CN102565224B - Method for screening and measuring active ingredients in Chinese medicine - Google Patents
Method for screening and measuring active ingredients in Chinese medicine Download PDFInfo
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Abstract
The invention discloses a method for screening active ingredients in a Chinese medicine, in particular a method for screening and detecting the active ingredients in the Chinese medicine by utilizing the interaction of active cells and the active ingredients in the Chinese medicine. The method comprises the following steps of: preparing the Chinese medicine or a Chinese medicinal compound extracting solution; putting hollow fibers in which the active cells are implanted or injected into a phosphate buffer solution of the Chinese medicinal extracting solution, screening the active ingredients from the Chinese medicine by simulating human physiological conditions, and performing blank control simultaneously; and collecting an acceptation phase in a hollow fiber pipe, disaggregating medicines combined to the cells, and detecting the free active ingredients in a disaggregating solution through chromatograph. Compared with the detection result of a blank fiber, structural information of an active cell combined compound is obtained. The method is efficient, simple, convenient, cheap and accurate and reflects the characteristic of multi-component and multi-target synergistic effect of the Chinese medicine.
Description
Technical field
The present invention relates to screening, detection and the hollow fiber micropore permeation technology of Effective Component of Chinese Medicine.
Background technology
The screening of the effective substance of Chinese medicine and evaluation are the important component parts of modernization of cmm.Chemical composition of Chinese materia medica is very complicated, and single medicinal material itself is namely complicated potpourri, and take several flavors even the Chinese medicine compound prescription that forms of tens flavor medicinal material compatibilities as the citation form of middle medical drugs.Thereby exploration Chinese medicine effect-material base is not only a very difficult task and is the needs of modern medical service.Wherein, the TCM in Vitro screening active ingredients research method that build various modern technology, high flux, meets character of traditional Chinese medicine seems particularly necessary and important.
The group effect relationship of Chinese medicine " multicomponent, many target spots, by all kinds of means " performance drug action has become common recognition.Based on this, the technology of nearly ten years multiple in-vitro screening active ingredient of Chinese herbs that grow up.
Biochromatography is with biomacromolecule (enzyme, acceptor, DNA, memebrane protein, membrane phospholipid, transport protein in blood plasma etc.), competent cell film/imitative biological membrane, even living cells is fixed on the carriers such as silica gel as chromatographic stationary phases, when Chinese medicine extract is flowed through the biological chromatography post, because the fixing biomaterial of going up mutually can be combined with active ingredient of Chinese herbs specifically, make optionally retention activity composition of chromatographic column, thereby can utilize various technical parameter quantitatively characterizing medicines and biomacromolecule in chromatogram, cell-cell interaction, according to the difference of chromatographic parameter, active component being carried out biological parameter measures, screening active ingredients and compartment analysis.With human serum albumins and α
1-acidoglycoprotein conduct fixedly the Biochromatography of phase is screened the active component in medicinal herbs most in use-Radix Angelicae Sinensis, Ligusticum wallichii, oriental wormwood, the Radix Astragali, the radix paeoniae rubrathe, ginkgo leaf, the red sage root etc., measured active component plasma protein binding rate (Mao Xiqin, Zou Hanfa, Luo Quanzhou, Kong Liang, Li Xin, Sun Naichang, lecithin coating biological membrane as chromatographic is preparation and the stable investigation thereof of phase fixedly, chromatogram, 2001,19 (5): 433 ~ 435; Kong Liang, Zou Hanfa, Wang Hailin, Ni Jianyi, Zhang Yukui is take human serum albumins as the fixedly research of several active ingredient of Chinese herbs of molecular biosciences stratographic analysis of phase, SCI, 2000,21 (1): 36 ~ 40).Use the active component that different tissues cell membrane Chromatography Models has screened the Chinese crude drugs such as the red sage root, Ligusticum wallichii, Radix Angelicae Sinensis, the root of Dahurain angelica, frutus cnidii, kuh-seng, barrenwort, Radix Caulophylli, notopterygium root, radix glehniae, and determine effective constituent and active component (Zhao Xiaojuan in conjunction with pharmacological evaluation, party's climax, Yang Guangde, the analysis and comparison of shorthorned epimedium root and leaf active component, analytical chemistry, 2002,30 (2): 195 – 197; Gao Kun, the He Lang punching, Yang Guangde is with the effective constituent in cellular membrane chromatography screening study Radix Caulophylli, Chinese Pharmaceutical Journal, 2003,38 (1): 14-16; Xiaofang Hou, Mingzhe Zhou, Qiao Jiang, Sicen Wang, Langchong He adopts the active component in vascular smooth muscle/cell membrane chromatogram-off-line gas chromatography/mass spectrometry method identification, separation, Identification chinese herbs medicine material, JournaL of Chromatography A, 2009,1216:7081 – 7087; Sicen Wang, Meng Sun, Yanmin Zhang, Hui Du, Langchong He adopts a kind of new A
431/ cell membrane chromatogram-online high performance liquid chromatography/mass spectrometry method screens epidermal growth factor receptor antagonists, JournaL of Chromatography A, 2010,1217:5246 – 5252 from kuh-seng).But the biological chromatography technology has the following disadvantages: be combined biological material (1) with carriers such as silica gel, not only biological property can be subject to certain impact, and can not get rid of the silica gel activated centre to the medicine non-specific binding; (2) biological material Stationary phase preparation and dress post difficulty, need special technology and equipment, and process is complicated, and condition is strict, and cost is higher, and serviceable life is short; (3) biological material and Effective Component of Chinese Medicine effect are synchronizeed with chromatographic process and are carried out, and are difficult to satisfy simultaneously the top condition of operation of nature condition and chromatographic resolution in biosome.
Dialysis and partition method are that imitative biological membrane (liposome), target cell or target protein and Chinese medical extract are hatched altogether, through/without after dialysing, compare chromatogram with blank Chinese medicine, the peak of peak area marked change, be and the effective composition of biomaterial, use these compositions of technical appraisement such as online LC-MS, just can filter out active component from the Chinese medicine system of complexity.Adopt this technology to carry out active ingredient screening and research (Li Ping, Sheng Lianghong, Li Ruiyan, Li Songlin, Wang Wei, a kind of detection method of Effective Component of Chinese Medicine, Chinese invention patent, ZL200410041024.8 to Chinese medicines such as Radix Angelicae Sinensis Hemostasis Decoction, short-tube lycoris, Shi Shan; Li Ping, Zhou Jianliang, Liu Peng, Liu Ying, a kind of method of estimating active Chinese drug component and the mechanism of action based on Overall View, Chinese invention patent, CN101793883A).Wherein, the dialysis Shortcomings: to reach equilibration time longer due to dialysis procedure, cause cell mortality higher, and under physiological condition cell easily stop up the dialysis membrane aperture, thereby make blank lose meaning.The partition method Shortcomings: competent cell is cultivated together with Chinese medicine extract, is difficult to avoid pollution and the impact of impurity on the selection result in Chinese medicine extract.
The problems such as no matter Biochromatography or dialysis and partition method ubiquity detection sensitivity are low, and the cell consumption is large, and analysis time is long.
Publication number is to disclose a kind of screening active ingredients of Chinese medicament in the Chinese invention patent of CN101852787A, that biomacromolecule, imitative biological membrane or biological membrane are injected hollow fiber conduit, Chinese medicine or traditional Chinese medicine compound extract that hollow fiber conduit is placed in after processing are screened, collect after screening finishes and receive phase, will be free out with the active component of biomacromolecule, imitative biological membrane or biological membrane specific binding, carry out chromatogram or Gas Chromatography/Mass Spectrometry Analysis.
The method is not enoughBe: (1) does not adopt the biological living cells directly related with drug action, and only uses the imitative biological membrane of liposome or biomacromolecule; (2) will not imitate biological membrane and be fixed in the hollow fiber inside pipe wall, only biomacromolecule or imitative biological membrane be injected fiber lumens, therefore, can not simulate better actual biological internal milieu.
Summary of the invention
The present invention causes effective constituent screening and the difference of measuring for fear of in Effective Component of Chinese Medicine screening because biological property is affected, provide a kind of efficiently, the Screening test method of Effective Component of Chinese Medicine accurately.
For addressing the above problem, the technical solution used in the present invention is:
A kind of Screening test method of Effective Component of Chinese Medicine.Comprise the steps: (1) preparation Chinese medicine extract; (2) hollow fiber is cleaned, naturally dried, then carry out sterilization treatment, competent cell is implanted or the injection hollow fiber; (3) hollow fiber that will implant or inject competent cell is placed in Chinese medicine extract and carries out active ingredient screening; (4) after screening finishes, the effective constituent of competent cell combination is dissociated, detect the effective constituent in dissociation solution.
Because most medicines only enter human body, see through cell and just can show activity, play a role, the activity of in vivo physiology course of medicine, medicine and toxicity etc. all depend on the effect situation of medicine and cell.Thereby the present invention as the living cells carrier, based on the effect of effective substance and all kinds of living cells, sets up a kind of method of screening and detection Effective Component of Chinese Medicine and active component with hollow fiber.The present invention carries out the pharmaceutically active screening take hollow fiber as the competent cell carrier, easy and simple to handle, be easy to grasp, with low cost, detection sensitivity is high, the biological internal milieu of more approaching reality, the selection result accuracy is high, can reflect preferably Chinese medicine multicomponent, many target spots, synergistic characteristic, and is significant to the Chinese medicine Effective Compounds that discloses real onset in body.
The method for preparing Chinese medicine extract in step (1) is to get Chinese medicine water to be measured or/and organic solvent extracts, namely can water extract, the mixed solution of organic solvent extraction or water and organic solvent extracts, the Chinese medicine extract that organic solvent is extracted need concentrate removes organic solvent.After extracting the effective constituent in Chinese medicine to be measured, the solubilizer dissolving that is 1%-3% with phosphate buffered solution or the mass concentration of pH7.0-7.4.
Described organic solvent is methyl alcohol, ethanol, the mixed solution of any one or a few in butanols, ether, ethyl acetate and chloroform; Solubilizer is any one or a few the mixed solution in tween, dimethyl sulfoxide (DMSO), polyglycol or Qu Latong.
In step (2), select polypropylene hollow fiber, length is 6cm, polypropylene hollow fiber is successively with acetone, methyl alcohol, acid, alkali, distilled water difference ultrasonic cleaning 5 ~ 10min, and after repeatedly cleaning with distilled water, at room temperature naturally dry, with high-pressure steam sterilizing pan, it is carried out sterilization treatment.
In step (2), the method of injecting competent cell is to obtain cell suspending liquid after the cell that digestion obtains is dissolved with nutrient solution or phosphate buffer, extract cell suspending liquid and it slowly, is evenly injected in the hollow fiber tube chamber with disposable asepsis injector, sealing, stand-by.
In step (2), the method of implanting competent cell is to obtain cell suspending liquid after the cell that digestion obtains is dissolved with nutrient solution or phosphate buffer, extract cell suspending liquid and slowly, evenly inject it in hollow fiber tube chamber with disposable asepsis injector, the fiber of filling with cell suspending liquid is put into the Tissue Culture Flask that cell culture fluid is housed, fiber with repopulating cell after 48h takes out, sealing, stand-by.Hollow fiber is in Tissue Culture Flask during repopulating cell, put fiber 6-10 root for every bottle, put smooth, do not contact mutually, can not stack, Tissue Culture Flask bottleneck, valve protection cap are overdoed after sterilization, lid lid, putting into incubator cultivates, change every other day nutrient solution, the fiber with repopulating cell after 48h takes out, sealing.
Should pull out syringe needle when extracting cell suspending liquid with disposable asepsis injector, in case competent cell sustains damage, again syringe needle be set up when hollow fiber injects cell suspending liquid.
In step (3), the hollow fiber of implanting cell or injecting cell suspending liquid is with common fine rule tying, the cell hollow fiber conduit of tying is placed in Chinese medicine extract screens, after screening finishes, take out fiber, cut two ends closure fine rule, liquid in fiber is blown in a little EP pipe.
In step (4), hollow fiber one end after the screening is blown into the other end and blows out the methyl alcohol that uses 20 μ L when the active component of Cell binding is dissociated with micro syringe, with blow out liquid in hollow fiber after screening and merge, at the centrifugal 20min of 10000rpm, get centrifugal rear supernatant 20 μ L and carry out high performance liquid chromatography, multi-wavelength high performance liquid chromatography, gas chromatography, liquid phase/mass spectrum, gas phase/mass spectrophotometry, the active effective constituent of screening and mensuration and competent cell specific binding.Carry out blank fiber contrast in the competent cell screening, after screening finishes, collect in hollow fiber conduit and accept phase, and the medicine that is incorporated on cell is dissociated, detect active component free in dissociation solution with chromatogram.Compare with blank fiber testing result, the peak area of cell fiber the selection result chromatogram increases, goes out peak number and increases corresponding compound and be with competent cell interactional active component is arranged, with Chromatography/Mass Spectrometry or (with) nuclear magnetic resonance is combined and can obtains structural information with the competent cell binding compounds
In the inventive method, cell is implanted or is injected in hollow fiber conduit, and cell does not directly contact with the Chinese medicine extract that contains solubilizer, and in screening process, the survival rate of cell is high, the one-tenth current is long, and the screening equilibration time shortens (3-6h).
Traditional Chinese medicine fingerprint is to be applicable to the gordian technique that traditional Chinese medicine quality is controlled, but existing finger-print exists an outstanding problem that the pharmacology information of (effect-material base mapping information) Chinese medicine can not be provided exactly.Medicine and competent cell effect are the prerequisites that produces drug effect, with the interactional composition of competent cell may be just the active substance basis of Chinese crude drug or Chinese medicine compound prescription, this constituents should be the object of primary study in quality assessment and in controlling.Therefore, set up the active component finger-print of Chinese medicine and competent cell effect, effective component group in Chinese medicine is carried out quality control and comprehensive evaluation simultaneously, to comprehensive control quality of medicinal material, illustrate traditional Chinese medicine complex system effect-material base and the effect essence have great importance.
Competent cell is stronger than the biomembranous specificity of simulation.Cell membrane is separated from cell, and the biological property of cell membrane may be affected.The present invention directly uses the living cells of implanting or injecting hollow fiber, under the stirring of simulation Human Physiology environment and certain rotating speed, carry out cell and drug interaction, more approaching with the cell as drug targets in human body, therefore, the active component that the present invention filters out is more reliable, and accuracy is higher.
In detection method of the present invention cell accept phase volume (
=10-20 μ L) very little, the sample phase volume (
=5-10 mL) larger, therefore, screening the comparing of system (
) less.Early-stage Study (Tian Jie, Chen Xuan, Bai Xiaohong. Chinese crude drug composition organic solvent and ionic liquid dispersive liquid-liquid microextraction method relatively reach Exploration of Mechanism, analytical chemistry, 2010,38 (11): 1593 ~ 1598) show: compare littlely, cycles of concentration is larger (that is:
), the sensitivity of method is higher.This shows, after screening, active component has to a certain degree concentrated, measures sensitivity and improves.
Below further describing of the present invention-Effective Component of Chinese Medicine screening and detection method:
The extraction of Chinese medicine:Described Chinese medicine comprises the compound that single compound, a class or a few compounds, single medicinal material or kinds of traditional Chinese medicines form.According to the physicochemical property of the contained effective constituent of Chinese medicine, select suitable solvent to extract.But when screening, the extract solvent should not destroy cytoactive, is generally water, buffer solution or contains solubilizer (tween, dimethyl sulfoxide (DMSO)) aqueous solution lower than 3%.Used if namely extract Effective Component of Chinese Medicine the organic solvent that cytoactive is had destruction, should be removed.Use to cell without the buffer solution of the low concentration solubilizer of the destruction medium as the screening active component.
The selection of hollow fiber:Hollow fiber conduit not only can be used as plantation, hold the carrier with the prolection cell, and for the invention provides the platform of cell and active component effect.Countless apertures on hollow fiber walls not only can make Medicine small molecule have free passage, increased the surface area that competent cell contacts with chemical composition, and Chinese medical extract is played the effect of microfiltration and purifying, removed sample purification process consuming time and loaded down with trivial details in other screening active ingredients of Chinese medicaments from.The hollow fiber at non-activity center in choice structure of the present invention (without the strong hetero atom substituents of electronegativity), and the hollow fiber of blank hollow fiber dissociation solution a little less than without chromatographic peak or chromatographic peak.The present invention adopts polypropylene hollow fiber.
The selection of competent cell:All cells from human body and animal all can be used as screening and measure the target of active ingredient of Chinese herbs.As endothelial cell, cardiac muscle cell, smooth muscle cell, tumour cell etc., commercial cell line, as: endothelial cell, tumour cell etc. or oneself separate and cultivate or plant at the hollow fiber inside pipe wall.The screening active ingredient of Chinese herbs can be selected suitable cell according to document, data and trial test result.For example: have antitumaous effect according to reported in literature Coumarins chemical composition, therefore, the present invention selects stomach cancer cell implant or inject hollow fiber conduit frutus cnidii and corylifolia L Coumarins chemical composition have been carried out screening active ingredients and mensuration in an embodiment.When with neural female oncocyte, the active component in schisandra chinensis medicinal material being screened, find that going out peak position in the Lignanoids compounds chromatogram is equipped with several and active component cell interaction.Therefore, Lignanoids compounds not only has and may also have antitumaous effect to neural active function.In order to verify the antitumaous effect of Lignanoids compounds, we screen the Lignanoids compounds active anticancer in schisandra chinensis medicinal material with stomach cancer cell and mononuclear macrophage leukaemia again, found that schizandrin, Schisantherin C, schizandrin A, deoxyschizandrin all can with cancer cell produce to interact.
The combination of active component in competent cell and Chinese medical extract:The hollow fiber of implantation or injection competent cell is placed in traditional Chinese medicine extraction solution, and cell and drug interaction are screened under the stirring of simulation Human Physiology environment and certain rotating speed, and generally 3-6h, carry out simultaneously blank polypropylene hollow fiber and screen.After screening finished, in hollow fiber, solution was blown in a little EP pipe, with the washed with methanol fiber lumens of 20 μ L, blow out with screen after fiber in blow out the liquid merging.
Dissociating of Cell binding effective constituent:For the character of different activities composition, select the solution of different solvents or different pH, polarity, ionic strength etc. that the effective constituent of specific binding on cell is dissociated.
Active component screens, chromatogram detects and structure is determined:Peak area, retention time and the peak number of the chromatographic peak by the solution that dissociates after cell fiber and blank fiber screening are relatively determined kind and the quantity of the active ingredient of Chinese herbs that preliminary screening goes out.Simultaneously, also can carry out HPLC, GC to the dissociation solution of same Chinese medical extract and measure, the active component of screening opposed polarity; Measure under the HPLC different wave length, screen different classes of uv absorption reactive compound, tentatively realize Chinese medicine multicomponent, many target spots, the research of synergistic material base.For the active component that standard items are arranged in chromatogram, adopt the method that contrasts with standard items retention time and spectrogram, determine the active component that keeps on cell.For the effective constituent that there is no standard items, utilize the contrast of Chromatography/Mass Spectrometry combination analysis and document unknown active component to be made the judgement of structure.
Ultimate principle of the present invention is that hollow fiber is the tubular structure of being made by the macromolecular material of porous, and on hollow fiber walls, micropore allows Medicine small molecule to pass through, and biomacromolecule or impurity are had barrier effect.Medicine and hollow fiber are implanted into or the competent cell target spot that injects interacts, medicine just can enter in fibre pipe by the hole on hollow fiber walls and be combined with competent cell, medicine with Cell binding is dissociated, free medicine carries out stratographic analysis, with chromatogram after the screening of blank fiber relatively, have medicine with Cell binding chromatographic peak can occur after screening or peak area increases.Simultaneously, cell of the present invention is accepted the smaller of phase volume and sample phase volume, and therefore, after screening, active component has certain concentrating, and measures sensitivity and improves.If specimen is single medicinal material or compound extract, obtain be exactly in whole Chinese medicine with the interactional active component of competent cell, if specimen is single or a class, a few compounds, what obtain is exactly single or a class, a few compounds and the interactional active component of competent cell, so to a single or class, a few compounds interact with competent cell or single medicinal material or compound extract in active component and competent cell interaction, between them, the whether difference of existence effect or competition, inhibition can further be studied.
Compared with prior art the present invention has following beneficial effect:(1) take hollow fiber as the competent cell carrier, pharmaceutically active is screened, the biological property of not only having avoided cell changes and affects the accuracy of active ingredient screening, and improved the sensitivity that detects, and reduced the use amount of cell, shortened analysis time; (2) needn't prepare the fixing luggage post of going forward side by side mutually of biological membrane, not only get rid of the non-specific binding of Si-OH to medicine in silica gel, and simple to operate, be easy to grasp, with low cost; (3) screening active ingredients and sample purification are completed simultaneously, have removed the necessary sample pretreatment process of other active Chinese drug component screening methods from, have improved Chinese medicine active ingredient screening and the definite efficient of structure; (4) screening active ingredients and chromatographic process are carried out respectively, can satisfy the most suitable or top condition that biology and chromatographia require; (5) Chinese medical extract without separating directly and the competent cell effect, can reflect Chinese medicine multicomponent, many target spots, synergistic characteristic preferably, and the Chinese medicine Effective Compounds that discloses real onset in body is had great importance.(6) this invention is adopted competent cell and with its implantation or inject in hollow fiber conduit.Therefore, can simulate better actual biological internal milieu, the active component that filters out is more reliable, and accuracy is higher.
Description of drawings
Fig. 1 active ingredient of Chinese herbs screening plant.
In Fig. 1, the 1-thermostatic mixer, the 2-stirrer, the 3-vial, 4-hollow fiber, the buffer solution of 5-Chinese medical extract, 6-are implanted or the injection cell.
Coumarins active component and stomach cancer cell interaction chromatography figure in Fig. 2 frutus cnidii extract.
In Fig. 2, a standard items, blank fiber screening sample, c stomach cancer cell phase fiber screening sample.
Wherein, 3-psoralen (6.0min), 5-Isopsoralen (7.0min), 7-bergapten (9.0min), 10-Imperatorin (15.0min), 11-Osthole (16.2min)
Coumarins active component and stomach cancer cell interaction chromatography figure in Fig. 3 Psoralea corylifolia extract.
In Fig. 3, a standard items, the blank fiber screening sample of b, c injects stomach cancer cell fiber screening sample, d implantable gastric cancer cell fiber screening sample.
Wherein, 4-psoralen (6.0min), 5-Isopsoralen (7.0min), 9-Isomperatorin (17.0min)
Lignanoids active component and neural female oncocyte interaction chromatography figure in Fig. 4 Fructus Schisandrae Chinensis extractive solution.
In Fig. 4, a standard items, the blank fiber screening sample of b, the neural female oncocyte fiber screening sample of c.
Wherein, 2-Schisantherin C, 3-schizandrin A, 6-deoxyschizandrin.
Lignanoids active component and stomach cancer cell interaction chromatography figure in Fig. 5 Fructus Schisandrae Chinensis extractive solution.
In Fig. 5, a standard items, the blank fiber screening sample of b, c injects stomach cancer cell fiber screening sample, d implantable gastric cancer cell fiber screening sample.
Wherein, 1-schizandrin, 4-Schisantherin C, 5-schizandrin A, 7-deoxyschizandrin.
Lignanoids active component and mononuclear macrophage leukaemia interaction chromatography figure in Fig. 6 Fructus Schisandrae Chinensis extractive solution.
In Fig. 6, a standard items, the blank fiber screening sample of b, c mononuclear macrophage leukaemia fiber screening sample.
Wherein, 1-schizandrin A.
Lignanoids active component and PERIPHERAL BLOOD MONONUCLEAR CELL interaction chromatography figure in Fig. 7 Fructus Schisandrae Chinensis extractive solution.
In Fig. 7, a standard items, the blank fiber screening sample of b, c PERIPHERAL BLOOD MONONUCLEAR CELL fiber screening sample.
Wherein, 4-schizandrin A.
Embodiment
Material and reagentHollow fiber (Tianjin MoTian Membrane Engineering Technology Co., Ltd), Chinese medicine standard reference material (Nat'l Pharmaceutical ﹠ Biological Products Control Institute), PERIPHERAL BLOOD MONONUCLEAR CELL (normal human cell), cell line (gastric carcinoma cells BGC7901, mononuclear macrophage white blood corpuscle RAW264.7, neural female oncocyte SHSY5Y).Experimental water is redistilled water, and it is pure that other reagent is analysis.
InstrumentAgiLent TechnoLogies 1200 Series high performance liquid chromatographs.
Experimental procedure
Get the vial of 6mL, add the pH7.4 phosphate buffered solution of the 500 certain density active ingredient of Chinese herbs standard items of μ L or Chinese medical extract, put into stirrer and be fixed on magnetic stirring apparatus, at 37 ℃ of lower constant temperature.The polypropylene hollow fiber pipe that 6cm is long is used acetone, methyl alcohol, acid, alkali, distilled water ultrasonic cleaning 5 ~ 10min respectively successively, and after repeatedly cleaning with distilled water, air-dry at room temperature, with high-pressure steam sterilizing pan, hollow fiber is carried out sterilization treatment.Hollow fiber after the tweezers gripping sterilization of crossing with the spirit lamp pyroprocessing is with disposable asepsis injector extraction cell suspending liquid and it slowly, is evenly injected in the hollow fiber tube chamber.The fiber of filling with cell suspending liquid is carefully put into the Tissue Culture Flask that the 4mL cell culture fluid is housed, put into incubator and cultivate.The fiber that covers with cell after 48h takes out, sealing.The hollow fiber of implanting or inject competent cell is placed in traditional Chinese medicine extraction solution to be screened.Carry out simultaneously blank hollow fiber screening.After screening finishes, take out fiber, cut the sealing two ends place, liquid in fiber is blown in a little EP pipe, with the washed with methanol fiber lumens of 20 μ L, blow out with screen after fiber in blow out the liquid merging, at the centrifugal 20min of 10000rpm.Get respectively cell/blank hollow fiber supernatant 20 μ L and carry out stratographic analysis, the peak area of chromatogram more both, go out the peak number, determine active component and its finger-print with cytosis; With LC/MS or (with) NMR is combined and can obtains structural information with the competent cell binding compounds.
Following examples are the invention group take lignanoids and Coumarins active component as the screening object, neural female oncocyte, stomach cancer cell, mononuclear macrophage leukaemia, PERIPHERAL BLOOD MONONUCLEAR CELL are target cell, screening and the detection method of Coumarins active component in lignanoids active component and Psoralea corylifolia, frutus cnidii extract in the Chinese medicine Schisandra chinensis extract of foundation.
1. the extraction of Coumarins chemical composition in Fructus cnidii
Smashed the Chinese crude drug frutus cnidii to pieces sieve No. three, accurately weighed 1.0g adds approximately 45mL of methyl alcohol, and heating water bath backflow 2h lets cool, and filters, and subsequent filtrate is placed in 50mL volumetric flask methanol constant volume, and 4 ℃ of preservations are stand-by.
2. Coumarins active component in stomach cancer cell hollow fiber screening study frutus cnidii
The accurate frutus cnidii extract 5mL that draws removes methanol solvate in extract in the vial of 10mL, uses the phosphate buffered solution dissolving of the pH7.4 of 5mL, puts into stirrer (8 mm * 4 mm) and is fixed on magnetic stirring apparatus.The buffer solution that active stomach cancer cell hollow fiber is placed in the frutus cnidii extract screens, open magnetic stirring apparatus, 37 ℃ of constant temperature, under 1500 rpm stir, screening 3 h, after screening finishes, take out fiber, cut the sealing two ends place, take out solution in fiber, washed with methanol fiber lumens with 20 μ L merges twice solution.At the centrifugal 20min of 10000rpm.Get supernatant 20 μ L and carry out the HPLC analysis.
Utilize the present invention to carry out screening to Coumarins active component in Fructus cnidii and see Fig. 2.Found to be combined with the implantable gastric cancer cell and to have 11 (peak 1-11) active components that have of obvious reservation by Fig. 2, contrast 5 compositions that have that carried out the structure Preliminary Identification by retention time, respectively 3-psoralen (6.0min), 5-Isopsoralen (7.0min), 7-bergapten (9.1min), 10-Imperatorin (15.0min), 11-Osthole (16.2min); Can be combined and have 8 ( peak 1,3,6 of having of obvious reservation with the injection stomach cancer cell, 7,8,9,10,11) active component, contrasting 4 compositions that have that carried out the structure Preliminary Identification by retention time, is respectively 3-psoralen (6.0min), 7-bergapten (9.1min), 10-Imperatorin (15.0min), 11-Osthole (16.2min).Can carry out Chromatography/Mass Spectrometry combination analysis and document contrast for the active component that there is no standard items that occurs in chromatogram, determine that it may structure.
1. the extraction of Coumarins chemical composition in corylifolia L
Smashed the Chinese crude drug Psoralea corylifolia to pieces sieve No. three, accurately weighed 2.0g adds approximately 45mL of methyl alcohol, and heating water bath refluxed 2 hours, lets cool, and filters, and subsequent filtrate is placed in 50mL volumetric flask methanol constant volume, and 4 ℃ of preservations are stand-by.
2. Coumarins active component in stomach cancer cell hollow fiber screening study corylifolia L
The accurate Psoralea corylifolia extract 5mL that draws removes methanol solvate in extract in the vial of 10mL, uses the phosphate buffered solution dissolving of the pH7.4 of 5mL, puts into stirrer (8 mm * 4 mm) and is fixed on magnetic stirring apparatus.The buffer solution that active stomach cancer cell hollow fiber is placed in the Psoralea corylifolia extract screens, open magnetic stirring apparatus, 37 ℃ of constant temperature, under 1500 rpm stir, screening 3 h, after screening finishes, take out fiber, cut the sealing two ends place, take out solution in fiber, washed with methanol fiber lumens with 20 μ L merges twice solution.At the centrifugal 20min of 10000rpm.Get supernatant 20 μ L and carry out the HPLC analysis.
Utilize the present invention to carry out screening to Coumarins active component in corylifolia L and see Fig. 3.Found to be combined with the implantable gastric cancer cell and to have 11 (1-11) active components that have of obvious reservation by Fig. 3, contrast 3 compositions that have that carried out the structure Preliminary Identification by retention time, respectively 4-psoralen (6.0min), 5-Isopsoralen (7.0min), 9-Isomperatorin (17.0min); Can be combined and have 6 (Isosorbide-5-Nitrae, 5 of having of obvious reservation with the injection stomach cancer cell, 7,8,11) active component, contrasting 2 compositions that have that carried out the structure Preliminary Identification by retention time, is respectively 4-psoralen (6.0min), 5-Isopsoralen (7.0min).Can carry out Chromatography/Mass Spectrometry combination analysis and document contrast for the active component that there is no standard items that occurs in chromatogram, determine that it may structure.
1. the extraction of lignanoids active component in schisandra chinensis medicinal material
Get the dry medicinal material of the fruit of Chinese magnoliavine, porphyrize is crossed 30 mesh sieves, and precision takes 10 g in 100 mL tool plug volumetric flasks, adds methyl alcohol 80 mL, and ultrasonic (power 100 W) extract 30 min, put to room temperature, with methanol constant volume to 100 mL.4 ℃ of Refrigerator stores, stand-by.
2. lignanoids active component in cell hollow fiber screening study schisandra chinensis medicinal material
The accurate fruit of Chinese magnoliavine Chinese medicine extract 500 μ L that draw remove methanol solvate in extract in the vial of 6mL, use the phosphate buffered solution dissolving of the pH7.4 of 4mL, put into stirrer (8 mm * 4 mm) and are fixed on magnetic stirring apparatus.The buffer solution that the competent cell hollow fiber is placed in Fructus Schisandrae Chinensis extractive solution screens, open magnetic stirring apparatus, 37 ℃ of constant temperature, under 900 rpm stir, screening 3 h, after screening finishes, take out fiber, cut the sealing two ends place, take out solution in fiber, washed with methanol fiber lumens with 20 μ L merges twice solution.At the centrifugal 20min of 10000rpm.Get supernatant 20 μ L and carry out the HPLC analysis.
Utilize the present invention to carry out screening to lignanoids active component in schisandra chinensis medicinal material and see Fig. 4-7.6 active components that have of obvious reservation (Fig. 4) can are combined and have to discovery with the neural female oncocyte of injection, contrast 3 compositions that have that carried out the structure Preliminary Identification by retention time, respectively 2-Schisantherin C (7.28min), 3-schizandrin A (8.72min) and 6-deoxyschizandrin (11.59min); With implantable gastric cancer cell (Fig. 5) in conjunction with and 7 active components that have of obvious reservation are arranged, contrast 4 compositions that have that carried out the structure Preliminary Identification by retention time, respectively 1-schizandrin (4.22min), 4-Schisantherin C (7.28min), 5-schizandrin A (8.72min), 7-deoxyschizandrin (11.59min), with inject stomach cancer cell (Fig. 5) in conjunction with and 3 active components that have of obvious reservation are arranged, contrast 1 composition that has that has carried out the structure Preliminary Identification, 5-schizandrin A (8.72min) by retention time; Can with inject mononuclear macrophage leukaemia (Fig. 6) in conjunction with and 1 active component that has of obvious reservation arranged, by retention time contrast carried out the structure Preliminary Identification only have 1-schizandrin A (8.72min); With inject PERIPHERAL BLOOD MONONUCLEAR CELL (Fig. 7) in conjunction with and 6 compositions that have of obvious reservation are arranged, by retention time contrast carried out the structure Preliminary Identification only have 4-schizandrin A (8.72min).Can carry out Chromatography/Mass Spectrometry combination analysis and document contrast for the active component that there is no standard items that occurs in chromatogram, determine that it may structure.
Claims (6)
1. the Screening test method of an Effective Component of Chinese Medicine, is characterized in that comprising the steps:
(1) preparation Chinese medicine extract;
(2) hollow fiber is cleaned, is naturally dried, then carry out sterilization treatment, competent cell is implanted or the injection hollow fiber,
The method of injecting competent cell is to obtain cell suspending liquid after the cell that digestion obtains is dissolved with nutrient solution or phosphate buffer, extracts cell suspending liquid and it slowly, evenly injected in the hollow fiber tube chamber with disposable asepsis injector, and sealing, stand-by,
The method of implanting competent cell is to obtain cell suspending liquid after the cell that digestion obtains is dissolved with nutrient solution or phosphate buffer, extract cell suspending liquid and slowly, evenly inject it in hollow fiber tube chamber with disposable asepsis injector, the fiber of filling with cell suspending liquid is put into the Tissue Culture Flask that cell culture fluid is housed, fiber with repopulating cell after 48h takes out, sealing, stand-by
Should pull out syringe needle when extracting cell suspending liquid with disposable asepsis injector, in case competent cell sustains damage, again syringe needle be set up when hollow fiber injects cell suspending liquid;
(3) hollow fiber that will implant or inject competent cell is placed in Chinese medicine extract and carries out active ingredient screening;
(4) after screening finishes, the effective constituent of competent cell combination is dissociated, detects the effective constituent in dissociation solution,
Hollow fiber one end after the screening is blown into the other end and blows out the methyl alcohol that uses 20 μ L when the active component of Cell binding is dissociated with micro syringe, with blow out liquid in hollow fiber after screening and merge, at the centrifugal 20min of 10000rpm, get centrifugal rear supernatant 20 μ L and carry out high performance liquid chromatography, multi-wavelength high performance liquid chromatography, gas chromatography, liquid phase/mass spectrum, gas phase/mass spectrophotometry, the active effective constituent of screening and mensuration and competent cell specific binding.
2. the Screening test method of Effective Component of Chinese Medicine according to claim 1, it is characterized in that: the method for preparing Chinese medicine extract in step (1) is to get Chinese medicine water to be measured or/and organic solvent extracts, after extracting the effective constituent in Chinese medicine to be measured, with the solubilizer dissolving that phosphate buffered solution or the mass concentration of pH7.0-7.4 is 1%-3%, make the Chinese medicine extract for the active ingredient of Chinese herbs Screening test.
3. the Screening test method of Effective Component of Chinese Medicine according to claim 2, it is characterized in that: in step (1), Chinese medicine to be measured is single compound, a few compounds, single medicinal material or Chinese medicine compound prescription; Described organic solvent is methyl alcohol, ethanol, the mixed solution of any one or a few in butanols, ether, ethyl acetate and chloroform; Solubilizer is any one or a few the mixed solution in tween, dimethyl sulfoxide (DMSO), polyglycol or Qu Latong.
4. the Screening test method of Effective Component of Chinese Medicine according to claim 1, it is characterized in that: in step (2), select polypropylene hollow fiber, length is 6cm, polypropylene hollow fiber is successively with acetone, methyl alcohol, acid, alkali, distilled water difference ultrasonic cleaning 5 ~ 10min, and after repeatedly cleaning with distilled water, at room temperature naturally dry, with high-pressure steam sterilizing pan, it is carried out sterilization treatment.
5. the Screening test method of Effective Component of Chinese Medicine according to claim 1, it is characterized in that: hollow fiber is in Tissue Culture Flask during repopulating cell, put fiber 6-10 root for every bottle, put smoothly, do not contact mutually, can not stack, with Tissue Culture Flask bottleneck, valve protection cap overdo the sterilization after, the lid lid is put into incubator and is cultivated, and changes every other day nutrient solution, fiber with repopulating cell after 48h takes out, sealing.
6. the Screening test method of Effective Component of Chinese Medicine according to claim 1, it is characterized in that: in step (3), the hollow fiber of implanting cell or injecting cell suspending liquid is with common fine rule tying, the cell hollow fiber conduit of tying is placed in Chinese medicine extract to be screened, after screening finishes, take out fiber, cut the sealing two ends fine rule, liquid in fiber is blown in a little EP pipe.
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