CN101793883A - Method for evaluating Traditional Chinese medicine activity and mechanism based on holistic approach - Google Patents
Method for evaluating Traditional Chinese medicine activity and mechanism based on holistic approach Download PDFInfo
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Abstract
The invention relates to the research field of traditional Chinese medicine effect material basis, particularly relating to a method for evaluating traditional Chinese medicine activity and mechanism based on holistic approach, which is characterized by comprising the following steps: passing traditional Chinese medicine extract through high efficient liquid to acquire a traditional Chinese medicine finger print; determining target peaks in the finger print; connecting the outlet pipeline of a high performance liquid chromatography detector with a six-way switch valve, and collecting single target peaks, target peak groups at random combination and residue component bundles after knockout of target peak groups by the switch valve; and performing pharmacodynamic evaluation on a collected sample to obtain the activity sequence of the whole activity of the target peak groups and single target peaks, contribution of each single target peak in the traditional Chinese medicine extract and mutual interaction relationship between components. The invention broadens the research direction of the traditional Chinese medicine effect material basis, and provides a research method of traditional Chinese medicine integral action mechanism with simple operation and low cost.
Description
Technical field
The present invention relates to Chinese medicine effector substance fundamental research field, be specifically related to a kind ofly estimate the method for its activity and the mechanism of action by knocking out and catch chemical composition of Chinese materia medica, be a kind of method of utilizing liquid chromatography-mass spectrography-valve handoff technique coupling research Chinese medicine heterogeneity to interaction mechanism between the influence of overall activity and each composition based on whole sight of Chinese medicine.
Background technology
For a long time, the prevailing model of domestic traditional Chinese medicine research mainly is to have used for reference the elementary tactics of west to natural product active ingredient research: utilize pharmacological model (models such as whole animal, isolated organ, cell and molecule) that the pure product of compound that obtain are carried out biological activity test (referring to Tu Pengfei on chemical constitution extraction, separation and structure evaluation of foundation, pharmacognostic development and research thinking thereof, China's natural drug, 2006,4:411-419).Research method has although it is so been illustrated the active component and the mechanism of action of some Chinese medicines, but has ignored the mass action characteristics of Chinese medicine.
Along with going deep into of research, the multicomponent of Chinese medicine, many target spots and mass action pattern progressively become the common recognition of Chinese scholars (referring to J.Qiu.A culture in the balance.Nature, 2007,448:126-128; J.Qiu.China plans tomodernize traditional medicine.Nature, 2007,446:590-591; T.H.Han, R.Roy.Studyingtraditional Chinese medicine.Science, 2003,300:740-741; Zhou Jun, Chinese medicine compound prescription-natural combinatorial chemical library and many targets mechanism of action, Chinese combination of Chinese tradiational and Western medicine magazine, 1998,18:67; Xiao Peigen etc., the 21 century and the modernization of Chinese medicine, CHINA JOURNAL OF CHINESE MATERIA MEDICA, 2000,25:67-70).Traditional Chinese medicine fingerprint can directly be controlled in the Chinese medical extract composition as much as possible, becomes the effective ways of present traditional Chinese medicine quality control.Traditional chemical fingerprint is by the peak area of estimating each chromatographic peak in the medicinal material or the relative content that peak height ratios is determined chemical constitution in the sample, and in conjunction with the quality of relatively estimating Chinese crude drug or the quality of the pharmaceutical preparations of mathematical statistics method by similarity (referring to Tu Pengfei, high performance liquid chromatography is formulated the discussion of Chinese crude drug and traditional Chinese medicine characteristic fingerprint pattern, Chinese patent drug, 2000,22:516.Fruit Dean etc., the thinking of traditional Chinese medicine quality Control Study and method, Chinese natural drug, 2005,4:332-337.Cai Shaoqing etc., based on the similarity analysis of the chromatographic fingerprints of Chinese materia medica of intending distributing, Peking University's journal, 2008,44:695-699).Though this method has related to the characteristics of Chinese medicine " multicomponent ", the relevance between each composition and its drug effect does not also obtain effectively expressing in present stage, is not enough to illustrate the mass action characteristics of Chinese medicine.
In recent years, the biotechnology fast development, a kind of new complex art-biological chromatography method that intersected to form of life science and chromatographic separation technology, this looks for another way for the fundamental research of Chinese medicine effector substance.This method is the biological fingerprint graphical spectrum technology of using the earliest, comprise molecular biosciences chromatogram, biological membrane as chromatographic and cell biological chromatogram etc., its ultimate principle be material with various biologically actives as stationary phase, big molecule of active bio or competent cell film are anchored on the chromatogram carrier as target.Different and different component separating is come according to the interactional power of Chinese medicine and targets such as enzyme, acceptor or cell membrane, can carry out rapid screening to the various active composition of Chinese medicine, remedy the deficiency that present chemical fingerprint control traditional Chinese medicine quality can break off relations with drug effect, had certain pharmacology or physiologic meaning.(referring to: Wang Hailin etc., the molecular biosciences chromatogram is used for the research of active ingredient of Chinese herbs screening and method of quality control, chromatogram, 1999,17 (2): 123-127.Mao Xiqin etc., biological membrane as chromatographic and the application in active constituents of medicine is analyzed thereof, analytical chemistry, 2002,30 (2): 231-236.Sheng Lianghong etc., the permeability of cell membrane composition and the quality control thereof of immobilized liposome body colour spectrum research Chinese medicinal formulae, analytical chemistry, 2004,32 (12): 1595-1598.Yuan Bingxiang etc., the biological affinity chromatography medicament sifting motion system of cell membrane, Chinese Pharmacological circular, 2003,20 (3): 68.) yet there are problems in actual applications in this method, combine its biological property of back as carriers such as enzyme, the big molecule of acceptor isoreactivity or cell membrane and silica gel and can be subjected to certain influence etc., and this method can only illustrate the active strong and weak difference between the Chinese medicine heterogeneity, can not explain between each composition and each composition and medicinal material overall activity between correlation.
The inventor has invented a kind of active component high-throughput screening method based on the affine selection of target protein in early-stage Study.Its ultimate principle is that target protein and Chinese medical extract are hatched in damping fluid altogether, this moment, activated compound and target protein were combined into target protein-active component compound, hatched to comprise target protein-active component compound and the free micromolecular compound that does not combine with target protein in the mixed solution that obtains.Hatch solution and be splined on molecular-exclusion chromatography post or first online solid-phase extraction column, use the buffer solution elution pillar, eluent stream detects and collects the eluent that at first goes out the peak through UV-detector under absorbing wavelength 280nm; Then eluent is mixed with dissociation solution and dissociate, the active component that combines with target protein in the solution that dissociates is dissociated into free cpds; The solution that will dissociate subsequently is splined on second online solid-phase extraction column, and target protein and buffer salt are by the aqueous solution wash-out of high flow rate, and active component then is retained on second online solid-phase extraction column; The active component wash-out that is retained that above-mentioned steps is obtained enters the analysis of high performance liquid chromatogram-mass spectrometer system again.So just can filter out the reactive compound in the Chinese medicine, the reactive compound that screens is carried out structural identification (seeing CN200810124611.1).But this technology can only filter out in the Chinese medicine isolated chemical active ingredient, can not analyze heterogeneity in the Chinese medicine on the whole to the contribution degree of overall activity, interaction and whole overall activity of active component groups between each composition.
Summary of the invention
The present invention has set up and a kind ofly based on whole the passing through of seeing of Chinese medicine chemical constitution has been knocked out and caught the research method of estimating the active and mass action characteristics of Chinese medicine.The present invention can knock out and catch any one chromatographic peak in the traditional Chinese medicine fingerprint, and the variation of analysis chromatographic peak and the degree of association between the Chinese medicine activity are to obtain the contribution degree of each chromatographic peak in the Chinese medicine overall activity; Can also utilize simultaneously active ingredient screening technology based on the affine selection of target protein, after rapid screening is determined the active component peak, it is knocked out and catches, with the relation between one or more active components and the Chinese medicine overall activity in the research Chinese medical extract, and can make up the collaborative or antagonism of further inquiring between each active component by different way by the active component of will catch.
The present invention is based on the detection method of the whole traditional Chinese medicine ingredients effect characteristics of seeing of Chinese medicine, comprise, obtain Chinese medicine extract, carry out as follows then medicinal solvent extraction in to be measured:
A. Chinese medicine extract detects through high performance liquid chromatography, obtains traditional Chinese medicine fingerprint;
B. determine the target peak in the finger-print;
C. after determining target peak, the outflow pipeline of high performance liquid chromatography detecting device is linked to each other with six direction changeover valves, switch by valve, eluent is collected in the following manner: the target peak group and 3 of combination in any 1) single target peak, 2)) knock out the residual components group behind the target peak (group) in the finger-print, in this three part, can three parts all collect, also can collect first and third part, or collect second and third part;
D. the sample that the c collection step is obtained carries out the pharmacodynamics evaluation, obtains target peak group's overall activity, the activity ordering at single target peak, the contribution degree of single target peak in Chinese medical extract, and the interaction relationship between each composition.
Be further elaborated below in conjunction with said method:
In the said method, Chinese medicine to be measured can be that single medicinal material also can be a Chinese medicine compound prescription.The solvent that extracts Chinese medicine extracts by this area method commonly used, can be water and/or organic solvent.Preferred solvent is to be selected from water, C
1-C
5Alcohol, ethyl acetate, ether, chloroform, sherwood oil, or their potpourri.Described C
1-C
5Pure particular methanol, ethanol, normal butyl alcohol.More preferably, prepare Chinese medicine extract with 70-100% ethanol or 70-100% methanol extraction.The solvent that extracts Chinese medicine is that neutrality, acidity or alkalescence all can; Can select for use in mineral acid, inorganic base, organic acid or the organic base and acid or alkaline Chinese medicine extract, carry heavy method of alkali or alkali extraction and acid precipitation as acid.
Owing to contain many compositions in the Chinese medicine, add that high performance liquid chromatography has very high separation efficiency, Chinese medicine extract many peaks can occur by high performance liquid chromatogram, generally speaking, under a kind of chromatographic condition, 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 chromatographic condition, can change a kind of chromatographic condition will be separately.Optimum chromatographic condition should make that peak shape is stable, degree of separation is good.In the actual mechanical process, the technician can consult data of literatures according to concrete Chinese medicine to be measured, and present most Chinese medicine all has the method that obtains traditional Chinese medicine fingerprint of comparative maturity.
In the finger-print target peak define following two kinds of methods: 1. obtain: Chinese medicine extract and target protein are hatched in damping fluid altogether through screening active ingredients, to not contain the Chinese medicine extract of target protein simultaneously as blank, use active component high-throughput screening method to detect respectively based on the affine selection of target protein, definite peak that can combine with target protein, be the active component peak, as target peak. 2. obtain without screening active ingredients: the forward chromatographic peak of ordering according to peak height value or the descending ordering of peak area value, is chosen as target peak in the finger-print peak, and promptly be advisable with 5-20 in the major component peak.
At present, the method for determining active component peak in the finger-print has multiple, such as the certain methods of exploitation before this laboratory (referring to Li Ping, Sheng Lianghong, Li Ruiyan, Li Songlin, neat refining literary composition, the detection method of pharmaceutically active ingredient in a kind of, Chinese invention patent, ZL200410041024.8; Li Ping, Sheng Lianghong, Li Ruiyan, Li Songlin, Wang Wei, the detection method of pharmaceutically active ingredient in a kind of, Chinese invention patent, ZL200410041115.1 and Li Ping, Zhou Jianliang, An Jingjing, Li Huijun is based on the active component high-throughput screening method of the affine selection of target protein, CN200810124611.1).Among the present invention, the preferred method of using the affine selection of target protein, be specially: Chinese medicine extract and target protein are hatched in damping fluid altogether, to not contain the Chinese medicine extract of target protein simultaneously as blank, detect with multidimensional liquid-matter coupling technique respectively, compare both collection of illustrative plates, the obviously peak of change takes place in peak area, promptly is target peak.
If the Chinese medicine extract in the above-mentioned active ingredient screening method comprises organic solvent, should concentrate and remove whole organic solvents, with the solubilizer of damping fluid dissolving or adding low concentration, in order to avoid destroy target protein.The phosphate buffer of the preferred pH6.0 of damping fluid～8.0, the TRIS buffer of pH6.0～8.0, pH6.0～8.0 ammonium acetate buffers or pH6.0～8.0 ammonium bicarbonate buffers.The selection of solubilizer is based on the activity of not destroying or do not destroy substantially target protein.In practical operation, the ratio of the solubilizer that target protein can bear can be checked by simple experiment.For example influenced hardly by Ellman spectrophotometric method proof acetylcholinesterase activity in the buffer salt system that contains 5% methyl alcohol.Described solubilizer is the mixed solution of one or more in tween, dimethyl sulfoxide (DMSO), polyglycol, Qu Latong, methyl alcohol, ethanol preferably.The ratio that preferred solubilizer volume accounts for the damping fluid volume is 0.1-5%.
All all can be used as the target that screens Chinese medicine from human body and animal with the closely-related activated protein of disease.Preferred target protein comes from enzyme or the acceptor relevant with senile dementia, angiocardiopathy or tumor disease of human body or animal tissue; preferred acetylcholinesterase, butyrylcholine esterase, angiotensin converting enzyme or histone deacetylase etc. among the present invention; there is a large amount of commercial activated proteins to buy at present; as acetylcholinesterase, butyrylcholine esterase etc., also can oneself carry out the purifying preparation.Can select suitable activated protein according to documents and materials and trial test when specifically screening Chinese medicine, show the significantly activity of acetylcholine esterase inhibition of maryllidaceous alkaloid position as document, in the screening maryllidaceous alkaloid, can select acetylcholinesterase as target protein during active component so.
Determining as mentioned above of major component peak, easy and simple to handle, do not need to use screening technique, only use each peak in the finger-print according to peak height value or the descending ordering of peak area value, choose the forward chromatographic peak of ordering as target peak, promptly be advisable with 5-20 in the major component peak.The operator can select suitable target peak to determine method according to the experiment needs.
After determining target peak, the outflow pipeline of high performance liquid chromatography detecting device is linked to each other with six direction changeover valves, concrete device as shown in Figure 1.Switch by valve, can collect the eluent separated into two parts: the target peak (group) that a part is determined for the b step, promptly target peak catches; Another part is the residual components after target peak (group) disappearance, and promptly target peak knocks out.The sample of collecting with Chinese medicine extract the same terms to be measured (concentration, chromatogram testing conditions etc.) under carry out efficient liquid phase chromatographic analysis whether detect knocking out of target peak complete.
In the above-mentioned steps, when going out the peak, the target chromatographic peak switches six-way valve immediately, six-way valve switches back to original position again when going out the peak end, obtain the target chromatographic peak thereby from eluent, catch, so the peek that the flow export of detecting device is little to the preferred caliber of the pipeline between the six-way valve, pipeline is short pipe is (as the red peek pipeline of Agilent, internal diameter 0.13mm, chromatographic peak only needs 0.02 second through this pipeline, almost can ignore time delay), thus the delay volume of minimizing chromatographic peak, the error when the reduction peak knocks out.
The sample that above-mentioned collection is obtained carries out the biologically active that chemical analysis was caught or knocked out to Biological Detection evaluation.Concrete biological assessment method can be selected suitable method of testing according to documents and materials and trial test.With the target protein is that cholinesterase is an example, can calculate by the Ellman spectrophotometric method of classics and catch the target peak (group) that obtains and become to hive off to the inhibiting rate of cholinesterase with target peak (group) disappearance back is remaining, with its with do not carry out any Chinese medicine eluent that knocks out and compare, can analyze and obtain contribution degree and the active ordering thereof of target peak in Chinese medical extract, thereby excavate the multicomponent effect characteristics of Chinese medicine.In addition, the single goal peak of catching is made up by different way, estimate the target peak group's who obtains activity, also can further inquire into the collaborative or antagonism between each composition.
Contain organic solvent because above-mentioned wash-out is collected the sample that obtains, preferably it is concentrated into do after, with the damping fluid dissolving or add the solubilizer of low concentration, in order to avoid when active testing, destroy target protein.Choosing of damping fluid and solubilizer is the same.
When target peak is caught, can obtain different single goal peaks respectively, the operator revalues its biologically active after can single target peak being combined into new sample according to the experiment needs, and then can make explanations to the collaborative or antagonism between the composition: due active sum when having surpassed their independent detections as if the activity that detects after two sample combination then may have synergy between the interpret sample; Activity when being lower than their independent detections as if the activity that detects after two sample combination then may have antagonism between the interpret sample.
Utilization the inventive method can be caught and knock out each target peak (group), and the activity that sample is collected in the test of utilization biological method utilizes mathematical statistics method to calculate the contribution degree and the active ordering of target peak (group).Carry out determination of activity after the single target peak that also collection can be obtained makes up in any way in addition, thereby can carry out further research collaborative, antagonism between each composition of Chinese medicine.This has widened Chinese medicine effector substance fundamental research direction greatly, be a kind of Chinese medicine mass action Mechanism Study method simple to operate, lower-cost, overcome classic method and can only filter out the deficiency that active ingredient of Chinese herbs but can not illustrate the mode of action between the active component.Use research method of the present invention, the inventor is definite four active component peaks of screening from the maryllidaceous alkaloid extract, after it is knocked out, make up catching the active component peak that obtains in a certain way again.The sample of collecting is carried out active testing, find that four active component peak disappearances can cause the active of residue significantly to reduce, and also have certain collaborative or antagonism between each active component peak.
Utilization this method, the inventor has chosen from stone China fir alkaloids extract and has determined 11 major component peaks, after it is knocked out and catches, the sample of collecting is carried out active testing.Test result shows that the big and active strong fingerprint peaks of active contribution might not be the high peak of peak area in the fingerprint collection of illustrative plates, and after the peak that activity is high knocks out, the activity of residue also can't significantly reduce, this shows that Chinese medicine is the integral body that multicomponent works, and also exists complex interactions between each composition.
More than two kinds of methods prove absolutely that the present invention is used for the fundamental research of Chinese medicine effector substance, disclose the feasibility of Chinese medicine multicomponent, many target spots, mass action characteristics.
Description of drawings
Fig. 1 is that target peak knocks out and acquisition equipment figure.A.C18 reverse-phase chromatography analytical column b. UV-detector c. six direction changeover valves.
Fig. 2 is the online screening plant figure of the affine selection active component of target protein.1.HLB solid-phase extraction column 2. coils 3. 6 direction changeover valve 4.C18 reverse-phase chromatography analytical columns 5. mass detector 6.MCX solid-phase extraction columns.
Be target protein-active component compound,
Be target protein,
It is active component.
Among Fig. 3, a is the finger-print of maryllidaceous alkaloid extract; B is that four active component peaks are by the uv-spectrogram after knocking out; C is the uv-spectrogram at four active component peaks of catching.
Fig. 4 is the finger-print of stone China fir alkaloids extract.
Fig. 5 is that the main major component peak of stone China fir alkaloid knocks out the checking collection of illustrative plates.A, No. 2 peaks are by the uv-spectrogram after knocking out; B, No. 3 peaks are by the uv-spectrogram after knocking out; C, No. 5 peaks are by the uv-spectrogram after knocking out.
Embodiment
The detection of maryllidaceous alkaloid active component and evaluation
The active component peak knocks out and catches:
Can (Acetylcholinesterase AChE) has interactional peak as target peak, i.e. active component peak, and it is knocked out and catch with acetylcholinesterase in the screening maryllidaceous alkaloid extract.
The preparation of maryllidaceous alkaloid: short-tube lycoris powder 5g, 70% methyl alcohol ultrasonic Extraction twice of 10 times of volumes, each 30min, the merging extract is concentrated into dried, adds 20mL2%HCl and regulates pH to 1, filters.Filtrate is transferred pH to 10 with 1MNaOH.The chloroform of 2 times of volumes is got the chloroform layer merging and is concentrated into dried its extraction 3 times.The 10mL dissolve with methanol, centrifugal back sample introduction, high-efficient liquid phase chromatogram technique analysis gets its finger-print.
The preparation of AChE: acetylcholinesterase freeze-dried powder 2000U (Acetylcholinesterase from electric eel, Sigma), with 2ml TRIS buffer (20mM, contain 100mMNaCl, pH7.5) fully dissolving, be mixed with 1000U/ml, it is standby to put in-20 ℃ of refrigerators packing.
The screening at active component peak: get 200 μ l maryllidaceous alkaloid extracts, Nitrogen evaporator is concentrated into it dried, uses 100 μ l TRIS buffer (containing 2% methyl alcohol as solubilizer) dissolving as liquid to be measured again.Get 10 μ l liquid to be measured and evenly mix, under 25 ℃ of environment, leave standstill 1h with 10 μ lAChE solution (1000U/ml).20 μ l are hatched solution and are splined on SPE1 (WatersOasis HLB), ammonium formate damping fluid (20mM, pH7.5) 3ml/min wash-out 30s, eluent (3ml/min) and dissociation solution (0.2% aqueous formic acid, 1ml/min) be mixed in coil (2ml) 30s that dissociates, the back sample that dissociates simultaneously is splined on SPE2 (Waters Oasis MCX), this moment, six direction changeover valves were in last sample (LOAD) position of accompanying drawing 2,4ml/min washes SPE21min with diethylamine aqueous solution (pH 10.0), subsequently six direction changeover valves are switched to sample introduction analysis (INJECT) position of accompanying drawing 2, be enriched in active component on the SPE2 and gone into the LC-MS system by wash-out and carry out compartment analysis.Simultaneously get 10 μ l liquid to be measured in addition and evenly mix, use the same method and analyze as blank with 10 μ l TRIS buffer (containing 2% methyl alcohol) as solubilizer.Both collection of illustrative plates relatively, peak area has the peak of obvious change, and promptly being has interactional peak with target cell, as target peak, i.e. active component peak.Utilization based on the active component high-throughput screening method of the affine selection of target protein (referring to Li Ping, Zhou Jianliang, An Jingjing, Li Huijun, active component high-throughput screening method based on the affine selection of target protein, CN200810124611.1) the maryllidaceous alkaloid extract is carried out screening active ingredients, determined four peaks can with the acetylcholinesterase combination, shown in a in the accompanying drawing 3.Through comparing with the galanthamine standard items, peak 4 is the active component galanthamine.
The knocking out and catching of active component peak: maryllidaceous alkaloid extract sample introduction 20 μ l, this moment, six direction changeover valves were in last sample (LOAD) position of accompanying drawing 1.Collect it sample introduction (INJECT) position that rapidly six direction changeover valves is switched to accompanying drawing 1 when target peak begins the peak.Go out to switch the supreme sample of six-way valve (LOAD) position when the peak finishes once more, target peak was collected and was finished this moment.And the gleanings of six-way valve when last sample (LOAD) position is the residual components group after target peak lacks.To knock out residual components group behind the target peak and maryllidaceous alkaloid extract in the same terms (concentration, chromatogram testing conditions etc.) sample introduction analysis down, checking knocks out effect, shown in b in the accompanying drawing 3.As can be seen from the figure, the present invention can knock out target peak.The target peak that captures is shown in c in the accompanying drawing 3.
Chromatogram and mass spectrophotometry condition: C18 chromatographic column, high performance liquid chromatography-electron spray level Four bar mass spectrum (HPLC-ESI/Quadrupole MS); Moving phase: acetonitrile: water (respectively containing 0.05% diethylamine) gradient elution; The ultraviolet detection wavelength is 280nm; The ESI ion gun; Positive ion mode, mass range: 100～1000m/z; Spray capillary voltage: 3500V; Dry gas (N
2) flow velocity: 9.0L/min; Dry gas temperature: 320 ℃; Atomizing pressure: 16psi; Fragment voltage: 100V.
Active testing: test system will be taken into account the dissolubility of the high activity and the micromolecular compound of target protein.The sample that obtains because of collection contains more organic solvent, so it is concentrated into dried back with the damping fluid dissolving, in order to avoid destroy target protein when active testing.Yet short-tube lycoris total alkali polarity is less, in buffer salt system, be difficult for dissolving, need to select suitable solvent to make the two coexistence, as add solubilizer of low concentration etc., the trishydroxymethylaminomethane buffer salt system that contains 2% methyl alcohol is used in this experiment, and acetylcholinesterase activity in system is influenced hardly.Also four active component peaks are made up (peak group 123, peak group 124, peak group 134, peak group 234) to investigate each peak-to-peak interaction in addition.The cholinesterase inhibition method of testing of sample is classical Ellman spectrophotometric method.Getting 4 μ l AChE (2U/ml), 80 μ lDTNB (1mM), 4 μ l samples and 10 μ l TRIS buffer mixes, incubated at room 10min, add 2 μ l ATCh initiation reactions subsequently, add 100 μ l methyl alcohol cessation reactions behind the reaction 10min, microplate reader is measured absorbance under the 412nm.Get simultaneously 4 μ l damping fluids in addition and replace sample to measure again, be the blank solution absorbance.
Testing result: three batch samples to parallel preparation are carried out active testing, and inhibiting rate mean value is as shown in the table:
| Sample | The total alkali extracting solution of short-tube lycoris | Peak group 1234 | Peak 1 | Peak 2 | Peak 3 | Peak 4 | |
| Inhibiting rate % | ??65.0 | ??63.9 | ??17.7 | ??35.4 | ??18.9 | ??63.5 | ??36.0 |
| Sample | Short-tube lycoris total alkali eluent | Peak group 1234 disappearances | Peak group 234 (lacking 1) | Peak group 134 (lacking 2) | Peak group 124 (lacking 3) | Peak group 123 (lacking 4) | |
| Inhibiting rate % | ??60.5 | ??28.8 | ??67.0 | ??63.2 | ??64.1 | ??30.1 | ??51.2 |
Consider extract through behind the chromatogram column analysis, composition has certain loss, and we also collect as a duplicate samples by the whole eluents after with the total alkali extracting solution sample introduction of short-tube lycoris, are short-tube lycoris total alkali eluent.We can draw by experimental data:
1, medicinal material is overall active quite active with active component peak group 1234, and knocks out residual components group behind the peak group 1234 and compare with the overall activity of medicinal material greatly and reduce, and illustrates that active component that the inventive method is drafted can represent the activity of medicinal material substantially.The residual components majority is the non-activity composition in the medicinal material simultaneously, or active relatively poor, is in a disadvantageous position when becoming swarming competition acetylcholinesterase binding site with the high activity that filters out, so do not sifted out.After removing active high composition, these active relatively poor compositions show more weak inhibiting effect to acetylcholinesterase.
2, four active component peak inhibiting effect orderings are 3＞peak, 2＞peak, 4＞peak, peak 1; Three at active peak is one group makes up back mensuration inhibiting rate, active ranking results is group 124＜peak, group 134＜peak, group 123＜peak, peak group 234, promptly lack 4＜scarce 2＜scarce 3＜scarce 1, corresponding contribution degree ordering illustrates that for 3＞peak, 2＞peak, 4＞peak, peak 1 contribution degree at each active peak is directly proportional with its activity.And after removing peak 4, the active of peak group 123 significantly descends, and illustrate that once more peak 4 is active best composition, and peak 4 galanthamines has been the listing anti senile dementia drug, thereby reconfirmed the accuracy and the reliability of this method.
3, peak group 123 activity is all high than peak 1, peak 3, but the activity when being lower than peak 2 individualisms illustrate the same binding site that peak 1,3 and peak 2 are competed in the target proteins.
4, peak group 124 has lacked a peak, but compare with full collection the in four peaks, its activity but raises slightly, may be exactly because eliminated of the competition effect of the low activity peak 3 of high-load to other peaks, because activity is low but compound that content is high can influence the combination of high activity composition to target protein, promptly influences it and suppress active.
Though 5 have removed activity peak 2 preferably, when collecting entirely than four peaks, peak group 134 activity only descends slightly, and may be because peak 4 active fine is in leading position in overall activity always, the back is active to descend and seldom so peak 2 is removed.Height when peak group 134 activity does not but have peak 4 individualisms simultaneously illustrates that there are the competition effect in peak 1,3 and peak 4, because peak 1,3 activity are lower, activity descends on the contrary when therefore finally causing making up.
Though 6 have lacked an active peak, peak group 234 activity increases when collecting entirely than four peaks on the contrary to some extent, and reason is with above-mentioned the 4th point.
Can find by above-mentioned data, some composition active though bad (as peak 1, peak 3), but content is higher, can occupy the binding site of some, therefore may influence combining of the stronger composition of affinity (as peak 2, peak 4) and albumen, make whole (four peaks are collected entirely) inhibition activity descend to some extent.This shows that Chinese medicine is the integral body that multicomponent works, each composition to the contribution of integral body be not simply add and, and also exist complex interactions between each composition.
The activity of stone China fir alkaloid major component detects and estimates
The major component peak knocks out and catches:
Determine the peak height value is bigger in the stone China fir alkaloids extract peak as target peak, i.e. major component peak, and it is knocked out and catch.
The alkaloidal preparation of Shi Shan: stone China fir powder 5g, immerse 10 times of volume pH values and be 3 hydrochloric acid, ultrasonic Extraction twice, each 30min.Merging filtrate, ammoniacal liquor adjust pH to 9 is with the chloroform extraction of 10 times of volumes 3 times.Getting the chloroform layer merging is concentrated into dried.The 10mL dissolve with methanol, centrifugal back sample introduction, high-efficient liquid phase chromatogram technique analysis gets its finger-print.
Determining of major component peak: the stone Shan Zhiwensepufeng that obtains according to the descending ordering of peak height value, is chosen the forward chromatographic peak of ordering as target peak, and promptly 11 major component peaks have finally been determined, as shown in Figure 4 in the major component peak.Through comparing with the huperzine standard items, No. 5 peaks are huperzine.
The knocking out and catching of major component peak: stone China fir alkaloids extract sample introduction 10 μ l, target peak is the major component peak, knocks out and catch step with embodiment 1.With knock out behind the target peak residual components group with stone China fir alkaloids extract the same terms (concentration, chromatogram testing conditions etc.) sample introduction analysis down, checking knocks out effect, shown in accompanying drawing 5 (A-C).As can be seen from the figure, the present invention can knock out target peak.
Chromatogram and mass spectrophotometry condition: C18 chromatographic column, high performance liquid chromatography-electron spray level Four bar mass spectrum (HPLC-ESI/Quadrupole MS); Moving phase: acetonitrile: water (containing the 50mM ammonium formate) gradient elution; The ultraviolet detection wavelength is 230nm; The ESI ion gun; Positive ion mode, mass range: 100～1000m/z; Spray capillary voltage: 3500V; Dry gas (N
2) flow velocity: 9.0L/min; Dry gas temperature: 320 ℃; Atomizing pressure: 16psi; Fragment voltage: 100V.
Active testing: investigated and caught the activity that 11 major component peaks obtaining and each major component peak are knocked out back residual components group.Sample preparation requirement and activity test method are with embodiment 1.
Testing result: five batch samples to parallel preparation are carried out active testing, and inhibiting rate mean value is as shown in the table:
| | Peak | 1 | |
|
|
|
Peak 6 | Peak 7 | |
|
|
|
Huperzine 0.05 μ M | |
| Inhibiting rate % | ??46.6 | ??49.8 | ??3.4 | ??36.1 | ??87.6 | ??13.3 | ??4.4 | ??10.3 | ??4.6 | ??63.0 | ??0.2 | ??48.4 | ||
| | Peak | 1 | Peak | 2 disappearances | Peak 3 disappearances | Peak 4 disappearances | Peak 5 disappearances | Peak 6 disappearances | Peak 7 disappearances | Peak 8 disappearances | Peak 9 disappearances | Peak 10 disappearances | Peak 11 disappearances | Stone China fir total alkali eluent |
| Inhibiting rate % | ??88.9 | ??88.7 | ??88.9 | ??89.4 | ??86.4 | ??89.3 | ??88.9 | ??89.5 | ??89.3 | ??89.6 | ??89.2 | ??89.2 |
We can draw by experimental data:
1, it is best to contain No. 5 peak activity of active component huperzine, and after it was knocked out, residual components group's activity descended to some extent, but does not but significantly reduce.Probably be because when No. 5 peaks exist, because the compatibility of itself and target spot is strong, though other become swarmings that the very difficult and targeted integration of certain choline esterase inhibition is also arranged, No. 5 the peak then plays active leading role.After No. 5 peaks disappearance, other peaks just can and targeted integration, thereby performance activity makes overall activity remarkable decline can not occur.
2, the activity at peak 10 is only second to peak 4, yet after it is knocked out, residual components group's activity raises on the contrary, even active taller than the total alkali extracting solution of stone China fir, this the explanation peak 10 probably with extract in a plurality of peaks antagonism is all arranged, after its removal, the activity at other peaks is brought into play.
3, the peak area height is that the composition that content is bigger in the total alkali extracting solution of stone China fir is not necessarily active good, as: peak 3 and peak 11; Peak area is low to be the less composition of content not necessarily a little less than the activity, as: peak 10.
Can find by above data, big and the active strong fingerprint peaks of active contribution might not be the high peak of peak area in the fingerprint collection of illustrative plates, otherwise, the peak that peak area is high might not be active all right, therefore only control traditional Chinese medicine quality by a certain or several major components in the simple analysis Chinese medicine and lose biasedly, ignored the characteristics of Chinese medicine multicomponent, many target spots, mass action.By the present invention, can investigate of the effect of Chinese medicine major component to Chinese medicine integral body.
Claims (9)
1. the detection method based on the traditional Chinese medicine ingredients effect characteristics of integral body sight comprises medicinal solvent extraction in to be measured, obtains Chinese medicine extract, carries out as follows then:
A. Chinese medicine extract detects through high performance liquid chromatography, obtains traditional Chinese medicine fingerprint;
B. determine the target peak in the finger-print;
C. after determining target peak, the outflow pipeline of high performance liquid chromatography detecting device is linked to each other with six direction changeover valves, switch by valve, collect following eluent: the target peak group of single target peak and/or combination in any, also collect knock out target peak or target peak group in the finger-print after remaining one-tenth hive off;
D. the sample that the c collection step is obtained carries out the pharmacodynamics evaluation, obtains target peak group's overall activity, the activity ordering at single target peak, the contribution degree of single target peak in Chinese medical extract, and the interaction relationship between each composition.
2. the detection method of claim 1, the method of wherein determining the target peak in the finger-print is as follows: with the finger-print peak according to peak height value or the descending ordering of peak area value, choosing the forward chromatographic peak of ordering is the major component peak, is advisable so that 5-20 is individual, as target peak.
3. the detection method of claim 1, the method of wherein determining the target peak in the finger-print is as follows: Chinese medicine extract and target protein are hatched in damping fluid altogether, hatch solution and be splined on online solid-phase extraction column, buffer solution elution, eluent mixed with dissociation solution dissociate, the solution that dissociates is splined on second online solid-phase extraction column, target protein and buffering salt flowage go out pillar, active component then is retained on the solid-phase extraction column, the active component wash-out that is retained is entered the analysis of liquid phase-mass spectrometer system, get do not contain target protein Chinese medicine extract as blank, detect with said method equally, compare both collection of illustrative plates, the peak that peak area takes place obviously to change is the active component peak, as target peak.
4. the detection method of claim 3, wherein damping fluid is selected from the phosphate buffer of pH6.0～8.0, TRIS buffer, pH6.0～8.0 ammonium acetate buffers or pH6.0～8.0 ammonium bicarbonate buffers of pH6.0～8.0; Target protein be from human body or animal with the closely-related activated protein of disease.
5. the detection method of claim 4, wherein being selected from of human body or animal: acetylcholinesterase, butyrylcholine esterase, angiotensin converting enzyme or histone deacetylase with the closely-related activated protein of disease.
6. the detection method of claim 1, wherein the flow export from detecting device is a chromatogram peek pipe to the pipeline the six-way valve in the valve switching device shifter.
7. the detection method of claim 1, the solvent that wherein is used to extract Chinese medicine is selected from water, C
1-C
5Alcohol, ethyl acetate, ether, chloroform, sherwood oil in one or more mixed solvent.
8. the detection method of claim 1, wherein the sample that obtains of c collection step concentrates it earlier before carrying out the d step, again with the damping fluid dissolving or add the solubilizer of low concentration.
9. the detection method of claim 8, wherein damping fluid is selected from the phosphate buffer of pH6.0～8.0, TRIS buffer, pH6.0～8.0 ammonium acetate buffers or pH6.0～8.0 ammonium bicarbonate buffers of pH6.0～8.0; Solubilizer is selected from one or more in tween, dimethyl sulfoxide (DMSO), polyglycol, Qu Latong, methyl alcohol, the ethanol.
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