CN101285799A - Cellular membrane chromatography for sifting analgesic traditional Chinese medicine active ingredient - Google Patents
Cellular membrane chromatography for sifting analgesic traditional Chinese medicine active ingredient Download PDFInfo
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Abstract
The invention provides a cell membrane chromatography capable of synchronously sieving active ingredients of antalgic traditional Chinese medicine and determining an opium receptor subtype combined with the active ingredients. The method comprises the following steps of: the establishment of a fingerprint spectrum of extract liquid of angelica dahurica; the primary cultivation and the serial subcultivation of smooth muscle cells in a vas deferens; the combination and the dissociation of effective ingredients; and high-efficient liquid chromatographic determination combining the change of the spectrum. By acting traditional Chinese medicine on different types of smooth muscle cells in the vas deferens, the behavior of dissociation liquid of the traditional Chinese medicine on a high-efficient liquid chromatography column is examined, and the affinity of active ingredients of the traditional Chinese medicine and certain opium receptor subtype can be examined by using opium receptor excitant as a reference to opium receptor antagonist. The method takes an opium receptor on the vas deferens as a sieving model of key links of analgesics effect, can make an active ingredient sieving to the traditional Chinese medicine with abirritation, can also directly determine whether the active ingredients have the affinity with certain opium receptor subtype, and have good application prospect.
Description
Technical field
The invention belongs to active ingredient of Chinese herbs screening and study on mechanism field, particularly a kind of method for building up that screens the Chinese medicine analgesic bioactive substance and the cell membrane chromatogram model of the opiate receptor hypotype of determining its combination.
Background technology
Traditional active ingredient of Chinese herbs filtering mode as whole animal, system, organ model, exists length research cycle, high, the inefficient shortcoming of cost; Enzyme, receptor model can't reflect the characteristics of Chinese medicine polycomponent, many target spots.In numerous models, cellular membrane chromatography is shown one's talent, and becomes the new method of efficient screening active ingredient of Chinese herbs.
Cellular membrane chromatography (Cell Membrane Chromatography, CMC) be that 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 cell membrane and membrane receptor specificity combine closely related, the active ingredient screening that successfully is used for plurality of Chinese, 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.But this method also exists requirements such as can not satisfying the needed temperature of cell membrane, pressure, ionic strength and pH value of solution simultaneously, and can not get rid of the problem of non-specific binding fully.
Modern study shows that opioid drug produces analgesic activity by acting on opiate receptor, there are 8 kinds of hypotypes at least in opiate receptor in the body, except that the opiate receptor that maincenter distributes, also contain abundant opiate receptor on the vas deferens, mainly contain the δ acceptor as mouse vas deferens, part μ and kappa receptor are also arranged, the rabbit vas deferens contains single kappa opioid receptor, golden hamster contains delta opiate receptor, and rat spermatic duct is considered to contain epsilon receptor.By making drug effect in different genera vas deferens smooth muscle cell, examine or check the behavior of its dissociation solution on performance liquid chromatographic column, and make reference with opioid receptor agonist and antagonist, can examine or check the affinity of this medicine and certain opiate receptor hypotype.At present, existing people successfully cultivates the rat spermatic duct smooth muscle cell (Deferential Smooth Muscle Cell, DSMC), but the in vitro culture of mouse DSMC and rabbit DSMC is not appeared in the newspapers.
Summary of the invention
In order to solve above-mentioned the deficiencies in the prior art part, the object of the present invention is to provide a kind of cellular membrane chromatography that can screen the analgesia active ingredient of Chinese herbs simultaneously and determine the opiate receptor hypotype of its combination.The present invention separates biological part retinal diseases and chemical part and carries out, and helps keeping the activity of target spot, can bring into play specificity combination and stratographic analysis strong point separately.The vas deferens cellular membrane chromatography use in conjunction that the present invention set up cell extraction, high performance liquid chromatography, for the screening of the middle pharmaceutically active substance of polycomponent, many target spots very high applicability is arranged.
Simultaneously, the present invention is a research object with mouse, the rabbit vas deferens cell membrane that is rich in different opiate receptor δ and κ hypotype, affinity with the vas deferens cellular membrane chromatography specific research Chinese medicine root of Dahurain angelica and opiate receptor, the performance that utilizes drug effect is by medicine and the principle that cell target spot (acceptor) specificity combines, and has confirmed that the vas deferens cellular membrane chromatography can reflect the specificity and the selectivity of the root of Dahurain angelica and opiate receptor combination really.It is selective to opiate receptor to have illustrated the root of Dahurain angelica analgesic bioactive substance that filters out, and only specific in conjunction with opiate receptor κ hypotype, and irrelevant with the δ hypotype.Utilize this model investigation wild aconite root to obtain identical result, so this model for providing new method, the high selectivity rapid screening that other ease pain Chinese medicines have vast potential for future development at material foundation of tcm and study on mechanism field.
Purpose of the present invention is achieved through the following technical solutions: a kind of cellular membrane chromatography that can screen the analgesia active ingredient of Chinese herbs simultaneously and determine the opiate receptor hypotype of its combination comprises the steps:
(1) foundation of root of Dahurain angelica alcohol extract finger-print: take by weighing the 20g angelica root, add 160ml 95% ethanol, refluxing extraction 1～2h, volatilize ethanol, residue is settled to 20ml with dissolve with methanol, therefrom get 250 μ l and be diluted to 7.5ml, cross 0.45 μ m miillpore filter, obtain filtrate and be root of Dahurain angelica alcohol extract with methyl alcohol; Draw filtrate 20 μ l, adopt high performance liquid chromatography (HPLC) method to measure, obtain root of Dahurain angelica alcohol extract effective component group fingerprint atlas, determine that its characteristic peak (specifically sees the applicant's patented claim formerly, application number is 200610036947.3), totally 13 total fingerprint peakses.
Described efficient liquid phase chromatographic analysis condition: with the methanol-water is flow phase system, 30 ℃ of column temperatures, sample size 20 μ l, wavelength 254nm, length is that 250mm, diameter are the Lichrospher5-C18 chromatographic column of 4.6mm, eluent gradient: 0～5min, 5%～20% methyl alcohol; 5～15min, 20% methyl alcohol; 15～20min, 20%～70% methyl alcohol; 20～30min, 70% methyl alcohol; 30～40min, 70%～90% methyl alcohol; The above-mentioned percent by volume that is;
Each relative retention time of 13 total fingerprint peakses (characteristic peak) of described root of Dahurain angelica alcohol extract finger-print is respectively (is reference with No. 11 peaks):
No. 1 peak: 0.031～0.034; No. 2 peaks: 0.086～0.088; No. 3 peaks: 0.607～0.649;
No. 4 peaks: 0.658～0.665; No. 5 peaks: 0.704～0.708; No. 6 peaks: 0.730～0.739;
No. 7 peaks: 0.749～0.755; No. 8 peaks: 0.810～0.816; No. 9 peaks: 0.842～0.847;
No. 10 peaks: 0.856～0.861; No. 11 peaks: 1; No. 12 peaks: 1.085～1.087;
No. 13 peaks: 1.181～1.186.
Each total fingerprint peaks relative peak area scope is respectively (is reference with No. 11 peaks):
No. 1 peak: 0.005～0.061; No. 2 peaks: 0.016～0.132; No. 3 peaks: 0.050～0.366;
No. 4 peaks: 0.010～0.135; No. 5 peaks: 0.03～0.148; No. 6 peaks: 0.006～0.153;
No. 7 peaks: 0.086～0.328; No. 8 peaks: 0.047～0.105; No. 9 peaks: 0.018～0.059;
No. 10 peaks: 0.030～0.113; No. 11 peaks: 1; No. 12 peaks: 0.251～0.492;
No. 13 peaks: 0.304～0.845.
(2) former being commissioned to train of vas deferens smooth muscle cell supported and the cultivation of going down to posterity: get the vas deferens of bull mouse or adult male rabbit, peel off interior adventitial tissue, take enzyme digestion cultured cell in culture flask; When cell covers with covering bottle floorage 80% left and right sides, go down to posterity.
(3) combination of effect components with dissociate: the root of Dahurain angelica alcohol extract equal-volume that the cell suspension that will obtain through the cell dissociation that 3～10 generations went down to posterity and step (1) obtain mixes, behind 36～38 ℃ of incubation 0.5～24h, the centrifugal supernatant that goes, with DMEM nutrient solution washed cell 3～5 times, obtain even suspension, centrifugal, abandon supernatant, get sedimentation cell; The PBS (phosphate buffer) that adds pH2～6 in sedimentation cell makes cell degeneration (also can adopt other similar approach to make effect target spot inactivation on the film), and is centrifugal, and the collection supernatant also volatilizes, and thing must dissociate.
(4) high-performance liquid chromatogram determination changes in conjunction with the front and back collection of illustrative plates: with step (3) the gained thing dissolve with methanol that dissociates, use efficient liquid phase chromatographic analysis, described efficient liquid phase chromatographic analysis condition is identical with the efficient liquid phase chromatographic analysis condition of step (1); By the variation of each absorption peak in thing finger-print and the root of Dahurain angelica alcohol extract finger-print of dissociating of active component relatively, and make reference with opiate receptor antagonist and opioid receptor agonist respectively, determine whether the root of Dahurain angelica combines with opiate receptor.With the characteristic peak of opiate receptor specific bond be No. 7 peaks, No. 8 peaks and No. 10 peaks; The root of Dahurain angelica analgesic bioactive substance that filters out is No. 7 peaks, No. 8 peaks and No. 10 peaks; Described opiate receptor antagonist is a naloxone hydrochloride, and described opioid receptor agonist is a pethidine hydrochloride.
With No. 7 peaks of characteristic peak, three characteristic peaks in No. 8 peaks and No. 10 peaks of opiate receptor specific bond are the active component groups that combine with opiate receptor.Described No. 7 peaks, No. 8 peaks, No. 10 peak representative material action sites are identical with Allylnoroxymorphone, can be by its antagonism; No. 7 peak representative material action site is different with pethidine hydrochloride; No. 8 peaks, No. 10 peak representative material action sites are identical with pethidine hydrochloride.The described root of Dahurain angelica analgesic bioactive substance that filters out (i.e. No. 7 peaks, No. 8 peaks, No. 10 peaks) is selective to opiate receptor, and only specific in conjunction with opiate receptor κ hypotype, and irrelevant with the δ hypotype.
The present invention compared with prior art has following advantage and beneficial effect:
(1) the present invention successfully cultivates mouse DSMC and rabbit DSMC first, and institute's cultured cells identifies that through α-SMA SABC endochylema is pale brown look, and nucleus is not colored, and the purity of smooth muscle cell is more than 99%.
(2) the present invention cultivates mouse DSMC and the used method of rabbit DSMC is an enzyme digestion, has the advantage of saving time, avoid heteroproteose cell to pollute.
(3) the vas deferens cellular membrane chromatography of the present invention's foundation, with the screening model of the opiate receptor on the vas deferens as the analgesic action key link, except that carrying out Chinese medicine the active ingredient screening with analgesic activity, because δ and kappa opioid receptor that the vas deferens of animals such as mouse, rabbit contains have nothing in common with each other, therefore select for use these two kinds of animal vas deferens to prepare vas deferens cell membrane chromatogram model, can determine directly that also this medicine and certain opiate receptor hypotype have affinity (mainly being opiate receptor κ, δ hypotype), have a good application prospect.
(4) the present invention sets up the research method of the screening antalgesic active component of high selectivity, good reproducibility as a result.
Description of drawings
Fig. 1 is a root of Dahurain angelica alcohol extract finger-print.
Fig. 2 is a rabbit vas deferens smooth muscle cell (200 *).
Fig. 3 is that rabbit vas deferens smooth muscle cell is identified figure (400 *).
Fig. 4 is mouse vas deferens smooth muscle cell (200 *).
Fig. 5 identifies figure (400 *) for the mouse vas deferens smooth muscle cell.
(A is a root of Dahurain angelica alcohol extract collection of illustrative plates to Fig. 6 for root of Dahurain angelica alcohol extract and rabbit DSMC effect collection of illustrative plates, B be root of Dahurain angelica alcohol extract with by the back collection of illustrative plates of the saturated rabbit DSMC effect of activator, C be root of Dahurain angelica alcohol extract with by the back collection of illustrative plates of the saturated rabbit DSMC effect of antagonist, D is root of Dahurain angelica alcohol extract and rabbit DSMC effect back collection of illustrative plates, and E is last eluent).
(A is a root of Dahurain angelica alcohol extract collection of illustrative plates to Fig. 7 for root of Dahurain angelica alcohol extract and mouse DSMC effect collection of illustrative plates, B be root of Dahurain angelica alcohol extract with by the back collection of illustrative plates of the saturated mouse DSMC effect of activator, C be root of Dahurain angelica alcohol extract with by the saturated mouse DSMC effect back collection of illustrative plates of antagonist, D is the back collection of illustrative plates of root of Dahurain angelica alcohol extract and mouse DSMC effect).
Embodiment
The present invention is described in further detail below in conjunction with embodiment and accompanying drawing, but embodiments of the present invention are not limited thereto.
Embodiment 1: the method for building up (rabbit vas deferens smooth muscle cell) of the vas deferens cellular membrane chromatography model of screening analgesia active ingredient of Chinese herbs
(1) foundation of angelica root finger-print
Precision takes by weighing the dry root of 20g Radix angelicae dahuricae Angelica dahurica (Fisch.ex Hoffm.) Benth.etHook.f.var.formosana (Boiss.) Shan et Yuan, add 160ml 95% ethanol, refluxing extraction 2h, volatilize ethanol, residue is settled to 20ml with dissolve with methanol, therefrom gets 250 μ l and is diluted to 7.5ml with methyl alcohol, crosses 0.45 μ m miillpore filter, the filtrate that obtains is root of Dahurain angelica alcohol extract, analyzes for HPLC as need testing solution.Draw filtrate, use high effective liquid chromatography for measuring, chromatographiccondition: Lichrospher-C18 (4.6 * 250mm), be moving phase with the methanol-water, detect wavelength 254nm, sample size 20 μ l, 30 ℃ of column temperatures.Eluent gradient: 0～5min, 5%～20% methyl alcohol; 5～15min, 20% methyl alcohol; 15～20min, 20%～70% methyl alcohol; 20～30min, 70% methyl alcohol; 30～40min, 70%～90% methyl alcohol (the above-mentioned percent by volume that is).Obtain root of Dahurain angelica alcohol extract effective component group fingerprint atlas, determine its characteristic peak, have 13 total fingerprint peakses.Specifically see the applicant's patented claim formerly, application number is 200610036947.3.
Each relative retention time of 13 total fingerprint peakses (characteristic peak) of angelica root (root of Dahurain angelica alcohol extract) finger-print is respectively (is reference with No. 11 peaks):
No. 1 peak: 0.031～0.034; No. 2 peaks: 0.086～0.088; No. 3 peaks: 0.607～0.649;
No. 4 peaks: 0.658～0.665; No. 5 peaks: 0.704～0.708; No. 6 peaks: 0.730～0.739;
No. 7 peaks: 0.749～0.755; No. 8 peaks: 0.810～0.816; No. 9 peaks: 0.842～0.847;
No. 10 peaks: 0.856～0.861; No. 11 peaks: 1; No. 12 peaks: 1.085～1.087;
No. 13 peaks: 1.181～1.186.
Each total fingerprint peaks relative peak area scope is respectively (is reference with No. 11 peaks):
No. 1 peak: 0.005～0.061; No. 2 peaks: 0.016～0.132; No. 3 peaks: 0.050～0.366;
No. 4 peaks: 0.010～0.135; No. 5 peaks: 0.03～0.148; No. 6 peaks: 0.006～0.153;
No. 7 peaks: 0.086～0.328; No. 8 peaks: 0.047～0.105; No. 9 peaks: 0.018～0.059;
No. 10 peaks: 0.030～0.113; No. 11 peaks: 1; No. 12 peaks: 0.251～0.492;
No. 13 peaks: 0.304～0.845.
(2) vas deferens smooth muscle cell former is commissioned to train foster
Get the vas deferens of bull rabbit, peel off interior adventitial tissue, take enzyme digestion cultured cell in culture flask.Concrete operations are as follows:
Get the bull rabbit, anesthesia is made the lower abdomen transverse incision and is advanced abdomen, and free bilateral vas deferens and excision are taken out, and put in the aseptic PBS liquid and clean 3 times, peel off interior adventitial tissue with dissecting forceps under dissecting microscope, and vas deferens is cut into 1mm
3The piece of tissue of size.With 0.1% (mass percent) type i collagen enzyme+0.2% trypsase (mass percent) digestion piece of tissue 0.5h.Abandon digestive juice, piece of tissue evenly is attached at the 25cm that handled through nutrient solution with curved mouth dropper with the 0.5cm spacing
2The culture flask bottom with the culture flask upset, is put 37 ℃ and is contained 95% air, 5% (percent by volume) CO
2The cellular incubation incubator in 1h, add after 5ml contains the DMEM nutrient solution of 20% hyclone, slowly culture flask is turned, put 37 ℃ and contain 95% air, 5%CO
2The cellular incubation incubator in cultivate.Observation of cell form and growing state were changed above-mentioned nutrient solution 1 time in 2～3 days.
(3) cultivation of going down to posterity of vas deferens smooth muscle cell
When cell covers with covering bottle floorage 80% left and right sides, go down to posterity.Concrete operations are as follows:
When cell covers with covering bottle floorage 80% left and right sides, remove cell culture fluid, 37 ℃ of PBS (phosphate buffer) liquid cleans 2 times, add the cell dissociation buffer that contains 0.2% (mass percent) trypsase+0.02% (mass percent) EDTA, leave standstill 1～3min, microscopically is observed the control cell free, is taken off the wall situation, add the DMEM nutrient solution that contains 10% (percent by volume) hyclone and stop digestion, piping and druming gently, the mixing cell, obtain cell suspension, cell suspension is sucked in the 15ml centrifuge tube, 1000 rev/mins centrifugal 5 minutes.Abandoning supernatant adds the above-mentioned nutrient solution of 2ml, blows and beats cell gently with dropper and makes it mixing, gets 1 and puts the microscopically cell count with blood counting chamber, by 1 * 10
5The cell density of/ml, (quantity ratio) was inoculated into 25cm in 1: 2
2In the culture flask, put 37 ℃ and contain 95% air, 5%CO
2The cellular incubation incubator in cultivate.Observation of cell form and growing state were changed above-mentioned nutrient solution 1 time in 2～3 days.The 3rd generation go down to posterity after cell purity higher, cellular morphology shows as spindle shape or star, cell stretches out projection and contacts with each other, and merges each other, subregion cell multilayer is overlapping, the subregion cell monolayer just rises and falls, and is " peak-paddy " shape growth (see figure 2).
(4) α of vas deferens smooth muscle cell-SMA SABC is identified
Microslide is prevented the flake processing with poly-D-lysine, places six orifice plates, drips cell suspension, and cultured cell makes cell cover with microslide.The SABC authentication method is by the kit instructions operation of doctor's moral company.Through α-SMA immunohistochemical staining, cell is pale brown look, and the muscle fibril of streak arrangement is high-visible in the kytoplasm, and purity reaches 99% above (see figure 3).
(5) combination of effect components with dissociate:
1) gets 1ml root of Dahurain angelica alcohol extract in the cell dissociation after 3 generations went down to posterity obtains in the step (3) cell suspension 1ml and the step (1), mix 35～38 ℃ of incubation 0.5～1h in back.
2) 1000 rev/mins of centrifugal 10min remove supernatant.
3) wash 3～5 times with 2ml DMEM nutrient solution, detecting in the supernatant up to HPLC does not have the material wash-out to come out.In each washing process, drip washing is clean gently with the test liquid of tube wall earlier, with dropper piping and druming repeatedly lightly, makes into even suspension then, and 1000 rev/mins of centrifugal 10min abandon supernatant, sedimentation cell.
4) sedimentation cell that obtains in the step (3) is added the PBS (phosphate buffer) of pH2～6, make effect target spot inactivation (also can adopt other similar approach) on the cell membrane, the root of Dahurain angelica active component that is combined on the target spot dissociates.After blowing and beating repeatedly with dropper, 2500 rev/mins of centrifugal 10min collect supernatant and also volatilize, and thing must dissociate.
(6) HPLC measures and changes in conjunction with the front and back collection of illustrative plates
With step (3) the gained thing 1ml dissolve with methanol that dissociates, to analyze for HPLC, the HPLC analysis condition is identical with the HPLC analysis condition of step (1).Each absorption peak ratio in the root of Dahurain angelica alcohol extract finger-print that obtains in thing and step (1) by analytical solution is determined the composition that combines with vas deferens smooth muscle cell specificity.Be reference with opiate receptor antagonist and opioid receptor agonist in addition, can determine whether the root of Dahurain angelica combines with opiate receptor, concrete grammar promptly is before cell suspension and root of Dahurain angelica alcohol extract incubation, with opiate receptor antagonist naloxone hydrochloride or opioid receptor agonist pethidine hydrochloride and 35～38 ℃ of incubation 0.5～1h of cell suspension, make the opiate receptor on the cell saturated earlier.
As seen from Figure 6, with the characteristic peak of opiate receptor specific bond be No. 7 peaks, No. 8 peaks, 3 characteristic peaks in No. 10 peaks, described 3 characteristic peaks are the active component groups that combine with opiate receptor.No. 7, No. 8, No. 10 the peak occurs in D, and does not all occur in C; No. 8, No. 10 the peak does not all occur in B, C.Illustrate that No. 7 peaks, No. 8 peaks, No. 10 peak representative material action sites are identical with Allylnoroxymorphone, can be by its antagonism; No. 7 peak representative material action site is different with pethidine hydrochloride; No. 8 peaks, No. 10 peak representative material action sites are identical with pethidine hydrochloride.
Embodiment 2: the method for building up (mouse vas deferens smooth muscle cell) of the vas deferens cellular membrane chromatography model of screening Chinese medicine analgesic bioactive substance
(1) foundation of angelica root finger-print
Method is with step (1) among the embodiment 1.
(2) vas deferens smooth muscle cell former is commissioned to train foster
Get the vas deferens of bull mouse, peel off interior adventitial tissue, take enzyme digestion cultured cell in culture flask.Method is with embodiment 1.
(3) cultivation of going down to posterity of vas deferens smooth muscle cell
When cell covers with covering bottle floorage 80% left and right sides, remove cell culture fluid, 37 ℃ of PBS (phosphate buffer) liquid cleans 2 times, add the cell dissociation buffer that contains 0.2% (mass percent) trypsase+0.02% (mass percent) EDTA, leave standstill 1～3min, microscopically is observed the control cell free, is taken off the wall situation, add the DMEM nutrient solution that contains 10% (percent by volume) hyclone and stop digestion, piping and druming gently, the mixing cell, obtain cell suspension, cell suspension is sucked in the 15ml centrifuge tube, 1000 rev/mins centrifugal 5 minutes.Abandoning supernatant adds the above-mentioned nutrient solution of 2ml, blows and beats cell gently with dropper and makes it mixing, gets 1 and puts the microscopically cell count with blood counting chamber, by 1 * 10
5The cell density of/ml, (quantity ratio) was inoculated into 25cm in 1: 2
2In the culture flask, put 37 ℃ and contain 95% air, 5%CO
2The cellular incubation incubator in cultivate.Observation of cell form and growing state were changed above-mentioned nutrient solution 1 time in 2～3 days.The 3rd generation go down to posterity after cell purity higher, cellular morphology shows as spindle shape or star, cell stretches out projection and contacts with each other, and merges each other, subregion cell multilayer is overlapping, the subregion cell monolayer just rises and falls, and is " peak-paddy " shape growth (see figure 4).
(4) α of vas deferens smooth muscle cell-SMA SABC is identified
Microslide is prevented the flake processing with poly-D-lysine, places six orifice plates, drips cell suspension, and cultured cell makes cell cover with microslide.The SABC authentication method is by the kit instructions operation of doctor's moral company.Through α-SMA immunohistochemical staining, cell is pale brown look, and the muscle fibril of streak arrangement is high-visible in the kytoplasm, and purity reaches 99% above (see figure 5).
(5) combination of effect components with dissociate:
1) gets 1ml root of Dahurain angelica alcohol extract in the cell dissociation after 3 generations went down to posterity obtains in the step (3) cell suspension 1ml and the step (1), mix 35～38 ℃ of incubation 24h in back.
2) 1000 rev/mins of centrifugal 10min remove supernatant.
3) wash 3～5 times with 2ml DMEM nutrient solution, detecting in the supernatant up to HPLC does not have the material wash-out to come out.In each washing process, drip washing is clean gently with the test liquid of tube wall earlier, with dropper piping and druming repeatedly lightly, makes into even suspension then, and 1000 rev/mins of centrifugal 10min abandon supernatant, sedimentation cell.
4) sedimentation cell that obtains in the step (3) is added the PBS (phosphate buffer) of pH2～6, make effect target spot inactivation (also can adopt other similar approach) on the cell membrane, the root of Dahurain angelica active component that is combined on the target spot dissociates.After blowing and beating repeatedly with dropper, 2500 rev/mins of centrifugal 10rnin collect supernatant and also volatilize, and thing must dissociate.
(6) HPLC measures and changes in conjunction with the front and back collection of illustrative plates
With step (3) the gained thing 1ml dissolve with methanol that dissociates, analyze for HPLC, it is identical that HPLC analysis condition and step (1) HPLC analyse condition.Each absorption peak ratio in the root of Dahurain angelica alcohol extract finger-print that obtains in thing and step (1) by analytical solution is determined the composition that combines with vas deferens smooth muscle cell specificity.Be reference with opiate receptor antagonist and opioid receptor agonist in addition, can determine whether the root of Dahurain angelica combines with opiate receptor, concrete grammar promptly is before cell suspension and root of Dahurain angelica alcohol extract incubation, with opiate receptor antagonist naloxone hydrochloride or opioid receptor agonist pethidine hydrochloride and 35～38 ℃ of incubation 24h of cell suspension, make the opiate receptor on the cell saturated earlier.
As seen from Figure 7, do not occur obvious peak among the D, and mainly contain the δ acceptor on the mouse vas deferens, illustrate that root of Dahurain angelica alcohol extract does not act on the δ acceptor.
It is selective to opiate receptor that the present invention has illustrated the root of Dahurain angelica analgesic bioactive substance that filters out, and only specific in conjunction with opiate receptor κ hypotype (see figure 6), and with the irrelevant (see figure 7) of δ hypotype.
The foregoing description is a preferred implementation of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and the principle, substitutes, combination, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (4)
1, a kind of cellular membrane chromatography that screens the analgesia active ingredient of Chinese herbs is characterized in that comprising the steps:
(1) foundation of root of Dahurain angelica alcohol extract finger-print: take by weighing the 20g angelica root, add 160ml 95% ethanol, refluxing extraction 1～2h, volatilize ethanol, residue is settled to 20ml with dissolve with methanol, therefrom get 250 μ l and be diluted to 7.5ml, cross 0.45 μ m miillpore filter, obtain filtrate and be root of Dahurain angelica alcohol extract with methyl alcohol; Draw filtrate 20 μ l, adopt efficient liquid-phase chromatography method to measure, obtain root of Dahurain angelica alcohol extract effective component group fingerprint atlas, determine 13 total fingerprint peakses;
Described efficient liquid phase chromatographic analysis condition: with the methanol-water is flow phase system, 30 ℃ of column temperatures, sample size 20 μ l, wavelength 254nm, length is that 250mm, diameter are the Lichrospher5-C18 chromatographic column of 4.6mm, eluent gradient: 0～5min, 5%～20% methyl alcohol; 5～15min, 20% methyl alcohol; 15～20min, 20%～70% methyl alcohol; 20～30min, 70% methyl alcohol; 30～40min, 70%～90% methyl alcohol; The above-mentioned percent by volume that is;
13 each relative retention times of total fingerprint peaks of described root of Dahurain angelica alcohol extract finger-print are respectively:
No. 1 peak: 0.031～0.034; No. 2 peaks: 0.086～0.088; No. 3 peaks: 0.607～0.649;
No. 4 peaks: 0.658～0.665; No. 5 peaks: 0.704～0.708; No. 6 peaks: 0.730～0.739;
No. 7 peaks: 0.749～0.755; No. 8 peaks: 0.810～0.816; No. 9 peaks: 0.842～0.847;
No. 10 peaks: 0.856～0.861; No. 11 peaks: 1; No. 12 peaks: 1.085～1.087;
No. 13 peaks: 1.181～1.186;
Each total fingerprint peaks relative peak area scope is respectively:
No. 1 peak: 0.005～0.061; No. 2 peaks: 0.016～0.132; No. 3 peaks: 0.050～0.366;
No. 4 peaks: 0.010～0.135; No. 5 peaks: 0.03～0.148; No. 6 peaks: 0.006～0.153;
No. 7 peaks: 0.086～0.328; No. 8 peaks: 0.047～0.105; No. 9 peaks: 0.018～0.059;
No. 10 peaks: 0.030～0.113; No. 11 peaks: 1; No. 12 peaks: 0.251～0.492;
No. 13 peaks: 0.304～0.845;
Be reference all below with No. 11 peaks;
(2) former being commissioned to train of vas deferens smooth muscle cell supported and the cultivation of going down to posterity: get the vas deferens of bull mouse or bull rabbit, peel off interior adventitial tissue, take enzyme digestion cultured cell in culture flask; When cell covers with covering bottle floorage 80% left and right sides, go down to posterity;
(3) combination of effect components with dissociate: the root of Dahurain angelica alcohol extract equal-volume that the cell suspension that will obtain through the cell dissociation that 3～10 generations went down to posterity and step (1) obtain mixes, behind 36～38 ℃ of incubation 0.5～24h, the centrifugal supernatant that goes, with DMEM nutrient solution washed cell 3～5 times, obtain even suspension, centrifugal, abandon supernatant, get sedimentation cell; The PBS that adds pH2～6 in sedimentation cell makes cell degeneration, and is centrifugal, and the collection supernatant also volatilizes, and thing must dissociate;
(4) high-performance liquid chromatogram determination changes in conjunction with the front and back collection of illustrative plates: with step (3) the gained thing dissolve with methanol that dissociates, use efficient liquid phase chromatographic analysis, described efficient liquid phase chromatographic analysis condition is identical with the chromatographiccondition of step (1); By the variation of each absorption peak in relatively dissociate thing finger-print and the root of Dahurain angelica alcohol extract finger-print, and make reference with opiate receptor antagonist and opioid receptor agonist respectively, determine whether the root of Dahurain angelica combines with opiate receptor; With the characteristic peak of opiate receptor specific bond be No. 7 peaks, No. 8 peaks and No. 10 peaks; The root of Dahurain angelica analgesic bioactive substance that filters out is No. 7 peaks, No. 8 peaks and No. 10 peaks; Described opiate receptor antagonist is a naloxone hydrochloride, and described opioid receptor agonist is a pethidine hydrochloride.
2, a kind of cellular membrane chromatography that screens the analgesia active ingredient of Chinese herbs according to claim 1, it is characterized in that: described No. 7 peaks of characteristic peak, three characteristic peaks in No. 8 peaks and No. 10 peaks with the opiate receptor specific bond are the active component groups that combine with opiate receptor.
3, a kind of cellular membrane chromatography that screens the analgesia active ingredient of Chinese herbs according to claim 1, it is characterized in that: described No. 7 peaks, No. 8 peaks, No. 10 peak representative material action sites are identical with Allylnoroxymorphone, can be by its antagonism; Described No. 7 peak representative material action sites are different with pethidine hydrochloride; Described No. 8 peaks, No. 10 peak representative material action sites are identical with pethidine hydrochloride.
4, a kind of cellular membrane chromatography that screens the analgesia active ingredient of Chinese herbs according to claim 1, it is characterized in that: the described root of Dahurain angelica analgesic bioactive substance that filters out is selective to opiate receptor, and only specific in conjunction with opiate receptor κ hypotype, and irrelevant with the δ hypotype.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102533912A (en) * | 2011-12-09 | 2012-07-04 | 江南大学 | Method for quickly and accurately discovering, identifying and preparing proteolysis provenance antibacterial peptide |
| CN106568879A (en) * | 2016-11-02 | 2017-04-19 | 西安交通大学 | Cell membrane chromatographic column for identifying microscale target component in complex sample and preparation method thereof, and identification method of target component |
| CN106990200A (en) * | 2017-05-16 | 2017-07-28 | 暨南大学 | A kind of cell membrane solid phase chromatography for screening Chinese medicine psychoactive ingredient and its application |
| CN107064394A (en) * | 2017-02-23 | 2017-08-18 | 西安交通大学 | A kind of dual-target membrane flexibility post and its preparation method and application |
| CN107607646A (en) * | 2017-09-14 | 2018-01-19 | 神威药业集团有限公司 | A kind of platelet cell membrane chromatography |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1904606B (en) * | 2006-08-04 | 2011-06-29 | 暨南大学 | Angelice dehurica crude drug alcoholic extract effective component group fingerprint map and its establishing method and application |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102533912A (en) * | 2011-12-09 | 2012-07-04 | 江南大学 | Method for quickly and accurately discovering, identifying and preparing proteolysis provenance antibacterial peptide |
| CN106568879A (en) * | 2016-11-02 | 2017-04-19 | 西安交通大学 | Cell membrane chromatographic column for identifying microscale target component in complex sample and preparation method thereof, and identification method of target component |
| CN107064394A (en) * | 2017-02-23 | 2017-08-18 | 西安交通大学 | A kind of dual-target membrane flexibility post and its preparation method and application |
| CN106990200A (en) * | 2017-05-16 | 2017-07-28 | 暨南大学 | A kind of cell membrane solid phase chromatography for screening Chinese medicine psychoactive ingredient and its application |
| CN107607646A (en) * | 2017-09-14 | 2018-01-19 | 神威药业集团有限公司 | A kind of platelet cell membrane chromatography |
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