claims (10)
1, a kind of middle pharmaceutically active ingredient detection method comprises the steps:
Get Chinese medicine to be measured, after solvent extraction, it is standby to make Chinese medicine extract;
Chinese medicine extract and biological membrane joined in the bag filter dialyse;
Detect the liquid that Chinese medicine extract was dialysed with the high efficiency chromatography method.
2, the detection method of claim 1 is characterized in that: Chinese medicine to be measured is single medicinal material or Chinese medicine compound prescription; The solvent that extracts Chinese medicine is water and/or organic solvent; To containing the high extract of organic solvent or organic solvent content, concentrated removal all or behind certain amount of organic solvent and/or the dilute with water, it is standby to make Chinese medicine extract.
3, the detection method of claim 2 is characterized in that: the solvent that extracts Chinese medicine is selected from water, C
1-C
5Lower alcohol, ethyl acetate, ether, chloroform, sherwood oil, or their potpourri; Add solubilizer in the Chinese medicine extract.
4, the detection method of claim 3 is characterized in that: C
1-C
5Lower alcohol be methyl alcohol, ethanol, normal butyl alcohol; The solvent that extracts Chinese medicine is neutral, acid or alkaline; Select for use in mineral acid, inorganic base, organic acid or the organic base and acid or alkaline Chinese medicine extract; Solubilizer is selected from tween, dimethyl sulfoxide (DMSO), polyglycol, Qu Latong or their potpourri; The volume of pressing Chinese medicine extract adds the solubilizer of 0.1-5% volume.
5, the detection method of claim 4 is characterized in that: Chinese medicine extract is to extract the Chinese medicine extract of preparation with containing the 1-30% concentration ethanol.
6, the detection method of claim 1 is characterized in that: biological membrane is the cell membrane of simulation biological membrane, human body or animal tissue cell.
7, the detection method of claim 6 is characterized in that: the simulation biological membrane is that the potpourri with lecithin or lecithin and natural phospholipid is prepared from; Cell membrane comprises endothelial cell membrane, erythrocyte membrane, myocardial cell membrane, smooth muscle cell film.
8, the detection method of claim 1, it is characterized in that: Chinese medicine extract and biomembranous bag filter will be housed place the damping fluid under the simulation physiological condition to dialyse, to not contain biomembranous Chinese medicine extract simultaneously as blank, pack into and dialyse in the bag filter, temperature and time is inversely proportional to during dialysis, temperature is low more, it is long more that the film both sides reach the needed time of balance, after treating film two lateral balances, detect the liquid of dialysing with the high efficiency chromatography method respectively, compare both finger-prints, the peak that peak area changes promptly is with biological membrane interactional composition to be arranged.
9, the detection method of claim 8 is characterized in that: biological membrane is a liposome; The damping fluid of the simulation physiological condition of dialysis usefulness is the phosphate of pH2.0-7.4 or the acetate buffer of pH2.0-7.4; When the dialysis temperature was 4-25 ℃, dialysis time was 12-24 hour.
10, the detection method of claim 1 is characterized in that: behind the liquid that usefulness high efficiency chromatography method detection Chinese medicine extract was dialysed, with liquid phase-mass spectrum and/or nuclear magnetic resonance coupling, obtain to have with film the structural information of interaction composition.
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| CN101865805B (en) * | 2009-04-14 | 2013-05-22 | 南京海昌中药集团有限公司 | Method for testing crispness of medicament calcined by Chinese medicament |
| CN101949899B (en) * | 2010-08-16 | 2013-09-11 | 四川省中医药科学院 | Quantitative measurement method of ligustilide |
| CN102242190B (en) * | 2011-04-25 | 2013-04-24 | 辽宁中医药大学 | Method for screening effective medicinal flavors of Chinese herbal compounds |
| CN107305181A (en) * | 2016-04-18 | 2017-10-31 | 重庆大学 | A kind of method for studying percutaneous dosing solvent penetration |
| CN109470762B (en) * | 2017-09-07 | 2020-12-29 | 中国科学院大连化学物理研究所 | Method for accurately identifying expiration of propofol injection |
| CN111044422A (en) * | 2020-01-11 | 2020-04-21 | 河南省中医院（河南中医药大学第二附属医院） | Quality control method and device for Chinese herbal compound |
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