CN101852787B - Method for screening active ingredients of Chinese medicament - Google Patents
Method for screening active ingredients of Chinese medicament Download PDFInfo
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- CN101852787B CN101852787B CN2010102049832A CN201010204983A CN101852787B CN 101852787 B CN101852787 B CN 101852787B CN 2010102049832 A CN2010102049832 A CN 2010102049832A CN 201010204983 A CN201010204983 A CN 201010204983A CN 101852787 B CN101852787 B CN 101852787B
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Abstract
The invention provides an efficient, rapid, simple and convenient method for screening active ingredients of a Chinese medicament. The method comprises the following steps of: preparing a Chinese medicament or a Chinese medicinal compound extract a; adding buffer solution of phosphate with pH value of 7.4 into the extract a; keeping a constant temperature of 37 DEG C and stirring the mixture to obtain solution b; performing inertia treatment on a hollow fiber pipe; injecting large biological molecules, bionic biomembranes or biomembranes into the hollow fiber pipe; placing the hollow fiber pipe into the solution b to screen; collecting receiving phases after screening; and freeing the active ingredients of the Chinese medicament which are combined with the specificity of the large biological molecules, the bionic biomembranes or the biomembranes to perform chromatographic or chromatographic/mass spectrographic analysis so as to screen the active ingredients which can be combined with the specificity of the large biological molecules, the bionic biomembranes or the biomembranes. The method has the characteristics of improving and ensuring the reliability and the high efficiency of screening the active ingredients of the Chinese medicament and having significance for disclosing the actually effective Chinese medicinal effect substance base in a human body, along with simple and rapid operation.
Description
Technical field
The present invention relates to pharmaceutically active screening and hollow fiber mocromembrane technology, be specially a kind of Chinese medicine method for screening active ingredients.
Background technology
The screening of Chinese medicine and natural drug effective substance and evaluation are the important component parts of modernization of cmm, people making great efforts to attempt always and research efficiently, external Chinese medicine drug screening and evaluation method fast and accurately.Chemical composition of Chinese materia medica is very complicated; Simple itself is exactly a complex mixture; And Chinese medicine tradition is particular about the compound medication that several flavors even tens flavor medicines are formed mostly, more in addition in the leaching process some chemical constitution interact, effective ingredient in Chinese often contains the compound of numerous similar performances.Possibly exist complex interactions such as drug effect complementation and toxicity disappear mutually simultaneously in the Chinese medicine between each component, this is not that the single receptor model can be accomplished.Therefore, the multicomponent of Chinese medicine, many target spots and globality make herbal medicine efficacy material base Research Advances in Evaluation slow.In recent years, the angle anthropomorphic dummy's body cell film from drug absorption carries out the focus that the active constituents of medicine analysis is effective substance research.Because the activity of in vivo physiology course of medicine, medicine and toxicity etc. all depend on the allocation situation of medicine on film, most medicines has only the human body of entering or sees through cell and just can show activity, plays a role.Therefore, the permeability of compound on cell membrane is most important to exciting pharmaceutically active, and the permeability of research compound cell membrane is screening and estimates reliably and directly method of pharmaceutically active.
Application at present biological chromatography method (biochromatography) more widely is to grow up for 20 end of the centurys, by chromatographic separation technology and life science a kind of pharmaceutically active screening that intersects to form and the chromatographic technique of estimating.It is that biomacromolecule (enzyme, acceptor, carrier protein), competent cell film (imitative biological membrane) are fixed on the carrier as the chromatographic stationary phase; When Chinese medicine extract is flowed through the biological membrane as chromatographic post; Because stationary phase can combine with active ingredient of Chinese herbs specifically; Make optionally retention activity composition of chromatographic column, different material shows the different retention properties on film with the difference of membrane interaction degree, according to this difference medicine is carried out biological parameter mensuration, screening active ingredients and compartment analysis.Active component in Chinese traditional medicine angelica, Ligusticum wallichii, oriental wormwood, the Radix Astragali, the radix paeoniae rubrathe, ginkgo leaf, the red sage root etc. is screened as the biological chromatography method of stationary phase with human serum albumins and acid seromucoid, measured active component plasma protein binding rate (Mao Xiqin, Zou Hanfa; Luo Quanzhou, Kong Liang, Li Xin; Sun Naichang. lecithin applies the preparation of biological membrane as chromatographic stationary phase and the investigation [J] of stability thereof. chromatogram. 2001; 19 (5): 433 ~ 435, Kong Liang, Zou Hanfa; Wang Hailin; Ni Jianyi, Zhang Yukui. with the human serum albumins is the research [J] of several kinds of active ingredient of Chinese herbs of molecular biosciences stratographic analysis of stationary phase. SCI. 2000,21 (1): 36 ~ 40).
Not enough below the biological chromatography technology exists: (1) is as a kind of stripped active component analysis means; And biological membrane and Chinese medical extract effect and chromatographic process carry out simultaneously, is difficult to provide simultaneously in the biosome absorption and the required condition of chromatographic separation process naturally.(2) preparation of biological membrane stationary phase and dress post difficulty need special technique and equipment, and the reappearance of experiment and precision can not guarantee.(3) biological chromatography has used silica gel with the Si-OH activated centre chromatographic column as biofilm carrier, can not get rid of the possibility of silica gel activated centre to the medicine non-specific binding.
Summary of the invention
It is different and can overcome the active ingredient screening method of the deficiency that existing biological chromatography technology exists to the purpose of this invention is to provide a kind of and existing biological chromatography technical operation; With hollow fiber biological membrane, imitative biological membrane or biomacromolecule micro-extraction is core, the screening technique of efficient, quick, easy active ingredient of Chinese herbs.
Solve above technical matters, a kind of Chinese medicine method for screening active ingredients of the present invention may further comprise the steps:
(1) preparation Chinese medicine or traditional Chinese medicine compound extract a; (2) PBS of adding pH7.4 in extract a in 37 ℃ of following constant temperature and stirring, gets solution b; (3) hollow fiber conduit is distinguished ultrasonic cleaning 5 ~ 10min with acetone, methyl alcohol, acid and/or alkali, distilled water successively; And after cleaning repeatedly with distilled water; Dry up, after in biomacromolecule, imitative biological membrane or the biological membrane injection hollow fiber conduit, place solution b to screen hollow fiber conduit; (4) screening finishes the big molecule of back collection of biological, imitative biological membrane or biological membrane reception phase; To dissociate out with the active ingredient of Chinese herbs that biomacromolecule, imitative biological membrane or biological membrane specificity combine, carry out chromatogram or chromatogram/mass spectrophotometry, thereby filter out the active component that can combine with biomacromolecule, imitative biological membrane or biological membrane specificity.
Hollow fiber biological membrane Microtraps choosing (hollow fiber biomembrance micro-screening of the present invention; HFBMMS) be at hollow fiber liquid-phase micro-extraction (hollow fiber 1iquid phase microextraction; HFLPME) a kind of that the binding molecule recognition principle sets up being gone up on active ingredient of Chinese herbs analysis and research basis be to imitate biological membrane, biological membrane or biomacromolecule carrier with hollow fiber, easy, quick, efficiently study the interactional new technology of active ingredient of Chinese herbs and bio-target molecule.HFBMMS injects competent cell film, imitative biological membrane, biomacromolecule (enzyme, acceptor, carrier protein) in the hollow fiber conduit and with it and is inserted into Chinese medicine extract or traditional Chinese medicine compound extract; Under the condition that pH3.4,37 ℃ approach to absorb naturally in the biosome, imitative biological membrane in the hollow fiber conduit or biomacromolecule produce specificity to active ingredient of Chinese herbs and combine.With certain solvent or solution pair active ingredient of Chinese herbs that combines with imitative biological membrane, biological membrane or biomacromolecule parsing carrying out chromatogram or chromatogram/mass spectrophotometry.Active ingredient of Chinese herbs shows the difference of chromatographic peak area, retention time or peak number then in the difference of imitating retention behavior on difference table imitative now biological membrane, biological membrane or the biomacromolecule that specificity combines on biological membrane, biological membrane or the biomacromolecule.Utilize HFBMMS to combine chromatographic technique to measure active ingredient of Chinese herbs and imitative biological membrane, biological membrane or biomacromolecule incorporating parametric, the interaction between research active ingredient of Chinese herbs and the bio-target molecule; Structure is the active ingredient of Chinese herbs screening technique of core with HFBMMS, and it is that the competent cell film of carrier screens active ingredient of Chinese herbs that the method is equally applicable to the hollow fiber.
The main Study on mechanism of HFBMMS method: (1) blank hollow fiber is to before and after the screening of active ingredient of Chinese herbs standard items, and the ability spectrogram and the electron-microscope scanning figure of hollow fiber surface diffusion layer do not have significant change; (2) contain imitative biological membrane, biological membrane or biomacromolecule hollow fiber and blank hollow fiber and respectively the active ingredient of Chinese herbs standard items are screened, the former desorbed solution has chromatographic peak or chromatographic peak stronger, a little less than latter's desorbed solution does not have chromatographic peak or chromatographic peak very.Explain do not have non-specific binding or binding ability very between hollow fiber activated centre and the active ingredient of Chinese herbs a little less than; Hollow fiber only plays biofilm carrier in HFBMMS; Do not participate in the screening of active ingredient of Chinese herbs; The bioactive molecule that obtains in the desorbed solution is the result of biological membrane or biomacromolecule and active component effect, and the effect between active component and the bio-target molecule is the dominant mechanism of HFBMMS method.
The selection of biological membrane or biomacromolecule carrier-hollow fiber: hollow fiber conduit not only can be used as the carrier that holds and protect imitative biological membrane or biomacromolecule, and for the HFBMMS method platform of bio-target molecule and active component effect is provided.Countless apertures on the hollow fiber walls not only can make the medicine micromolecule have free passage; Increase the surface area that imitative biological membrane, biological membrane or biomacromolecule contact with chemical constitution; And Chinese medical extract played the effect of microfiltration and purifying, removed consuming time and loaded down with trivial details sample purge process in other Chinese medicine method for screening active ingredients from.In HFBMMS, select (1) blank hollow fiber active ingredient screening front and back ability spectrogram and electron-microscope scanning figure not to have the hollow fiber that changes; (2) desorbed solution does not have chromatographic peak or the weak hollow fiber of chromatographic peak in the blank hollow fiber.Satisfy above condition and can select the hollow fiber that does not contain strong reactive group for use, the present invention preferably adopts polypropylene, polysulfones, Kynoar or polyethersulfone hollow fiber conduit.
The reappearance of the reappearance of HFBMMS method and stability: HFBMMS and stability are whether Chinese medicine or Chinese medicine compound prescription active ingredient screening result be basic reliably.Non-specific binding directly influences the reappearance and the stability of The selection result between hollow fiber activated centre and the chemical composition of Chinese materia medica, is the principal element that causes the bioactivity screening mistake.In order to solve non-specific binding problem between hollow fiber and the chemical composition of Chinese materia medica; The present invention at first carries out the inertization of deactivation to hollow fiber; Be about to hollow fiber conduit successively with acetone, methyl alcohol, acid and/or alkali, distilled water difference ultrasonic cleaning 5 ~ 10min; And clean repeatedly with distilled water, dry up, make hollow fiber conduit itself not with Chinese medicine in active component react; Wherein used bronsted lowry acids and bases bronsted lowry is knownly to be used to clean and the solution of inertization alkali such as acid such as example hydrochloric acid, nitric acid, sulfuric acid, acetic acid and NaOH, ammoniacal liquor, sodium methoxide.Simultaneously, biological membrane in the hollow fiber conduit or biomacromolecule produce absorption and immobilized there is certain inhibition in the hollow fiber activated centre and seals effect on its surface.Under certain HFBMME and HPLC condition; Repeatedly parallel laboratory test; Mensuration is after HFBMME method screening, and the withinday precision of the active component standard items recovery, retention time and peak area and day to day precision (RSD) prove the reappearance and the stability of HFBMME method.
The HFBMMS specificity combines the parsing of effective constituent: the method for among the present invention the middle pharmaceutically active ingredient of specific bond on biomacromolecule, biological membrane, the imitative biological membrane in the hollow fiber conduit being dissociated out is to select for use different active ingredient with the solution of different solvents or different pH, polarity, ionic strength etc. the effective constituent that specificity on biological membrane or the biomacromolecule combines to be resolved to different active ingredient and different biomacromolecules, biological membrane, imitative biological membrane.Desorbed solution is used efficient liquid phase chromatographic analysis, and chromatographic peak retention time, peak area are stable, and highly sensitive analysis condition is resolved for HFBMMS effective constituent or condition.
Utilize the HFBMMS method that active component in Chinese medicine and the traditional Chinese medicine compound extract is screened and structure is confirmed: through peak area, retention time and the peak number of HFBMME screening back chromatographic peak; Confirm the kind and the quantity of the active ingredient of Chinese herbs that preliminary screening goes out, the top condition of experiment is undertaken confirming after the shaker test by the active ingredient of Chinese herbs standard items.For the active component that standard items are arranged in the chromatogram, adopt the method that contrasts with standard items retention time and spectrogram, confirm the active component that keeps on the bio-target molecule.For the effective constituent that does not have standard items; Utilize the contrast of chromatogram/mass spectrometry analysis and document; In conjunction with the LC/MS coupling technique unknown active component is made the accurate judgement of structure, realize the screening and the evaluation of active ingredient of Chinese herbs, have great importance finding new active component.
The detection sensitivity that we find HFBMMS method identification effective constituent in research of the present invention is research biological membrane or biomacromolecule and the key of active ingredient of Chinese herbs interaction, high flux screening.The invention group is found in the research of HFBMME because Chinese medicine or traditional Chinese medicine compound extract matrix are complicated; And active component content is lower; The HPLC-UV signal that provides after active component and the bio-target molecule effect a little less than; Particularly those trace active compositions or with the bio-target molecule binding ability a little less than the active component signal more a little less than, be difficult to even detect.In order to improve hollow fiber trace biological membrane or biomacromolecule detection sensitivity to the identification active component; We can (1) according to the character of active component in the Chinese medicine select that the aperture is little, the hollow fiber of wall thickness to be to increase the detergent power of hollow fiber, reduces detectability; (2) use for reference to improve among the prior art HFLPME and compare the character that (ratio of Chinese medicine extract volume and organic reception phase volume) can improve the enrichment multiple; (3) use chromatograph to analyze to the active component that filters out known structure, to improve the detection sensitivity of HFBMMS to active ingredient of Chinese herbs identification with high sensitivity detector (detecting devices such as fluorescence, galvanochemistry).
Compared with prior art the present invention has following beneficial effect: (1) needn't prepare the biological membrane stationary phase luggage post of going forward side by side.Therefore, do not need special technique and equipment, got rid of in the silica gel Si-OH the non-specific binding of medicine, simple to operate, with low cost, be easy to grasp.(2) screening active ingredients and chromatographic process are carried out respectively, can satisfy the most suitable or top condition that biology and chromatographia require; Chinese medical extract reflects Chinese medicine multicomponent, the synergistic characteristic of many target spots preferably without separating directly and the biological membrane effect, improves and guaranteed the reliability and the high efficiency of active ingredient of Chinese herbs screening.(3) screening active ingredients and sample purification are accomplished simultaneously, have removed the necessary sample pretreatment process of other Chinese medicine screening active ingredients methods from, have improved Chinese medicine active ingredient screening and the definite efficient of structure.(4) Chinese medical extract can reflect Chinese medicine multicomponent, many target spots, synergistic characteristic preferably without separating directly and biological membrane or biomacromolecule effect, and the Chinese medicine effector substance basis of disclosing real onset in the body is had great importance.
Description of drawings
Fig. 1 is the active ingredient of Chinese herbs screening plant.
Among the figure, the 1-micro syringe, the 2-micro syringe, the 3-vial, the 4-hollow fiber, 5-imitates biological membrane, biological membrane or biomacromolecule, 6-stirrer, 7-thermostatic mixer.
Fig. 2 is flavonoids active component and biological membrane or interaction of biomacromolecules chromatogram in the HFBMMS research ginkgo extract.
Among the figure, A-liposome hollow fiber screening blank solution, A '-bovine serum albumin(BSA) hollow fiber screening blank solution, the blank hollow fiber screening sample of B-liquid, C-liposome hollow fiber screening sample liquid, D-bovine serum albumin(BSA) hollow fiber screening sample liquid
Wherein, (1) liposome: the 6-red bayberry is plain, 7-Quercetin, 8-Kaempferide, 9-Isorhamnetin; (2) bovine serum albumin(BSA): 8-Quercetin, 10-Kaempferide, 11-Isorhamnetin.
Fig. 3 is anthraquinone active component and biological membrane or interaction of biomacromolecules chromatogram in the HFBMMS research giant knotweed extract.
Among the figure, A-liposome hollow fiber screening blank solution, A '-bovine serum albumin(BSA) hollow fiber screening blank solution, the blank hollow fiber screening sample of B-liquid, C-liposome hollow fiber screening sample liquid, D-bovine serum albumin(BSA) hollow fiber screening sample liquid
Wherein, (1) liposome: 3-aloe-emodin, 4-archen; (2) bovine serum albumin(BSA): 3-aloe-emodin, 4-archen.
Fig. 4 is anthraquinone active component and biological membrane or interaction of biomacromolecules chromatogram in the HFBMMS research cassia seed extract.
Among the figure, A-liposome hollow fiber screening blank solution, A '-bovine serum albumin(BSA) hollow fiber screening blank solution, the blank hollow fiber screening sample of B-liquid, C-liposome hollow fiber screening sample liquid, D-bovine serum albumin(BSA) hollow fiber screening sample liquid
Wherein, (1) liposome: 1-aloe-emodin, 3-Rhein, 5-Chrysophanol; (2) bovine serum albumin(BSA): 1-aloe-emodin, 3-Rhein, 5-Chrysophanol.
Fig. 5 is anthraquinone active component and biological membrane or interaction of biomacromolecules chromatogram in the HFBMMS research multiflorum extracting solution.
Among the figure, A-liposome hollow fiber screening blank solution, A '-bovine serum albumin(BSA) hollow fiber screening blank solution, the blank hollow fiber screening sample of B-liquid, C-liposome hollow fiber screening sample liquid, D-bovine serum albumin(BSA) hollow fiber screening sample liquid
Wherein, (1) liposome: 2-aloe-emodin, 3-archen, 4-Physcion; (2) bovine serum albumin(BSA): 2-aloe-emodin, 3-archen, 4-Physcion.
Embodiment
In conjunction with following experiment the present invention is further specified, its experimental technique is:
Material and reagent hollow fiber (Tianjin MoTian Membrane Engineering Technology Co., Ltd), Chinese medicine standard reference material (Nat'l Pharmaceutical & Biological Products Control Institute), bovine serum albumin matter (sky, Shanghai is Science and Technology Ltd.), lecithin.Experimental water is a redistilled water, and it is pure that other reagent is analysis.
Instrument Agilent Technologies 1200 Series high performance liquid chromatographs, self-control hollow fiber liquid phase Microtraps screening device.
Experimental procedure is as shown in Figure 1, gets the vial of 10ml, adds the PBS (confession phase) of certain density active ingredient of Chinese herbs standard items of 5ml or Chinese medical extract pH7.4, puts into stirrer and is fixed on the magnetic stirring apparatus, at 37 ℃ of following constant temperature.The hollow fiber (being hollow fiber conduit) of certain-length after inertization, is cleaned, dried up.Biomacromolecule (enzyme, acceptor, carrier protein) or imitative biological membrane are injected in the hollow fiber conduit and it is bent to U-shaped with micro syringe and place and supply the phase bottle.Under the condition that absorbs naturally of simulation biological membrane, chemical constitution interaction in imitative biological membrane or biomacromolecule and active component standard items or the Chinese medicine extract filters out the active component that can act on biological membrane or biomacromolecule.With the active component parsing of specificity combination on biological membrane or the biomacromolecule or with biological membrane or biomacromolecule fragmentation, under the chromatographic condition of the best, carry out chromatogram or chromatogram/mass spectrophotometry.
Following examples are that the invention group serves as the screening object with flavonoids, anthraquinone active component standard items; Stability and reappearance that the imitative biological membrane of trace bovine serum albumin(BSA) and liposome is the HFBMMS method of target molecule are studied; Set up flavonoids active component in ginkgo, the sophora bud, the arbor-vitae extract, the HFBMMS screening technique of anthraquinone active component in giant knotweed, cassia seed, the RADIX POLYGONI MULTIFLORI PREPARATA extract.
1. the extraction of flavonoids chemical constitution in the ginkgo medicinal material
Get dry medicinal material, porphyrize is crossed 60 mesh sieves, and precision takes by weighing ginkgo 3 g, puts to add methyl alcohol 20 ml in the tool plug conical flask, weighs, and soaks 10min, and ultrasonic Extraction (power 100W) 30 min are put to room temperature, supply the weight that subtracts mistake with methyl alcohol.Add 25% HCl 5 ml again, 30 min that reflux are put coldly, filter, with methanol constant volume to 25 ml.4 ℃ of refrigerators are preserved, and are for use.
2. flavonoids active component in the ginkgo extract is studied in the choosing of hollow fiber biological membrane liquid phase Microtraps
Draw ginkgo Chinese medicine extract 0.3ml and clean in the cuvette that dries up in 2ml, add phosphate buffer solution 1.2 ml of pH7.4 again, making liquor capacity is 1.5ml.The Kynoar hollow fibers (inner/outer diameter: 0.5/0.8mm, the aperture is 0.2 μ m) that three 10 cm are long are distinguished ultrasonic cleaning 5 ~ 10min with acetone, methyl alcohol, nitric acid, NaOH, distilled water, and clean repeatedly with distilled water and to dry up.Hollow fiber after the processing immerses 1 min in liposome solutions (0.01g/ml) or the protein solution (0.1g/ml); Treat to take out after its hole is full of biological membrane; U-shaped is dried and be bent into to the liposome solutions or the protein solution of fiber surface; With micro syringe 20 μ l liposome solutions or protein solution are injected in the hollow fiber conduit, the U-shaped hollow fiber conduit is immersed in the sample solution.Open magnetic stirring apparatus, 3 ℃ of constant temperature, 1500 rpm stir down, screen 30 min, and screening end back is collected and received in the little EP pipe of 1ml, adds 50 μ l methyl alcohol, leaves the heart 20 min 12000, and the absorption supernatant injects HPLC and analyzes.
Utilize the HFBMMS-HPLC method to have the composition that combines to carry out screening and see Fig. 2 ginkgo extract and liposome and bovine serum albumin(BSA).Find to combine with liposome and to have 9 compositions of obvious reservation by Fig. 2, combine and have 11 compositions of obvious reservation with bovine serum albumin(BSA).4 known activity composition red bayberry elements, Quercetin, Kaempferide and Isorhamnetins with a grain of salt in the ginkgo have been carried out the structure Preliminary Identification through the retention time contrast; For the active component that does not have standard items that occurs in the chromatogram; Can carry out the contrast of chromatogram/mass spectrometry analysis and document, confirm their possible structure.
1. the extraction of anthraquinone class chemical constitution in the giant knotweed medicinal material
The crude drug giant knotweed is smashed to pieces, and powder (crossing sieve No. three) 0.5g, accurate methenyl choloride 25 ml and 2.5 mol/L sulfuric acid solutions, 20 ml of adding decided in accurate title; Claim to decide weight, put to add in 80 ℃ of water-baths and refluxed 2 hours, be cooled to room temperature; Claim again to decide weight, supply the weight that subtracts mistake, shake up with methenyl choloride.Obtain methenyl choloride liquid, precision is measured 20 ml, steams appearance at 40 ℃ of following evaporates to dryness with revolving, and residue adds dissolve with methanol, is transferred in the 25 ml measuring bottles, adds methyl alcohol to scale, shakes up, and gets subsequent filtrate, promptly gets.4 ℃ of refrigerators are preserved, and are for use.
2. anthraquinone active component in the giant knotweed extract is studied in the choosing of hollow fiber biological membrane liquid phase Microtraps
Get giant knotweed extract 2ml and clean in the cuvette that dries up in 5ml, add the phosphate buffer solution 3ml of pH7.4 again, making liquor capacity is 5ml.The polysulfone hollow fibre that three 10 cm are long (inner/outer diameter: 0.8/1.2 mm mm, the aperture is 0.2 μ m) is distinguished ultrasonic cleaning 5 ~ 10min with acetone, methyl alcohol, nitric acid, NaOH, distilled water, and cleans repeatedly with distilled water and to dry up.Hollow fiber after the processing immerses 1 min among liposome solutions 0.01g/ml or the protein solution 0.1g/ml; Treat to take out after its hole is full of biological membrane; U-shaped is dried and be bent into to liposome solutions or protein solution that fiber surface is overflowed; With micro syringe 20 μ l liposome solutions or protein solution are injected in the hollow fiber conduit, the U-shaped hollow fiber conduit is immersed in the sample solution.Open magnetic stirring apparatus, 37 ℃ of constant temperature, 900 rpm stir down, screen 40 min, and screening end back is collected and received in the little EP pipe of 1ml, adds 50 μ l methyl alcohol, leaves the heart 20 min 12000, and the absorption supernatant injects HPLC and analyzes.
Utilize the HFBMMS-HPLC method to have the composition that combines to carry out screening and see Fig. 3 giant knotweed extract and liposome and bovine serum albumin(BSA).Find that by Fig. 3 giant knotweed extracts fluid power combines and have obvious reservation with liposome or bovine serum albumin(BSA) 5 compositions that have.2 known activity composition 3-aloe-emodins with a grain of salt; The 4-archen has carried out the structure Preliminary Identification through the retention time contrast; For the active component that does not have standard items that occurs in the chromatogram, can carry out the contrast of chromatogram/mass spectrometry analysis and document, confirm their possible structure.
1. the extraction of anthraquinone class chemical constitution in the cassia seed medicinal material
The crude drug cassia seed is smashed to pieces, accurate claimed decide powder (crossing sieve No. three) 0.5 g, put in the tool plug conical flask, accurate methyl alcohol 25 ml that add claim decide weight, put in the water-bath reflux 30 minutes, put coldly, and weight decided in title again, supplies with methyl alcohol to subtract weight loss, shakes up filtration.Precision is measured subsequent filtrate 5ml, adds 10% hydrochloric acid solution, 20 ml, puts in the water-bath reflux 1 hour, immediately cooling; Extract four times with methenyl choloride, each 20 ml merge methenyl choloride liquid, steam appearance at 40 ℃ of following evaporates to dryness with revolving; Residue is used dissolve with methanol, is transferred in the 25 ml measuring bottles, and is diluted to scale; Shake up, get subsequent filtrate, promptly get.4 ℃ of refrigerators are preserved, and are for use.
2. anthraquinone active component in the cassia seed extract is studied in the choosing of hollow fiber biological membrane liquid phase Microtraps
Depend on that pine torch extract 2ml cleans in the cuvette that dries up in 5ml, add phosphate buffer solution 3 ml of pH7.4 again, making liquor capacity is 5ml.The polypropylene hollow fiber that three 10 cm are long (inner/outer diameter: 0.6/1.0 mm, the aperture is 0.2 μ m) is distinguished ultrasonic cleaning 5 ~ 10min with acetone, methyl alcohol, nitric acid, NaOH, distilled water, and cleans repeatedly with distilled water and to dry up.Hollow fiber after the processing immerses 1 min among liposome solutions 0.01g/ml or the protein solution 0.1g/ml; Treat to take out after its hole is full of biological membrane; U-shaped is dried and be bent into to liposome solutions or protein solution that fiber surface is overflowed; With micro syringe 20 μ l liposome solutions or protein solution are injected in the hollow fiber conduit, the U-shaped hollow fiber conduit is immersed in the sample solution.Open magnetic stirring apparatus, 37 ℃ of constant temperature, 900 rpm stir down, screen 40 min, and screening end back is collected and received in the little EP pipe of 1ml, adds 50 μ l methyl alcohol, leaves the heart 20 min 12000, and the absorption supernatant injects HPLC and analyzes.
Utilize the HFBMMS-HPLC method to have the composition that combines to carry out screening and see Fig. 4 cassia seed extract and liposome and bovine serum albumin(BSA).Find that by Fig. 4 cassia seed extracts fluid power combines and have obvious reservation with liposome 6 compositions that have, and combines and have 5 compositions of obvious reservation with bovine serum albumin(BSA).3 known activity composition 1-aloe-emodins wherein with a grain of salt; The 3-Rhein; The 5-Chrysophanol has carried out the structure Preliminary Identification through the retention time contrast; For the active component that does not have standard items that occurs in the chromatogram, can carry out the contrast of chromatogram/mass spectrometry analysis and document, confirm their possible structure.
1. the extraction of anthraquinone class chemical constitution in the RADIX POLYGONI MULTIFLORI PREPARATA medicinal material
The crude drug RADIX POLYGONI MULTIFLORI PREPARATA is smashed to pieces, accurate claimed customization tuber of multiflower knotweed powder (crossing sieve No. three) 0.5 g, put in the tool plug conical flask, the accurate methyl alcohol 25ml that adds claims decide weight, and reflux 1 hour is put coldly, claims to decide weight again, supplies with methyl alcohol to subtract weight loss, shakes up filtration.Precision is measured subsequent filtrate 5ml, puts in the flask, steams appearance at 40 ℃ of following evaporates to dryness with revolving, and adds 8% hydrochloric acid solution 10ml, sonicated 2 minutes; Add methenyl choloride 10 ml again, reflux 1 hour is put coldly, puts in the separating funnel, with a small amount of methenyl choloride washing container; Incorporate in the separating funnel, obtain the methenyl choloride layer, acid solution is extracted three times with methenyl choloride again, each 10 ml; Merge methenyl choloride liquid, steam appearance at 40 ℃ of following evaporates to dryness with revolving, residue is used dissolve with methanol, is transferred in the 25ml measuring bottle; Add methyl alcohol to scale, shake up, get subsequent filtrate, promptly get.4 ℃ of refrigerators are preserved, and are for use.
2. anthraquinone active component in the RADIX POLYGONI MULTIFLORI PREPARATA extract is studied in the choosing of hollow fiber biological membrane liquid phase Microtraps
Get RADIX POLYGONI MULTIFLORI PREPARATA extract 2ml and clean in the cuvette that dries up in 5ml, add phosphate buffer solution 3 ml of pH7.4 again, making liquor capacity is 5ml.The polyethersulfone hollow fiber that three 10 cm are long (inner/outer diameter: 0.8/1.32 mm, the aperture is 0.2 μ m) is distinguished ultrasonic cleaning 5 ~ 10min with acetone, methyl alcohol, nitric acid, NaOH, distilled water, and cleans repeatedly with distilled water and to dry up.Hollow fiber after the processing immerses 1 min among liposome solutions 0.01g/ml or the protein solution 0.1g/ml; Treat to take out after its hole is full of biological membrane; U-shaped is dried and be bent into to liposome solutions or protein solution that fiber surface is overflowed; With micro syringe 20 μ l liposome solutions or protein solution are injected in the hollow fiber conduit, the U-shaped hollow fiber conduit is immersed in the sample solution.Open magnetic stirring apparatus, 37 ℃ of constant temperature, 900 rpm stir down, screen 40 min, and screening end back is collected and received in the little EP pipe of 1ml, adds 50 μ l methyl alcohol, leaves the heart 20 min 12000, and the absorption supernatant injects HPLC and analyzes.
Utilize the HFBMMS-HPLC method to have the composition that combines to carry out screening and see Fig. 5 RADIX POLYGONI MULTIFLORI PREPARATA extract and liposome and bovine serum albumin(BSA).Find that by Fig. 5 RADIX POLYGONI MULTIFLORI PREPARATA extracts fluid power combines and have obvious reservation with liposome or bovine serum albumin(BSA) 4 compositions that have.3 known activity composition 2-aloe-emodins wherein with a grain of salt; The 3-archen; The 4-Physcion has carried out the structure Preliminary Identification through the retention time contrast; For the active component that does not have standard items that occurs in the chromatogram, carrying out the contrast of chromatogram/mass spectrometry analysis and document, confirm their possible structure.
Claims (3)
1. a Chinese medicine method for screening active ingredients is characterized in that may further comprise the steps: (1) preparation Chinese medicine or traditional Chinese medicine compound extract a; (2) PBS of adding pH7.4 in extract a gets solution b; (3) with hollow fiber conduit successively with acetone, methyl alcohol, acid and/or alkali, distilled water ultrasonic cleaning 5 ~ 10min respectively, and after cleaning repeatedly with distilled water, dry up; After injecting biomacromolecule, imitative biological membrane or biological membrane in the hollow fiber conduit; Place solution b in 37 ℃ of following constant temperature and stirring hollow fiber conduit, screen, (4) screening finishes the big molecule of back collection of biological, imitative biological membrane or biological membrane reception phase; To dissociate out with the active ingredient of Chinese herbs that biomacromolecule, imitative biological membrane or biological membrane specificity combine; Carry out chromatogram or chromatogram/mass spectrophotometry, thereby filter out the active component that can combine with biomacromolecule, imitative biological membrane or biological membrane specificity, described active ingredient of Chinese herbs is flavonoids and anthraquinone class; The method that active ingredient of Chinese herbs is dissociated out is to collect to receive in the little EP pipe of 1ml; Add 50 μ l methyl alcohol, leave the heart 20 min, inject HPLC after the absorption supernatant and analyze 12000.
2. a kind of Chinese medicine method for screening active ingredients according to claim 1 is characterized in that: described hollow fiber conduit adopts Kynoar, polypropylene, polysulfones, polyethersulfone hollow fiber conduit.
3. a kind of Chinese medicine method for screening active ingredients according to claim 2 is characterized in that: the hollow fiber conduit aperture of selection is 0.2 μ m.
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| CN102824756B (en) * | 2012-09-07 | 2015-06-03 | 浙江大学 | Method for screening triglyceride enzyme inhibitor from plant extract |
| CN103884811B (en) * | 2014-03-04 | 2016-01-27 | 中国人民解放军第二军医大学 | A kind of biological chromatography compares screening system and application thereof |
| CN107192778B (en) * | 2017-06-06 | 2020-03-03 | 郭嘉亮 | Method for separating bacterial biofilm inhibiting components based on target protein affinity monolithic chromatographic column |
| CN110018265A (en) * | 2019-01-24 | 2019-07-16 | 河北医科大学第二医院 | A kind of " integration " active ingredient of Chinese herbs screening technique based on target compatibility |
| CN111122759A (en) * | 2019-12-30 | 2020-05-08 | 浙江工业大学 | Target protein-based liposome biological membrane chromatographic column and application thereof in screening active components in natural products |
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