CN100429517C - Setting-up method for cell membrance solid phase chromatography model for sieving Chinese medicine platelet aggregation resisting active component - Google Patents
Setting-up method for cell membrance solid phase chromatography model for sieving Chinese medicine platelet aggregation resisting active component Download PDFInfo
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Abstract
The process of setting up cell membrane solid phase chromatographic model for screening active platelet aggregation components from Chinese medicine includes the following steps: establishing the fingerprint of ginger oleoresin solution as the Chinese medicine to be screened, separating chicken platelet suspension, immobilizing the effective component of ginger oleoresin solution with the platelet cell membrane, eluting the uncombined components, dissociating the platelet immobilized the effective component of ginger oleoresin, and the HPLC analysis of the dissociated solution of platelet immobilized effective component. The present invention has rich material source, low cost and excellent application foreground.
Description
Technical field
The invention belongs to active ingredient of Chinese herbs screening and Study on mechanism field, the method for building up of the cell membrane solid phase chromatography model of particularly a kind of screening Chinese medicine antiplatelet aggregative activity (anti thrombotic action) active component.
Background technology
The research mode of traditional active ingredient of Chinese herbs, promptly to Chinese medicine extract, separation and purification single component and drug effect Validation Mode, exist workload huge, research cycle is long, and can not react the characteristics of Chinese medicine multicomponent, many target spots and integration comprehensively, make the research of herbal medicine efficacy material base make slow progress always.
Cell membrane solid phase chromatographic technique (Cell Membrane Immobilized Chromatography, CMIC), it is a kind of new bio affinity chromatography technology, overcome and from Chinese medicine, separated active component or monomer in the past earlier, analyze its drug effect again, thereby the drawback that the screening of component separating and effect is disconnected is the drug research method of a kind of chemical constitution-effect-mechanism of action interlock.
CMIC is that (Cell Membrane Chromatography grows up on basis CMC), and both all belong to novel biological affinity chromatography technology at cellular membrane chromatography.CMC is that the active mass's cell membrane with the human or animal is fixed on the specific support surface, be prepared into the cell membrane stationary phase, detect the retention characteristic of analyzed composition on the cell membrane stationary phase by on-line ultraviolet, gained chromatographic parameter and medicine and film and membrane receptor specificity combine closely related, the active ingredient screening that successfully is used for plurality of Chinese, as with the CMC model discrimination of different tissues preparation the active component in the Chinese medicines such as barrenwort, Radix Caulophylli, stellate cladonia fruticose thallus, Ligusticum wallichii, for the research of innovation material foundation of tcm is laid a good foundation.
But there is the researcher to think, active component with CMC research Chinese medicine, the cell membrane bag is by the difficult control of the uniformity coefficient of silica gel in the coating rate of silica gel and the post, particularly be difficult in the HPLC biological chromatography post, and can not get rid of the problem of non-specific binding fully with respect to requirements such as the needed temperature of cell membrane, pressure, ionic strength and pH value of solution.In order to solve above-mentioned the deficiencies in the prior art part, Fan Hongwei has set up Platelet Membrane Immobilized Chromatography (Platelet Membrane Immobilized Chromatography, PMIC), utilize the composition in the platelet cell film immobilization danshen injections, obtained protocatechualdehyde, a group compounds such as chlorogenic acid, this group compound can platelet aggregation-against, think that PMIC can reflect compound and cell membrane biological targets (acceptor substantially, passage, enzyme etc.) interaction, reservation composition in this system and its pharmacological action have significant correlativity, can have the screening of the active component of anti thrombotic action Chinese medicine.But utilize PMIC to set up the active constituents of medicine empirical model that screening has anti thrombotic action, whole experiment needs a large amount of blood platelets to experimentize, hematoblastic activity is particularly crucial to experimental result, the blood platelet of animals such as many at present employing pigs, dog experimentizes, the cost height is once gathered biologically active pdgf in a large number and is difficult to keep.And the PC in the chicken blood is higher than mammal, and function is similar to mammal and active stronger, and external easy the gathering is desirable high activity blood platelet source, do not appear in the newspapers at present but adopt the chicken blood platelet to set up the blood platelet solid phase chromatography model.
Summary of the invention
In order to solve the weak point that above-mentioned prior art exists, the object of the present invention is to provide a kind of method for building up that screens the cell membrane solid phase chromatography model of Chinese medicine antiplatelet aggregative activity (anti thrombotic action) active component cheaply.The present invention utilizes the chicken blood platelet as the cell model of setting up PMIC, and the source is abundant, cheap, and draws materials conveniently.
The present invention adopts the chicken blood platelet to set up the blood platelet solid phase chromatography model, to the rhizoma zingiberis supercritical CO
2Extraction position---oleoresin ginger carries out active ingredient screening and structure is identified, by the composition that filters out being carried out the pharmacology checking, determine the feasibility of this method, can be used for the active ingredient screening of other antiplatelet aggregative activities (anti thrombotic action) medicine.
Cell membrane solid phase chromatography model---chicken Platelet Membrane Immobilized Chromatography (the Chicken ThrombocytesMembrane Immobilized Chromatography of screening Chinese medicine antiplatelet aggregative activity (anti thrombotic action) active component that the present invention sets up, CTMIC), can carry out active ingredient screening to Chinese medicine with antiplatelet aggregative activity (anti thrombotic action).
Purpose of the present invention is achieved through the following technical solutions: a kind of method for building up that screens the cell membrane solid phase chromatography model of Chinese medicine antiplatelet aggregative activity active component comprises the steps:
(1) wait to screen the foundation of Chinese medicine oleoresin ginger test liquid finger-print: precision takes by weighing the 5mg oleoresin ginger, with the dissolving of 2% Tween-80, get the oleoresin ginger test liquid, filter through the water filter membrane, filtrate is analyzed for HPLC, determines the total fingerprint peaks in the oleoresin ginger test liquid.
(2) separation of chicken platelet suspension: with chicken whole blood collection (volume ratio of blood and anti-coagulants is 9: 1) in the plastic centrifuge tube of the anti-coagulants that contains sodium citrate, again the anticoagulation branch is filled in the plastic centrifuge tube, at 22 ℃ with 680 rev/mins of centrifugal 10min, draw upper strata platelet rich plasma (PRP), again with PRP with 2800 rev/mins of centrifugal 10min, make the blood platelet precipitation, after blood platelet is washed with pH 7.4PBS, furnishing 10
10Individual/the ml platelet suspension.
(3) platelet cell film immobilization waits to screen the effect components in the Chinese medicine oleoresin ginger test liquid: get the oleoresin ginger test liquid that platelet suspension 5ml adds preparation in the 1ml step (1), mixing is placed on the combination of vibrating in 37 ℃ of water-baths, makes the abundant and platelet membrane receptors bind of active component in the oleoresin ginger.
The wash-out of (4 non-specific binding composition) (promptly not binding constituents): with the platelet suspension after the immobilization effect components in the PBS washing step (3) of the pH7.4 of 22 ℃ of 2 times of amounts, make into even suspension, 2800 rev/mins of centrifugal 10min, abandon supernatant, take off a layer blood platelet washing, make non-specific binding composition wash-out.
(5) the dissociating of active component in the platelet immobilization oleoresin ginger: the PBS solution that in the platelet suspension that is combined with the drug effect composition after step (4) washing, adds 3ml pH 4, make into even suspension, after placing 37 ℃ of water-bath vibration 30min, 2800 rev/mins of centrifugal 10min, supernatant are the active component dissociation solution.
(6) the HPLC stratographic analysis of platelet immobilization active component dissociation solution: the active component dissociation solution is passed through Solid-Phase Extraction (SPE) post, keep liquid and cross organic filter membrane, the nitrogen volatilization is concentrated into 200 μ l, HPLC analyzes, by comparing the ratio of each absorption peak in active component dissociation solution and the oleoresin ginger finger-print, determine the characteristic peak with the blood platelet specific bond, obtain the characteristic peak of active component.
Described and the characteristic peak blood platelet specific bond are No. 2 peaks, No. 3 peaks, No. 4 peaks, No. 5 peaks, five characteristic peaks in No. 6 peaks, and described five characteristic peaks are the effect components groups with the platelet membrane targeted integration.
Determine to wait to screen each characteristic peak material chemical constitution in the traditional Chinese medicine fingerprint, described No. 2 peak materials are that 6-gingerol, No. 3 peak materials are that 8-gingerol, No. 4 peak materials are that 6-ginger brain, No. 6 peak materials are the 10-gingerol.
In order to realize the present invention better, the HPLC analysis condition is as follows in described step (1), the step (6): chromatographic column: Agilent Zorbax Eclipse XDB-C18 150 * 4.6mm, 5 μ m; Chromatographic condition: adopt gradient elution, moving phase: A is a water, and B is an acetonitrile; A+B=100%, 0～8min, 55%A becomes 50%A; 8～15min, 50%A becomes 45%A; 15～40min, 45%A becomes 10%A; 40～45min, the 90%B gradual change is to 45%B (the above-mentioned percent by volume that is); Flow velocity 1ml/min; Column temperature: 25 ℃; DAD detects wavelength 280nm.
Step (4) determines that according to the variation that keeps ingredient in the liquid the preferred washing times of lower floor's blood platelet is 3 times under the situation of getting rid of the interference of non-specific binding composition.
The present invention compared with prior art, its advantage and beneficial effect are:
1) the chicken Platelet Membrane Immobilized Chromatography of the present invention's foundation, advantage with cell membrane solid phase chromatographic technique, " non-specific binding " and the problems such as " residing biologically active state of cell membrane stationary phase and chromatographic analysis system can't coexist as in the same system " that exist in the conventional cell membrane chromatography technology have promptly been solved, and take the cell model of chicken blood platelet solid phase chromatographic technique as the antithrombotic reagent primary dcreening operation, the source is abundant, cost is low, can carry out active ingredient screening to Chinese medicine, have favorable application prospect with antiplatelet aggregative activity (anti thrombotic action).
2) the effector substance 8-gingerol that filters out from oleoresin ginger has the external platelet aggregation inhibitory activity (IC of report 8-gingerol
50=6 ± 1 μ M) more be better than aspirin (IC50=20 ± 11 μ M), prompting 8-gingerol is expected to be developed as the antithrombotic reagent of a new generation.
3) effector substance that filters out from oleoresin ginger by model of the present invention---gingerol class material is to the influence of heatstroke endotoxemia model mice; its pharmacological mechanism is by alleviating the damage of heatstroke to liver; improve heatstroke endotoxemia model mice peritoneal macrophage energetic supersession level; strengthen its phagocytic activity; increase activity of plasma SOD, reduce the MDA generation; heatstroke endotoxemia model mice is had protective effect, may be the effective constituent of ginger control heatstroke.
4) CMIC of the present invention's foundation not only has the advantage of cell membrane solid phase chromatographic technique, as the epicyte protein of directly taking the biological targets enrichment optionally in conjunction with the active component in (immobilization) Chinese medicine extract, behind wash-out non-specific binding composition, capable again isolation identification effector substance, biological part retinal diseases partly separated with chemical analysis carry out, can bring into play the strong point separately of specificity combination and stratographic analysis, and carrier and acceptor in the molecular biosciences chromatogram have been saved, the process that passage is connected with protein such as enzymes, reduction of expenditure and time, especially help keeping the activity of biological targets, and utilizing the chicken blood platelet to set up screening model, the PMIC cost of Jian Liing was low more in the past.
Description of drawings
Fig. 1 is the oleoresin ginger finger-print.
Fig. 2 separates the chicken blood platelet Wright's staining figure of acquisition for the present invention.
Fig. 3 is oleoresin ginger blood platelet immobilon-p dissociation solution and blank group film dissociation solution, the HPLC contrast collection of illustrative plates of last eluent.
Fig. 4 is the HPLC collection of illustrative plates of the last eluent of chicken platelet immobilization oleoresin ginger test liquid.
Fig. 5 is the HPLC contrast collection of illustrative plates of oleoresin ginger chicken blood platelet immobilon-p dissociation solution and oleoresin ginger test liquid.
Fig. 6 is a platelet membrane dissociation solution collection of illustrative plates (reappearance).
Fig. 7 is each group mouse platelets smear comparison diagram.
Fig. 8 is Turnover of Mouse Peritoneal Macrophages Wright's staining figure.
Fig. 9 is each group ultrastructure of hepatic cell variation diagram.
Figure 10 is a chicken cell film solid phase chromatography model experimental road line chart of the present invention.
Embodiment
The present invention is further described in detail below in conjunction with embodiment and accompanying drawing, but embodiments of the present invention are not limited thereto.
Embodiment one: the method for building up of the cell membrane solid phase chromatography model of screening active ingredient of Chinese herbs
As shown in figure 10, chicken cell film solid phase chromatography model experiment route of the present invention is as follows:
(1) oleoresin ginger test liquid and the immobilised research and analysis of chicken platelet cell film
(1) wait to screen the foundation of Chinese medicine oleoresin ginger test liquid finger-print: precision takes by weighing the 5mg oleoresin ginger, and with the dissolving of 2% Tween-80, the eddy mixer hydrotropy gets the oleoresin ginger test liquid, filters through 0.45 μ m water filter membrane, and filtrate is analyzed for HPLC.Analysis condition is as follows: chromatographic column: Agilent Zorbax EclipseXDB-C18150 * 4.6mm, 5 μ m.Chromatographic condition: adopt gradient elution, moving phase: A is a water, and B is an acetonitrile; A+B=100%, 0～8min, 55%A becomes 50%A; 8～15min, 50%A becomes 45%A; 15～40min, 45%A becomes 10%A; 40～45min, the 90%B gradual change is to 45%B (the above-mentioned percent by volume that is).Flow velocity 1ml/min; Column temperature: 25 ℃.DAD detects wavelength 280nm.According to the finger-print of the chromatographic condition of list of references preparation, precision and stability are all good, each peak-to-peak shape symmetry in the collection of illustrative plates, and degree of separation is good, determines 1～8 total fingerprint peaks in the oleoresin ginger test liquid, as shown in Figure 1.
(2) separation of chicken platelet suspension: with 20ml chicken whole blood collection (volume ratio of blood and anti-coagulants is 9: 1) in the 50ml of the anti-coagulants that contains sodium citrate plastic centrifuge tube, with plastic suction pipe the anticoagulation branch is filled in the 15ml plastic centrifuge tube again, every pipe 6.5ml, in 22 ℃ of horizontal freezing hydro-extractors with 680 rev/mins of centrifugal 10min, draw upper strata platelet rich plasma (PRP), again with PRP with 2800 rev/mins of centrifugal 10min, make the blood platelet precipitation, after blood platelet usefulness pH 7.4PBS washing 2 times, furnishing 5ml blood platelet 10
10Individual/the ml suspension, as shown in Figure 2.Fig. 2 (a) is the chicken whole blood figure of Wright's staining, and chicken red blood cell is the haemocyte that sharp-edged ellipse has nuclear as seen from the figure, nuclear hyperchromatism, kytoplasm understain (shown in black arrow).The white arrow indication is for assembling agglomerating chicken blood platelet.Fig. 2 (b) is Wright's staining chicken blood platelet figure, and Fig. 2 (c) is the chicken blood platelet enlarged drawing of white arrow indication among Fig. 2 (b), and visible chicken platelet nucleus is oval, and engrain, periphery have transparent foaming sample film.
(3) experiment grouping: test is divided into oleoresin ginger group and blank group.The oleoresin ginger group is for getting the oleoresin ginger test liquid that platelet suspension 5ml adds preparation in the 1ml step (1), and the final concentration that the blank group promptly adds with volume in platelet suspension is 0.3% Tween-80 solvent.By Fig. 3 oleoresin ginger chicken blood platelet immobilon-p dissociation solution (b) and blank group dissociation solution (a), the HPLC contrast collection of illustrative plates of last eluent (c) as seen, each group is thought of as the impurity peaks that in the solvent ultraviolet is had absorption all occurring an impurity peaks (shown in arrow among Fig. 3) about 1.2min.Because therefore the appearance of each main peak material in oleoresin ginger thought that solvent is noiseless substantially to this experimental result before its appearance time was equal.
(4) effect components in the platelet immobilization oleoresin ginger test liquid: get three parts of platelet suspension 5ml, the oleoresin ginger test liquid that adds preparation in the 1ml step (1) respectively, mixing is placed in 37 ℃ of water-baths and vibrates in conjunction with 30min, makes the abundant and platelet membrane receptors bind of active component in the oleoresin ginger.The blank group, then the final concentration that adds with volume in the platelet suspension is 0.3% Tween-80 solution, surplus operation is all identical.
(5) wash-out of binding constituents (being the non-specific binding composition) not: the PBS with the pH7.4 of 22 ℃ of 2 times of amounts wash every duplicate samples, in each washing process, earlier drip washing is clean gently with the oleoresin ginger test liquid of tube wall, then with plastic suction pipe piping and druming repeatedly lightly, make into even suspension, 2800 rev/mins of centrifugal 10min, abandon supernatant, take off a layer blood platelet repeated washing, according to the variation that keeps ingredient in the liquid, determine that the best washing times under the situation of getting rid of the interference of non-specific binding composition is 3 times.As Fig. 4 is the HPLC collection of illustrative plates of the last eluent of chicken platelet immobilization oleoresin ginger test liquid, as seen in the oleoresin ginger active component with after blood platelet combines, PBS through utilization and body fluid pH value suitable (pH=7.4) washs that absorption affinity in the test liquid is more weak No. 7 peaks and No. 8 peak composition wash-outs 3 times.
(6) the dissociating of active component in the platelet immobilization oleoresin ginger: in the blood platelet that is combined with the drug effect composition, add the PBS solution of 3ml pH=4, make effect target spot inactivation on the platelet membrane.Blow and beat repeatedly with plastic suction pipe and to make into even suspension, place 37 ℃ of water-baths vibration 30min after, 2800 rev/mins of centrifugal 10min, supernatant are the active component dissociation solution.
(7) the HPLC stratographic analysis of biologically active pdgf composition dissociation solution: with Solid-Phase Extraction (SPE) post of 3 pipe active component dissociation solution by activating through methyl alcohol, ultrapure water, after the SPE post washes with the 0.5mI ultrapure water, keep with 1ml methyl alcohol, keep liquid and cross the organic filter membrane of 0.45 μ m, the nitrogen volatilization is concentrated into 200 μ l, and sample introduction is analyzed for HPLC.Analysis condition is identical with the chromatographiccondition of finger-print (in the step 1).By comparing the ratio of each absorption peak in dissociation solution and the oleoresin ginger finger-print, determine characteristic peak with the blood platelet specific bond, obtain No. 2 peaks, No. 3 peaks, No. 4 peaks, No. 5 peaks, five characteristic peaks in No. 6 peaks in the finger-print, be effect components group with the platelet membrane targeted integration, wherein No. 3 peaks, No. 4 peak materials and platelet cell binding force of membrane are stronger, and No. 3 peak materials are the strongest compositions of antiplatelet effects in five characteristic peak materials.The HPLC of oleoresin ginger chicken blood platelet immobilon-p dissociation solution and oleoresin ginger test liquid contrasts collection of illustrative plates as shown in Figure 5, the composition at No. 3 peaks, No. 4 peaks content in test liquid is not high, and content obviously increases (shown in arrow among Fig. 5) in blood platelet immobilon-p dissociation solution, illustrating that the specific binding capacity of these two kinds of compositions on platelet membrane is stronger, is to the stronger material of chicken blood platelet effect.Relatively respectively keep the data of the relative retention time at peak, the proportion in total peak area again in the chromatogram of oleoresin ginger blood platelet immobilon-p dissociation solution, last eluent, as shown in table 1:
In the chromatogram of table 1 blood platelet immobilon-p dissociation solution, last eluent
Each keeps the relative retention time at peak, the proportion in total peak area
Substantially be consistent by each material peak area ratio in dissociation solution and the last eluent, wherein No. 3 peak materials increase in the last eluent of the shared percentage of blood platelet immobilon-p dissociation solution, and No. 3 peaks of shared percentage the highest (16.38%) in the dissociation solution total peak area, explanation is by making No. 3 peak materials that discharge from the platelet membrane target spot behind the platelet membrane target spot inactivation maximum, illustrating that the specific binding capacity of this composition on platelet membrane is the strongest, is to the strongest material of chicken blood platelet effect in these five characteristic peak materials.
(8) repeated experiment: institute is all identical with above operation in steps.Effect components (repeated experiment) in different time usefulness chicken platelet cell film solid phase chromatographic technique model discrimination oleoresin ginger, found that, do not changed by the immobilised effect components of platelet membrane acceptor, and the peak area ratio basically identical (as shown in Figure 6) of each composition, the composition that is the blood platelet specific bond is identical, No. 3 the peak material all shows very strong binding ability, illustrate with antiplatelet aggregative activity (anti thrombotic action) active component in this chicken Platelet Membrane Immobilized Chromatography screening oleoresin ginger test liquid, method is reliable and stable, good reproducibility.
(2) the determining of characteristic peak effector substance chemical constitution in the oleoresin ginger finger-print: owing to gingerol class boiling point substance height, be difficult to volatilization, and have character such as thermally labiles, should not adopt gas-matter method for combined use to carry out qualitative and measure its molecular weight, so the present invention adopts the liquid-matter coupling technique with effects such as sample is few with the sample amount, good separating effect, electro-spray ionizations to carry out each effector substance of qualitative evaluation and measure its molecular weight.In conjunction with oleoresin ginger finger-print information (according to relative retention value) and the LC-MS technology reported, identify that wherein four characteristic peak compositions are gingerol class material, be respectively 6-gingerol, 8-gingerol, 6-ginger brain and 10-gingerol, wherein No. 3 characteristic peak materials are the 8-gingerol.Each characteristic peak effector substance chemical constitution is as follows respectively:
(3) effector substance group's pharmacology activity research: according to the literature, 6-gingerol, 8-gingerol, 6-ginger brain and 10-gingerol, these four compounds all show external antiplatelet aggregative activity to a certain degree under the concentration of 10 μ M, the external anti-platelet activity the strongest (96%) of 8-gingerol wherein, its external platelet aggregation inhibitory activity (IC
50=6 ± 1 μ M) is better than aspirin (IC
50=20 ± 11 μ M), No. 4 peak 6-ginger brain activity is taken second place (91%), and this is consistent with the conclusion that the present invention draws.The present invention adopt the one-tenth of the specific bond that platelet aggregation-against experiment in the mouse body obtains by the CTMIC screening oleoresin ginger hive off-gingerol carries out activity checking (method with the results are shown in embodiment three), gingerol is by increasing circle tree type blood platelet ratio in the mouse body, reduce expansion type blood platelet ratio and reach the antiplatelet aggregative activity similar, so experiment in vivo and vitro results suggest gingerol is expected to be developed as the antithrombotic reagent of a new generation to aspirin with the platelet aggregation number.
Embodiment two: the separation and Extraction of gingerol
The present invention extracts gingerol as pharmacological evaluation by chemical method, verifies effect components---the platelet aggregation inhibitory activity of gingerol class material that obtains in the chicken platelet cell film solid phase chromatographic technique screening oleoresin ginger test liquid of setting up by the present invention.
Commercially available ginger is that yellow snow pine professor identifies by the Institute of Technology of Ji'nan University Food Science and Engineering.Fresh ginger is cleaned earth, be cut into ginger splices, dry, wear into 100 order ginger powder then, use the acetone extract ginger powder in aeration-drying place; Every batch of ginger powder extracts three times; Three extracts are collected in together.Reclaim solvent being lower than under 35 ℃ the temperature, the gingerol crude extract.Get 2 parts of gingerol crude extracts, with the n-hexane extraction gingerol of 1 part of volume, continuous extraction 3 times; After merging the normal hexane extract, reclaim normal hexane, get thick gingerol.With above-mentioned thick gingerol silica gel adsorption, be loaded on the silicagel column; With the continuous wash-out of normal hexane ether, and place crystallization being lower than under 0 ℃ the environment, the gingerol coarse crystallization.With gingerol coarse crystallization n-hexane dissolution, recrystallization repeatedly, must make with extra care, (data such as mass spectrum, nuclear magnetic resoance spectrum, ultraviolet spectrum, infrared spectrum are confirmed that it is gingerol in a high-purity bunch shape gingerol crystallization, be respectively the 6-gingerol, 8-gingerol and 10-gingerol) (the yellow snow pine. separating and evaluation of crystallization gingerol. food and fermentation industries 2005; 31 (10): 71～72.Huang XS.Crystalgingerol of isolation and identification.Food and Fermentation Industries 2005; 31 (10): 71～72.), dissolve for reagent with 0.5% sucrose ester.
Embodiment three: platelet aggregation-against experiment in the gingerol body
Mouse is divided into three groups at random, is respectively solvent control group, aspirin group and gingerol group.The gingerol group is irritated stomach with 75mg/kg dosage, and the aspirin group is irritated stomach with 30mg/kg dosage, and the solvent control group irritates stomach for equal-volume 0.5% sucrose ester solution.Continuous gastric infusion 5d, 1 time/d, eyeball is got blood 1ml (3.8% liquor sodii citratis was by anti-freezing in 1: 9) behind the last administration 1h, places 5min for 4 ℃ and induces gathering, draws anticoagulation 300 μ l and drops on the dry slide that scribbles collodion solution about 2 * 2cm
2The blood sheet is put in 37 ℃ of constant temperature air dry ovens behind the incubation 8min, washes away red blood cell, leucocyte with pH=7.4PBS, and room temperature is air-dry.With saturated liquor potassic permanganate dyeing 8min, take out the blood sheet, distilled water washes down, natural air drying.10 * 40 power microscopes are observed also down and are taken a picture, and data show, aspirin group, the hematoblastic ratio of gingerol group circle tree type be all greater than the solvent control group, and the hematoblastic ratio of expansion type, a hematoblastic gathering number average are less than solvent control group (P＜0.01); Relatively gingerol group and all types of blood platelets of aspirin group and gathering number, result's there are no significant difference (P＞0.05).The blood platelet smear as seen, smooth surface, peripheral complete blood platelet are round; The edge is whole, and the blood platelet of the dendron shape pseudopodium that stretch out different in size, plucked, comes in every shape is the tree type; Round and tree type is collectively referred to as round tree type (Fig. 7 .1), is blood platelet form under the normal condition.The blood platelet of tree type is in contact with one another, or contacts with the blood platelet of its alloytype, very easily assembles.Thisly being merged mutually by two or more blood platelet edges, by the blood platelet form that the mutual commissure of dendron shape pseudopodium forms, be called expansion type (Fig. 7 .2), is that hematoblastic cell membrane shape or structure change and the adhesion accumulation shape that forms.Visible blood platelet is intensive under the blood platelet picture mirror of solvent control group, and form is irregular, assembles obviously, a large amount of expansion type blood platelets occur, and circle tree type less relatively (Fig. 7 .3); Compare with the solvent control group, the obvious density of blood platelet reduces in aspirin group (Fig. 7 .4) and gingerol group (Fig. 7 .5) the blood platelet smear, blood platelet form rule, and circle tree type blood platelet occupies the majority, and clustering phenomena is not obvious.Calculate under the visual field, each organizes circle tree type, the shared number percent of expansion type blood platelet in the blood platelet smear, and the hematoblastic gathering number of expansion type, the results are shown in Table shown in 2.
Table 2 is respectively organized in the mouse platelets smear circle tree type, the shared percentage of expansion type blood platelet and is assembled the comparison of number (X ± s)
Annotate: compare with the solvent control group,
*P＜0.05,
*P＜0.01.The result shows the hematoblastic ratio of aspirin group, gingerol group circle tree type all greater than the solvent control group, and the hematoblastic ratio of expansion type, a hematoblastic gathering number average are less than solvent control group (P＜0.01); Relatively gingerol group and all types of blood platelets of aspirin group and gathering number, result's there are no significant difference (P＞0.05).
Embodiment four: the foundation of heatstroke endotoxemia model
40 mouse are divided into physiological saline group, heatstroke endotoxemia model group (model group), normal temperature control group, gingerol group and solvent control group, 8 every group at random.The not administration of normal temperature control group exposes 2h under room temperature (25 ± 0.5) ℃, relative humidity (43 ± 5) % condition; The gingerol group is irritated stomach with gingerol, presses the administration of 75mg/kg body weight, and the solvent control group irritates stomach for isopyknic 0.5% sucrose ester, and the physiological saline treated animal is irritated stomach equal-volume physiological saline, not administration of model group.Behind the administration 30min 4 treated animals are put into simultaneously artificial hot weather boiler-plate, under the condition of dry-bulb temperature (35 ± 0.5) ℃, relative humidity (65 ± 5) %, carry out heat and expose 2h, induce it to produce the endogenous endotoxemia.
Embodiment five: the collection of peritoneal macrophage and evaluation
Animal takes off marrow and puts to death, 75% alcohol-pickled 5 seconds, be fixed in surgical console, the ice-cold 10U/ml heparin RPMI-1640 nutrient solution 5ml that contains of lumbar injection collects peritoneal lavage fluid, the centrifugal 10min of eluate 1000r/min behind the soft 1min, remove supernatant, precipitation is resuspended twice with serum-free RPMI-1640 nutrient solution, the trypan blue dyeing counting, and adjusting cell number is 1 * 10
6Individual/ml, expect the blue cytoactive of checking for 2%, Switzerland's dyeing identification of cell purity＞95%.As shown in Figure 8, Wright's staining is peritoneal macrophage under this visual field.
Embodiment six: MTT (tetramethyl azo azoles salt) method is measured macrophage energetic supersession level
The cell suspension of drawing 100 μ l of preparation adds to 96 porocyte culture plates, puts 37 ℃ and contains 95%O
2, 5%CO
2Incubator continues to cultivate 2h, add MTT (final concentration 5g/L), 4h is hatched, centrifugal 5 minutes of 1000r/min in 37 ℃ again in 20 μ l/ holes, discard liquid in the hole, add DMSO (150 μ l/ hole), fully mixing is put the 30min that vibrates on the microoscillator, on Bio-Rad680 type microplate reader, read the light absorption value at wavelength 570nm place, with A
1The mensuration of value representation macrophage energetic supersession level the results are shown in Table shown in 3.
Embodiment seven: the dimethyl diaminophenazine chloride method is measured macrophage phagocytic function
The cell suspension of drawing preparation 100 μ l adds to 96 porocyte culture plates, puts 37 ℃ and contains 95%O
2, 5%CO
2After incubator continues to cultivate 2h, remove nutrient solution, add 0.072% neutral red solution (100 μ l/ hole), 37 ℃ of incubators continue to cultivate 1h, abandon supernatant, 37 ℃ of physiological saline of pre-temperature fully wash 2 times, add 200 μ l lysates (absolute ethyl alcohol: 0.1mmol/L acetate volume ratio is 1: 1), the room temperature concussion fully dissolved it in 30 minutes, fragmentation, putting Bio-Rad680 type microplate reader detection wavelength is the absorbance at 570nm place, with A
2The power of value representation macrophage phagocytic function the results are shown in Table shown in 3.
Table 3 gingerol is to the influence of each group Turnover of Mouse Peritoneal Macrophages energetic supersession level and phagocytic function (X ± s)
Annotate: compare with model group,
*P＜0.01.Normal temperature control group mice peritoneal macrophage energetic supersession level and phagocytic function are higher than model group (P＜0.01) respectively, point out this modelling success; Gingerol group Turnover of Mouse Peritoneal Macrophages energetic supersession level and phagocytic function are significantly higher than model group (P＜0.01).
Embodiment eight: blood plasma superoxide dismutase (SOD) determination of activity
The employing xanthine oxidase is measured, and gets mice plasma 30 μ l, and the application of sample amount is 20 μ l, is undertaken by kit instructions method of testing, measures the 555nm absorbance, records the control tube absorbance, presses
Calculate the SOD activity.To reach 50% o'clock pairing SOD amount be a SOD unit of activity (U) to the SOD inhibiting rate in every milliliter of reactant liquor, the results are shown in Table shown in 4.
Embodiment nine: MDA (MDA) assay
Adopt thiobarbituricacid method (TBA) method to measure, undertaken, get mice plasma 100 μ l by kit instructions method of testing, application of sample amount 100 μ l, standard MDA concentration is 10nmol/L, measures the absorbance of the long 532nm of each tube wave, bioassay standard pipe average A value, by formula calculate:
MDA content (unit is mmol/ml) the results are shown in Table shown in 4.
Table 4 gingerol is to the influence of each group mice plasma SOD activity, MDA content (X ± s)
Annotate: compare with model group,
*P＜0.01.Normal temperature control group mice activity of plasma SOD is higher than, MDA content then is lower than model group (P＜0.01), points out this modelling success; Gingerol group mice plasma SOD activity is higher than, MDA content then is lower than model group (P＜0.01).
Embodiment ten: the preparation of electron microscope specimen and observation
Animal is taken out liver rapidly after collecting peritoneal macrophage, and put 2.5% glutaraldehyde solution and fix, fixing 1h behind 1% osmic acid, 70%～100% gradient alcohol, acetone dewater in proper order; Epoxy resin Epon812 embedding becomes piece, makes 50～70nm ultra-thin section, through uranium acetate, the two dyeing of lead citrate, puts under the PHILIPS TECNAI-10 type transmission electron microscope and observes, takes a picture.In Fig. 9, the mitochondrial swelling of figure (1) physiological saline group (* 8900), mitochondrial cristae reduces, and endoplasmic reticulum swelling is the cavity shape, and glycogenosome reduces; Figure (2) heatstroke endotoxemia model group (* 6200) arrow indication part of hepatocytes nucleus distortion, irregular; Lysosome increases, and glycogenosome reduces, and visible a large amount of fat drip, and the picture left above is the mitochondrial enlarged drawing of this model group (* 15000), visible mitochondrial swelling, and mitochondrial cristae reduces; Figure (3) normal temperature control group (* 8900) liver cell is clear, and nucleus is circle or oval, and nuclear membrane is clear, does not have deformity and changes, and mitochondria does not have swelling, and mitochondrial cristae is abundant, and glycogenosome is abundant; Scheme (4) gingerol group (* 6000) liver cell form near normal group, nucleus distortion, irregular, mitochondria does not have swelling, and ridge is normal, and a small amount of cavity is arranged in the endochylema, does not see that fat drips; The mitochondrial swelling of figure (5) solvent sucrose ester group (* 15000), mitochondrial cristae reduces, and endoplasmic reticulum swelling is the cavity shape, and lysosome increases, and glycogenosome reduces.
The effector substance that the model of setting up by the present invention filters out from oleoresin ginger---gingerol class material is to the influence of heatstroke endotoxemia model mice; its pharmacological mechanism is by alleviating the damage of heatstroke to liver; administration's endotoxemia model mice peritoneal macrophage energetic supersession level in the raising; strengthen its phagocytic activity; increase activity of plasma SOD, reduce the MDA generation; heatstroke endotoxemia model mice is had protective effect, is the effective constituent of ginger control heatstroke.
As mentioned above, can realize the present invention preferably.
Claims (3)
1, a kind of method for building up that screens the cell membrane solid phase chromatography model of Chinese medicine antiplatelet aggregative activity active component is characterized in that comprising the steps:
(1) wait to screen the foundation of Chinese medicine oleoresin ginger test liquid finger-print: precision takes by weighing the 5mg oleoresin ginger, with the dissolving of 2% Tween-80, get the oleoresin ginger test liquid, filter through the water filter membrane, filtrate is analyzed for HPLC, determines the total fingerprint peaks in the oleoresin ginger test liquid;
(2) separation of chicken platelet suspension: with the chicken whole blood collection in the plastic centrifuge tube of the anti-coagulants that contains sodium citrate, again the anticoagulation branch is filled in the plastic centrifuge tube, at 22 ℃ with 680 rev/mins of centrifugal 10min, draw the upper strata platelet rich plasma, again with platelet rich plasma with 2800 rev/mins of centrifugal 10min, make the blood platelet precipitation, after blood platelet is washed with pH 7.4PBS, furnishing 10
10Individual/the ml platelet suspension;
(3) platelet cell film immobilization waits to screen the effect components in the Chinese medicine oleoresin ginger test liquid: get the oleoresin ginger test liquid that platelet suspension 5ml adds preparation in the 1ml step (1), mixing is placed on the combination of vibrating in 37 ℃ of water-baths, makes the abundant and platelet membrane receptors bind of active component in the oleoresin ginger;
(4) wash-out of non-specific binding composition: with the platelet suspension after the immobilization effect components in the PBS washing step (3) of pH7.4 of 22 ℃ of 2 times of amounts, make into even suspension, 2800 rev/mins of centrifugal 10min abandon supernatant, take off a layer blood platelet washing, make non-specific binding composition wash-out;
(5) the dissociating of active component in the platelet immobilization oleoresin ginger: the PBS solution that in the platelet suspension that is combined with the drug effect composition after step (4) washing, adds 3ml pH 4, make into even suspension, after placing 37 ℃ of water-bath vibration 30min, 2800 rev/mins of centrifugal 10min, supernatant are the active component dissociation solution;
(6) the HPLC stratographic analysis of platelet immobilization active component dissociation solution: the active component dissociation solution is passed through solid-phase extraction column, keep liquid and cross organic filter membrane, the nitrogen volatilization is concentrated into 200 μ l, HPLC analyzes, by comparing the ratio of each absorption peak in active component dissociation solution and the oleoresin ginger finger-print, determine the characteristic peak with the blood platelet specific bond, obtain the characteristic peak of active component;
The HPLC analysis condition is as follows in described step (1) and the step (6): chromatographic column: Agilent ZorbaxEclipse XDB-C18150 * 4.6mm, 5 μ m; Chromatographic condition: adopt gradient elution, moving phase: A is a water, and B is an acetonitrile; A+B=100%, 0～8min, 55%A becomes 50%A; 8～15min, 50%A becomes 45%A; 15～40min, 45%A becomes 10%A; 40～45min, the 90%B gradual change is to 45%B; Flow velocity 1ml/min; Column temperature: 25 ℃; DAD detects wavelength 280nm.
2, the method for building up of the cell membrane solid phase chromatography model of screening Chinese medicine antiplatelet aggregative activity active component according to claim 1, it is characterized in that: described and the characteristic peak blood platelet specific bond are No. 2 peaks, No. 3 peaks, No. 4 peaks, No. 5 peaks, five characteristic peaks in No. 6 peaks, and described five characteristic peaks are the effect components groups with the platelet membrane targeted integration; Described No. 2 peak materials are that 6-gingerol, No. 3 peak materials are that 8-gingerol, No. 4 peak materials are that 6-ginger brain, No. 6 peak materials are the 10-gingerol.
3, the method for building up of the cell membrane solid phase chromatography model of screening Chinese medicine antiplatelet aggregative activity active component according to claim 1 is characterized in that: in the described step (4), lower floor's blood platelet washing times is 3 times.
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| CN1247510C (en) * | 2004-09-08 | 2006-03-29 | 北京矿冶研究总院 | Method for separating 6-gingerol from ginger |
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