CN1958650A - Molecular engram polymer of vinblastine, and preparation method - Google Patents
Molecular engram polymer of vinblastine, and preparation method Download PDFInfo
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- CN1958650A CN1958650A CNA2006101230608A CN200610123060A CN1958650A CN 1958650 A CN1958650 A CN 1958650A CN A2006101230608 A CNA2006101230608 A CN A2006101230608A CN 200610123060 A CN200610123060 A CN 200610123060A CN 1958650 A CN1958650 A CN 1958650A
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Abstract
This invention discloses a vinblastine molecular imprinted polymer, which has the imprinting sites of the template molecule vinblastine. The vinblastine molecular imprinted polymer is prepared by heat-initiated polymerization of vinblastine 1 part, functional monomer 3-10 parts, crosslinker 10-50 parts and appropriate pore-forming agent at 40-80 deg.C. The obtained vinblastine molecular imprinted polymer has unique affinity, high selectivity and excellent molecular reorganization to vinblastine, and can be used as separation filler for extracting, separating and enriching vinblastine from natural plants and biological samples.
Description
Technical field
The present invention relates to a kind of organic high molecular compound, be specifically related to a kind of alkaloidal molecularly imprinted polymer, also relate to the preparation method of this polymkeric substance.
Background technology
Vinealeucoblastine(VLB) is to separate the dimeric indole Alkaloid with obvious anti-tumor activity that obtains from apocynaceae plant Vinca (Vinca rosea L. (Catharanthus roseus G.Don)), can suppress the tubulin assembling, prevent that spindle fibre from forming, thereby make mitotic division stop at mid-term.Clinical treatment Hokdkin disease and the chorioepithelium cancer of being mainly used in also has certain curative effect to lymphosarcoma, melanoma, ovarian cancer, leukemia etc., is one of present widely used cancer therapy drug.The source of vinealeucoblastine(VLB) mainly is a natural phant, and the content in natural phant is extremely low, only is ppm to ten ten thousand/several, though utilize chemosynthesis and semisynthetic method to obtain, cost is higher; Tissue culture, cell cultures are still immature; Method by the regulation and control pathways metabolism also is in conceptual phase, so extraction separation is still the main path that obtains vinealeucoblastine(VLB) from natural phant.
Obtaining vinealeucoblastine(VLB) from natural phant, mainly is to separate through column chromatography after adopting solvent method or supercritical methanol technology to extract to obtain again.Gunasekera etc. [Gunasekera SP.Method of isolating vinblastine[P] .WO:8803135,1988-05-05. (CA 1988,109:134967)] with the sour water lixiviate of Vinca herb, acid liquid is used dichloromethane extraction after the ammoniacal liquor alkalization, the total alkaloids extraction yield is 0.36%, and wherein vinealeucoblastine(VLB) is 8.3%.[Choi YH such as Choi, Yoo KP, Kim J.Supercritical fluid extraction and liquidchromatography-electrospray mass analysis of vinblastine from Catharanthus roseus.[J] .Chem Pharm Bull, 2002,50 (9): 1294-1296.] make the entrainment agent carbon dioxide upercritical fluid extraction with methyl alcohol, triethylamine, can extract the vinealeucoblastine(VLB) in the Vinca preferably.The extract that obtains in order to last method is the demand pole chromatographic separation and purification also, could obtain highly purified vinealeucoblastine(VLB).Post is once crossed in the normal employing of column chromatography separation, secondary is crossed post and reached repeatedly post.Once crossing post, to select alkali alumina for use be column packing, general degree of grade or the gradient elution in varing proportions such as benzene-sherwood oil, benzene-chloroform, benzene-methylene dichloride, methylene dichloride-chloroform, ethyl acetate-sherwood oil used, this law is the method that is used for scale operation at present, shortcoming is undesirable [the Gedeon R of the purity of product, Vegyeszeti GRT.Isolation of vinblastine and leurosine[P] .Hung:153200,1966-10-22. (CA 1967,66:118854)].It is that crude extract is successively handled with sephadex column and silicagel column that secondary is crossed the post method, product purity is [Gunasekera SP.Method of isolating vinblastine[P] .WO:8803135 better, 1988-05-05. (CA 1988,109:134967)], but the used dextrane gel price of this method is more expensive, and repeatedly use can cause irreversible adsorption, the separating effect variation.[Atta-ur-Rahman such as Atta-ur-Rahman, Bashir M, Hafeez M, et al.A rapid procedure for the isolation of catharanthine, vindoline andvinblastine[J] .Planta Med, 1983,47 (4): 246-247.] with total alkaloids successively after alumina column, silicagel column and preparation HPLC separate, can obtain highly purified vinealeucoblastine(VLB), but this law complex steps, yield is very low, is not suitable for enlarging preparation.[Renault JH such as Renault, Nuzillard JM, Le CG, et al.Isolation of indole alkaloids from Catharanthus roseus by centrifugalpartition chromatography in the pH-zone refining mode[J] .J Chromatogr A, 1999,849 (2): 421-431.] with high performance countercurrent chromatography method catharanthus roseus total alkaloids separation, leave standstill the organic phase that obtains and water two-phase with methyl tertiary butyl ether-acetonitrile-water (4: 1: 5) as adverse current, can be with vinealeucoblastine(VLB) and separated from impurities, but fractional dose is little, and is difficult to keep the long-term stability of post effect.
In sum, the column chromatography of conventional fillers such as employing aluminum oxide, silica gel is still the main method of present separation and purification vinealeucoblastine(VLB).But because vinealeucoblastine(VLB) is extremely low at the plant materials intensive amount, there be simultaneously a large amount of chemical structures compound close again with character, column chromatography with routine is separated, not only difficulty is big, and loss is also many in purge process, be difficult to obtain simultaneously the product of high purity, high yield, it is very necessary therefore to seek more effective parting material.
Molecularly imprinted polymer (MIP) is a kind of high molecular polymer that template molecule is had high selectivity, can use the molecular imprinting preparation, committed step comprises: (1) makes template molecule combine by non covalent bond with selected function monomer, thereby forms complementary template-monomer complex on the space structure; (2) make template-monomer complex and linking agent copolymerization, thereby form crosslinked porous reticular polymer (also claiming Molecularly Imprinted Polymer); (3) from Molecularly Imprinted Polymer, extract template molecule, thereby obtain having the molecularly imprinted polymer of template molecule binding site.These binding sites on molecularly imprinted polymer, similar antibody-antigen can be with high affinity and special selectivity again in conjunction with the template corresponding molecule, or the analogue of approximate construction.Have not yet to see the relevant report of molecular engram polymer of vinblastine.
Summary of the invention
In view of there is above-mentioned deficiency in prior art, technical problem to be solved by this invention provides a kind of molecular engram polymer of vinblastine.This polymkeric substance has special affinity and high selectivity to vinealeucoblastine(VLB).
The technical scheme that the present invention solves the problems of the technologies described above is:
A kind of molecular engram polymer of vinblastine, this polymkeric substance has the imprinted sites of template molecule vinealeucoblastine(VLB), and it was made 40～80 ℃ of following thermal-initiated polymerization reactions by following molar part monomer component in 10～24 hours:
1 part of template molecule vinealeucoblastine(VLB), 3～10 parts of function monomer methacrylic acid or methyl methacrylates, 10～50 parts of linking agent ethylene glycol dimethacrylate or trimethylolpropane trimethacrylates.
Make in the polyreaction of molecular engram polymer of vinblastine of the present invention used function monomer preferable methyl vinylformic acid; The preferred ethylene glycol dimethacrylate of used linking agent.
The preparation method of molecular engram polymer of vinblastine of the present invention is made up of following steps:
1) vinealeucoblastine(VLB), function monomer are pressed 1: 3～10 mol ratio mixings in pore-creating agent toluene, acetonitrile or chloroform, press vinealeucoblastine(VLB) again: linking agent: initiator=1: 10～50: 0.9 adds linking agent and initiator Diisopropyl azodicarboxylate, be mixed, deoxygenation, vacuumize, 40～80 ℃ of following thermal-initiated polymerization reactions 10～24 hours, obtain containing the high molecular polymer of vinealeucoblastine(VLB) then;
2) the step 1) resulting polymers grinds to form fine powder, again with acetone repeatedly sedimentation remove fine particle, remove the vinealeucoblastine(VLB) molecule with the washing of 10～40% Glacial acetic acid methyl alcohol, methanol solution successively then, vacuum-drying obtains molecular engram polymer of vinblastine.
Among the preparation method of molecular engram polymer of vinblastine of the present invention, preferred toluene of described pore-creating agent or acetonitrile.
Molecular engram polymer of vinblastine of the present invention can be used as separating filler and is used for extracting vinealeucoblastine(VLB) from natural phant, also can be used for the pre-treatment of analytic sample, the preparation separation in the chromatographic technique etc.The advantage that molecular engram polymer of vinblastine of the present invention is used to extract, separation, enrichment vinealeucoblastine(VLB) molecule have the affinity height, selectivity is strong.
Description of drawings
Fig. 1 is the concentration C of the testing sample of vinealeucoblastine(VLB) reference substance preparation and the relation curve of absorbance A;
Fig. 2 is the high-efficient liquid phase chromatogram of the solution of vinealeucoblastine(VLB) reference substance preparation;
Fig. 3 is the high-efficient liquid phase chromatogram of the solution of vincristine(VCR) reference substance preparation;
Fig. 4 is the high-efficient liquid phase chromatogram of leacheate in the following experiment three;
Fig. 5 is the high-efficient liquid phase chromatogram of elutriant in the following experiment three;
Fig. 6 is the infrared spectrogram of the prepared non-imprinted polymer of following experiment four;
Fig. 7 is the infrared spectrogram of preparation example 1 gained molecular engram polymer of vinblastine.
Embodiment
One, preparation example
Preparation example 1:
Earlier the 0.1mmol vinealeucoblastine(VLB) is added in the ampoule, add methacrylic acid 0.5mmol and toluene 1.5ml, on vibrator, vibrated repeatedly 60 minutes.Add ethylene glycol dimethacrylate 4.5mmol and Diisopropyl azodicarboxylate 15mg again, after mixing in ultrasonic 10 minutes,, vacuumize and use the alcohol blast burner tube sealing with nitrogen purging 5 minutes.This polyreaction of thermal initiation, and made it in 60 ℃ of water-baths polyreaction 18 hours.After polyreaction is finished, in polymerizing pipe, obtain material all in one piece, break up this pipe and this material all in one piece is ground into smaller particles, and be 25～75 microns the screening of the irregularly shaped particles of gained.In order to remove 25～75 microns fine particles (＜25 microns) in the part, use acetone this material of sedimentation repeatedly, become transparent up to supernatant liquid.Then with the transfer of granules that obtains in paper sleeve and be placed in the apparatus,Soxhlet's device, successively with 10% Glacial acetic acid methanol wash 48 hours, methanol solution washing 4 hours, up to elutant through UV (hewlette-packard production, HP8453 type ultraviolet-visible spectrophotometer, the detection wavelength is 264nm) detect, do not see till the absorption peak that vinealeucoblastine(VLB) is arranged that vacuum-drying promptly gets molecular engram polymer of vinblastine of the present invention.
Preparation example 2:
Earlier the 0.1mmol vinealeucoblastine(VLB) is added in the ampoule, add methacrylic acid 0.5mmol and toluene 1.5ml, on vibrator, vibrated repeatedly 60 minutes.Add ethylene glycol dimethacrylate 4.5mmol and Diisopropyl azodicarboxylate 15mg again, after mixing in ultrasonic 10 minutes,, vacuumize and use the alcohol blast burner tube sealing with nitrogen purging 5 minutes.This polyreaction of thermal initiation, and made it in 40 ℃ of water-baths polyreaction 24 hours.After polyreaction is finished, in polymerizing pipe, obtain material all in one piece, break up this pipe and this material all in one piece is ground into smaller particles, and be 25～75 microns the screening of the irregularly shaped particles of gained.In order to remove 25～75 microns fine particles (＜25 microns) in the part, use acetone this material of sedimentation repeatedly, become transparent up to supernatant liquid.Then with the transfer of granules that obtains in paper sleeve and be placed in the apparatus,Soxhlet's device, successively with 10% Glacial acetic acid methanol wash 48 hours, methanol solution washing 4 hours, detect through UV up to elutant, do not see till the absorption peak that vinealeucoblastine(VLB) is arranged, vacuum-drying promptly gets molecular engram polymer of vinblastine of the present invention.
Preparation example 3:
Earlier the 0.1mmol vinealeucoblastine(VLB) is added in the ampoule, add methacrylic acid 0.5mmol and toluene 1.5ml, on vibrator, vibrated repeatedly 60 minutes.Add ethylene glycol dimethacrylate 4.5mmol and Diisopropyl azodicarboxylate 15mg again, after mixing in ultrasonic 10 minutes,, vacuumize and use the alcohol blast burner tube sealing with nitrogen purging 5 minutes.This polyreaction of thermal initiation, and made it in 80 ℃ of water-baths polyreaction 10 hours.After polyreaction is finished, in polymerizing pipe, obtain material all in one piece, break up this pipe and this material all in one piece is ground into smaller particles, and be 25～75 microns the screening of the irregularly shaped particles of gained.In order to remove 25～75 microns fine particles (＜25 microns) in the part, use acetone this material of sedimentation repeatedly, become transparent up to supernatant liquid.Then with the transfer of granules that obtains in paper sleeve and be placed in the apparatus,Soxhlet's device, successively with 10% Glacial acetic acid methanol wash 48 hours, methanol solution washing 4 hours, detect through UV up to elutant, do not see till the absorption peak that vinealeucoblastine(VLB) is arranged, vacuum-drying promptly gets molecular engram polymer of vinblastine of the present invention.
Preparation example 4:
Earlier the 0.1mmol vinealeucoblastine(VLB) is added in the ampoule, add methacrylic acid 0.3mmol and toluene 1.5ml, on vibrator, vibrated repeatedly 60 minutes.Add ethylene glycol dimethacrylate 3.0mmol and Diisopropyl azodicarboxylate 15mg again, after mixing in ultrasonic 10 minutes,, vacuumize and use the alcohol blast burner tube sealing with nitrogen purging 5 minutes.This polyreaction of thermal initiation, and made it in 60 ℃ of water-baths polyreaction 24 hours.After polyreaction is finished, in polymerizing pipe, obtain material all in one piece, break up this pipe and this material all in one piece is ground into smaller particles, and be 25～75 microns the screening of the irregularly shaped particles of gained.In order to remove 25～75 microns fine particles (＜25 microns) in the part, use acetone this material of sedimentation repeatedly, become transparent up to supernatant liquid.Then with the transfer of granules that obtains in paper sleeve and be placed in the apparatus,Soxhlet's device, successively with 25% Glacial acetic acid methanol wash 48 hours, methanol solution washing 4 hours, detect through UV up to elutant, do not see till the absorption peak that vinealeucoblastine(VLB) is arranged, vacuum-drying promptly gets molecular engram polymer of vinblastine of the present invention.
Preparation example 5:
Earlier the 0.1mmol vinealeucoblastine(VLB) is added in the ampoule, add methacrylic acid 0.6mmol and toluene 1.5ml, on vibrator, vibrated repeatedly 60 minutes.Add ethylene glycol dimethacrylate 5.0mmol and Diisopropyl azodicarboxylate 15mg again, after mixing in ultrasonic 10 minutes,, vacuumize and use the alcohol blast burner tube sealing with nitrogen purging 5 minutes.This polyreaction of thermal initiation, and made it in 60 ℃ of water-baths polyreaction 24 hours.After polyreaction is finished, in polymerizing pipe, obtain material all in one piece, break up this pipe and this material all in one piece is ground into smaller particles, and be 25～75 microns the screening of the irregularly shaped particles of gained.In order to remove 25～75 microns fine particles (＜25 microns) in the part, use acetone this material of sedimentation repeatedly, become transparent up to supernatant liquid.Then with the transfer of granules that obtains in paper sleeve and be placed in the apparatus,Soxhlet's device, successively with 40% Glacial acetic acid methanol wash 48 hours, methanol solution washing 4 hours, detect through UV up to elutant, do not see till the absorption peak that vinealeucoblastine(VLB) is arranged, vacuum-drying promptly gets molecular engram polymer of vinblastine of the present invention.
Preparation example 6:
Earlier the 0.1mmol vinealeucoblastine(VLB) is added in the ampoule, add methacrylic acid 1.0mmol and acetonitrile 1.5ml, on vibrator, vibrated repeatedly 60 minutes.Add ethylene glycol dimethacrylate 1.2mmol and Diisopropyl azodicarboxylate 15mg again, after mixing in ultrasonic 10 minutes,, vacuumize and use the alcohol blast burner tube sealing with nitrogen purging 5 minutes.This polyreaction of thermal initiation, and made it in 60 ℃ of water-baths polyreaction 24 hours.After polyreaction is finished, in polymerizing pipe, obtain material all in one piece, break up this pipe and this material all in one piece is ground into smaller particles, and be 25～75 microns the screening of the irregularly shaped particles of gained.In order to remove 25～75 microns fine particles (＜25 microns) in the part, use acetone this material of sedimentation repeatedly, become transparent up to supernatant liquid.Then with the transfer of granules that obtains in paper sleeve and be placed in the apparatus,Soxhlet's device, successively with 10% Glacial acetic acid methanol wash 48 hours, methanol solution washing 4 hours, detect through UV up to elutant, do not see till the absorption peak that vinealeucoblastine(VLB) is arranged, vacuum-drying promptly gets molecular engram polymer of vinblastine of the present invention.
Preparation example 7:
Earlier the 0.1mmol vinealeucoblastine(VLB) is added in the ampoule, add methyl methacrylate 0.7mmol and toluene 1.5ml, on vibrator, vibrated repeatedly 60 minutes.Add ethylene glycol dimethacrylate 4.5mmol and Diisopropyl azodicarboxylate 15mg again, after mixing in ultrasonic 10 minutes,, vacuumize and use the alcohol blast burner tube sealing with nitrogen purging 5 minutes.This polyreaction of thermal initiation, and made it in 60 ℃ of water-baths polyreaction 24 hours.After polyreaction is finished, in polymerizing pipe, obtain material all in one piece, break up this pipe and this material all in one piece is ground into smaller particles, and be 25～75 microns the screening of the irregularly shaped particles of gained.In order to remove 25～75 microns fine particles (＜25 microns) in the part, use acetone this material of sedimentation repeatedly, become transparent up to supernatant liquid.Then with the transfer of granules that obtains in paper sleeve and be placed in the apparatus,Soxhlet's device, successively with 10% Glacial acetic acid methanol wash 48 hours, methanol solution washing 4 hours, detect through UV up to elutant, do not see till the absorption peak that vinealeucoblastine(VLB) is arranged, vacuum-drying promptly gets molecular engram polymer of vinblastine of the present invention.
Preparation example 8:
Earlier the 0.1mmol vinealeucoblastine(VLB) is added in the ampoule, add methacrylic acid 0.5mmol and toluene 1.5ml, on vibrator, vibrated repeatedly 60 minutes.Add trimethylolpropane trimethacrylate 3.5mmol and Diisopropyl azodicarboxylate 15mg again, after mixing in ultrasonic 10 minutes,, vacuumize and use the alcohol blast burner tube sealing with nitrogen purging 5 minutes.This polyreaction of thermal initiation, and made it in 60 ℃ of water-baths polyreaction 24 hours.After polyreaction is finished, in polymerizing pipe, obtain material all in one piece, break up this pipe and this material all in one piece is ground into smaller particles, and be 25～75 microns the screening of the irregularly shaped particles of gained.In order to remove 25～75 microns fine particles (＜25 microns) in the part, use acetone this material of sedimentation repeatedly, become transparent up to supernatant liquid.Then with the transfer of granules that obtains in paper sleeve and be placed in the apparatus,Soxhlet's device, successively with 10% Glacial acetic acid methanol wash 48 hours, methanol solution washing 4 hours, detect through UV up to elutant, do not see till the absorption peak that vinealeucoblastine(VLB) is arranged, vacuum-drying promptly gets molecular engram polymer of vinblastine of the present invention.
Preparation example 9:
Earlier the 0.1mmol vinealeucoblastine(VLB) is added in the ampoule, add methacrylic acid 0.5mmol and chloroform 1.5ml, on vibrator, vibrated repeatedly 60 minutes.Add ethylene glycol dimethacrylate 4.5mmol and Diisopropyl azodicarboxylate 15mg again, after mixing in ultrasonic 10 minutes,, vacuumize and use the alcohol blast burner tube sealing with nitrogen purging 5 minutes.This polyreaction of thermal initiation, and made it in 60 ℃ of water-baths polyreaction 18 hours.After polyreaction is finished, in polymerizing pipe, obtain material all in one piece, break up this pipe and this material all in one piece is ground into smaller particles, and be 25～75 microns the screening of the irregularly shaped particles of gained.In order to remove 25～75 microns fine particles (＜25 microns) in the part, use acetone this material of sedimentation repeatedly, become transparent up to supernatant liquid.Then with the transfer of granules that obtains in paper sleeve and be placed in the apparatus,Soxhlet's device, successively with 10% Glacial acetic acid methanol wash 48 hours, methanol solution washing 4 hours, detect through UV up to elutant, do not see till the absorption peak that vinealeucoblastine(VLB) is arranged, vacuum-drying promptly gets molecular engram polymer of vinblastine of the present invention.
Two, molecular engram polymer of vinblastine of the present invention is to vinealeucoblastine(VLB) adsorption effect test experience
(1) experiment equipment and working parameter thereof:
Used luminosity is counted the HP8453 type ultraviolet-visible spectrophotometer that hewlette-packard is produced in this experiment, and the detection wavelength is 264nm.
(2) experimental technique: set up the typical curve equation of vinealeucoblastine(VLB) reference substance concentration earlier to absorbancy, be that sorbent material carries out Solid-Phase Extraction to the target compound vinealeucoblastine(VLB) with molecular engram polymer of vinblastine of the present invention respectively then, measure the absorbancy of extraction back leacheate and elutriant then, substitution typical curve equation is calculated the concentration of vinealeucoblastine(VLB) in leacheate and the elutriant again, and then calculates the adsorption rate of sorbent material.
The concrete operations step is as described below:
1, sets up the typical curve equation
Weighing vinealeucoblastine(VLB) reference substance 25mg places the 25ml volumetric flask, adds chloroform dissolving, and constant volume is made into the solution of 1mg/ml, pipette 1,2,3,4 more respectively, 5ml in the 100ml volumetric flask, use the chloroform constant volume, as testing sample.Then, described testing sample is carried out absorbance detection successively, get absorbancy and be respectively 0.14859,0.30959,0.47659,0.63454,0.78538.After obtaining the absorbancy of each testing sample, be ordinate zou with the absorbance A, concentration C (μ g/ml) is an X-coordinate, sets up system of coordinates, finds out the pairing coordinate of each testing sample, connects each coordinate point line linearity of going forward side by side and returns, and obtains the straight line shown in Fig. 1.This collinear equation of linear regression: C=0.5486+62.5370A, coefficient R=0.99984.
2, the measuring and calculating of adsorption rate
(1) the molecular engram polymer of vinblastine 400mg with preparation example 1 gained is filled in the polypropylene shell solid phase extraction column that internal diameter is 1.5cm, with wetting, the balance of toluene 20ml; Vinealeucoblastine(VLB) is made into the solution that concentration is 0.2mg/ml with toluene, gets sample on this solution of 1ml.Solid phase extraction column behind the last sample with 3ml chloroform drip washing 1 time, is collected leacheate earlier; The extraction pillar is used 10% Glacial acetic acid methanol solution wash-out 2 times again, each 3ml, and flow velocity is 0.5ml/min, collects elutriant.
(2) do not measure the absorbancy of leacheate, elutriant with luminosity score, the chloroform leacheate does not have absorption peak as a result, and the elutriant absorption peak of 10% Glacial acetic acid methyl alcohol is obvious, and absorbancy is 0.4297, illustrates to contain vinealeucoblastine(VLB) in the elutriant.With the equation of linear regression in the absorbancy 0.4297 value substitution step 1 of the elutriant of resulting 10% Glacial acetic acid methyl alcohol, just the vinealeucoblastine(VLB) concentration that obtains the elutriant of 10% Glacial acetic acid methyl alcohol is 27.42 μ g/ml, further calculate again in the elutriant of 10% Glacial acetic acid methyl alcohol the content of vinealeucoblastine(VLB) be 164.54 μ g.In view of the above, adopt conventional method of calculation, i.e. the amount of vinealeucoblastine(VLB) in the amount/sample solution of vinealeucoblastine(VLB) in adsorption rate=elutriant, calculating adsorption rate is 82.27%.
Adopt aforesaid method that the molecular engram polymer of vinblastine of preparation example 2～9 gained is calculated one by one, its result is: the adsorption rate of preparation example 2 gained molecular engram polymer of vinblastine is 70.94%; The adsorption rate of preparation example 3 gained molecular engram polymer of vinblastine is 68.20%; The adsorption rate of preparation example 4 gained molecular engram polymer of vinblastine is 51.33%; The adsorption rate of preparation example 5 gained molecular engram polymer of vinblastine is 82.63%; The adsorption rate of preparation example 6 gained molecular engram polymer of vinblastine is 86.70%; The adsorption rate of preparation example 7 gained molecular engram polymer of vinblastine is 42.35%; The adsorption rate of preparation example 8 gained molecular engram polymer of vinblastine is 78.23%; The adsorption rate of preparation example 9 gained molecular engram polymer of vinblastine is 73.68%.
Three, molecularly imprinted polymer selective adsorption effect experiment of the present invention
(1) experiment equipment and condition
1, highly effective liquid phase chromatographic system, manufacturer: hewlette-packard; Model: HP 1100.
2, chromatographic condition: select Agilent C for use
18Chromatographic column (250 * 4.6mm, 5 μ m); In methyl alcohol: acetonitrile: the ratio preparation moving phase of 1% triethylamine aqueous solution=60: 25: 15 is adjusted to 7.2 with phosphoric acid with its pH value again; The detection wavelength is 262nm, and flow velocity is 1.0ml/min, and sample size is 5 μ l.
(2) experimental technique
Earlier reference substance is carried out efficient liquid phase chromatographic analysis, obtain the standard colour chart of reference substance, make sorbent material with molecular engram polymer of vinblastine of the present invention again the mixture that contains target compound is carried out Solid-Phase Extraction, and resulting Solid-Phase Extraction liquid carried out efficient liquid phase chromatographic analysis, obtain the chromatogram of target compound, at last the chromatogram of target compound and the standard colour chart of reference substance are carried out feature relatively, judge whether the chromatogram of gained target compound is identical with reference substance.
Concrete experimental procedure is as follows:
1, getting commercially available vinealeucoblastine(VLB) and vincristine(VCR) is reference substance, with toluene is that solvent makes vinealeucoblastine(VLB) solution and the vincristine(VCR) solution that concentration is 0.1mg/ml respectively, and carry out efficient liquid phase chromatographic analysis, obtain the high performance liquid chromatography (see figure 2) of vinealeucoblastine(VLB) solution and the high performance liquid chromatography (see figure 3) of vincristine(VCR) solution.The retention time of vinealeucoblastine(VLB) reference substance (0.1mg/ml) is 8.76min, and the retention time of vincristine(VCR) reference substance (0.1mg/ml) is 7.36min.
2, the molecular engram polymer of vinblastine 400mg that gets preparation example 1 gained is filled in the solid phase extraction column that the polypropylene internal diameter of outer cover is 1.5cm, with wetting, the balance of toluene 20ml.Vinca extract (mainly containing vinealeucoblastine(VLB) and vincristine(VCR)) is made into the solution that concentration is 0.1mg/ml with toluene, gets 1ml and go up sample.Solid phase extraction column behind the last sample with chloroform drip washing 2 times (each 2ml, flow velocity is 0.5ml/min), is collected leacheate earlier; Use 10% Glacial acetic acid methanol solution wash-out 2 times (each 2ml, flow velocity is 0.5ml/min) again, collect elutriant.Leacheate, the elutriant collected are carried out efficient liquid phase chromatographic analysis, obtain the high performance liquid chromatography (see figure 4) of leacheate and the high performance liquid chromatography (see figure 5) of elutriant.
3, Fig. 4 is compared with Fig. 2, Fig. 3, visible Fig. 3 and Fig. 4 are that the 7.36min place has tangible chromatographic peak in retention time all, and waveform similarity; Fig. 5 is compared with Fig. 2, Fig. 3, visible Fig. 5 and Fig. 2 are that the 8.76min place has tangible chromatographic peak in retention time all, and waveform similarity again.Therefore can conclude that contained material is a vincristine(VCR) in the leacheate, and contained material is a vinealeucoblastine(VLB) in the elutriant, and then proof molecularly imprinted polymer of the present invention has the selective adsorption effect to vinealeucoblastine(VLB).
Four, molecular engram polymer of vinblastine of the present invention and non-imprinted polymer are to the adsorbing comparative experiments of vinealeucoblastine(VLB)
(1) preparation of non-imprinted polymer
Methacrylic acid 0.5mmol and toluene 1.5ml are added in the ampoule, on vibrator, vibrated repeatedly 60 minutes, add ethylene glycol dimethacrylate 4.5mmol and Diisopropyl azodicarboxylate 15mg again, after mixing in ultrasonic 10 minutes, with nitrogen purging 5 minutes, vacuumize and use the alcohol blast burner tube sealing.This polyreaction of thermal initiation, and made it in 60 ℃ of water-baths polyreaction 18 hours.After polyreaction is finished, in polymerizing pipe, obtain material all in one piece, break up this pipe and this material all in one piece is ground into smaller particles, and be 25～75 microns the screening of the irregularly shaped particles of gained.In order to remove 25～75 microns fine particles (＜25 microns) in the part, use acetone this material of sedimentation repeatedly, become transparent up to supernatant liquid.Then with the transfer of granules that obtains in paper sleeve and be placed in the apparatus,Soxhlet's device, successively with 10% Glacial acetic acid methanol solution washing 48 hours, methanol solution washing after 4 hours, vacuum-drying promptly gets non-imprinted polymer.
The EQUINOX 55 types infrared spectra of producing with German Bruker company that Fourier transform infrared spectroscopy-the infrared microscope combined instrument detects as shown in Figure 6 to non-imprinted polymer, with same instrument, same condition the vinealeucoblastine(VLB) Molecularly Imprinted Polymer of embodiment 1 gained is detected again, infrared spectrogram as shown in Figure 7.By Fig. 6 and Fig. 7 as seen, compare with non-imprinted polymer, molecular engram polymer of vinblastine has tangible vinealeucoblastine(VLB) molecule functional group's absorption peak: 3540cm
-1Be the absorption peak of secondary amine, 3000cm
-1Be the absorption peak of proton on two key unsaturated carbons, 1740cm
-1Be the absorption peak of ester carbonyl group, 1480cm
-1Be the skeletal vibration absorption of phenyl ring, 1250cm
-1Be the stretching vibration absorption of carbon-oxygen bond and carbonnitrogen bond, 1000-700cm
-1Absorption for formation vibration outside the face of phenyl ring and amido.
(2) non-imprinted polymer is to the adsorbing mensuration of vinealeucoblastine(VLB)
Employed photometer and working parameter thereof are identical with above-mentioned " adsorption effect detection " experiment in this mensuration process.
Get the prepared non-imprinted polymer 400mg of step () and be filled in the polypropylene shell solid phase extraction column that internal diameter is 1.5cm, with wetting, the balance of toluene 20ml; Vinealeucoblastine(VLB) is made into the solution that concentration is 0.2mg/ml with toluene, gets 1ml and go up sample respectively.Solid phase extraction column behind the last sample with 3ml chloroform drip washing 1 time, is collected leacheate earlier; The extraction pillar is used 10% Glacial acetic acid methanol solution wash-out 2 times again, each 3ml, and flow velocity is 0.5ml/min, collects elutriant.Leacheate, elutriant with collecting carry out absorbance measurement (laboratory apparatus is identical with experiment two with method) with photometer respectively, and chloroform leacheate absorption peak is obvious as a result, and the elutriant of 10% Glacial acetic acid methyl alcohol does not then have absorption peak.
Because used non-imprinted polymer is compared with molecular engram polymer of vinblastine of the present invention in this experiment, the synthetic method of the two is identical with condition, unique difference does not add template molecule---vinealeucoblastine(VLB) when synthetic exactly, therefore according to this experiment and in conjunction with above-mentioned experiment two and three result, provable molecular engram polymer of vinblastine of the present invention has the imprinted sites of template molecule vinealeucoblastine(VLB), and vinealeucoblastine(VLB) is had good identification and adsorptive power.
Claims (5)
1, a kind of molecular engram polymer of vinblastine, this polymkeric substance has the imprinted sites of template molecule vinealeucoblastine(VLB), and it is made 40～80 ℃ of following thermal-initiated polymerization reactions by following molar part monomer component:
1 part of template molecule vinealeucoblastine(VLB), 3～10 parts of function monomer methacrylic acid, methyl methacrylates, 10～50 parts of linking agent ethylene glycol dimethacrylate or trimethylolpropane trimethacrylates.
2, a kind of molecular engram polymer of vinblastine according to claim 1, it is characterized in that making function monomer used in the polyreaction of this polymkeric substance is methacrylic acid.
3, a kind of molecular engram polymer of vinblastine according to claim 1, it is characterized in that making linking agent used in the polyreaction of this polymkeric substance is ethylene glycol dimethacrylate.
4, the preparation method of claim 1,2 or 3 described a kind of molecular engram polymer of vinblastine is made up of following steps:
1) vinealeucoblastine(VLB), function monomer are pressed 1: 3～10 mol ratio mixings in pore-creating agent toluene, acetonitrile or chloroform, press vinealeucoblastine(VLB) again: linking agent: initiator=1: 10～50: 0.9 adds linking agent and initiator Diisopropyl azodicarboxylate, be mixed, deoxygenation, vacuumize, 40～80 ℃ of following thermal-initiated polymerization reactions 10～24 hours, obtain containing the high molecular polymer of vinealeucoblastine(VLB) then;
2) the step 1) resulting polymers grinds to form fine powder, again with acetone repeatedly sedimentation remove fine particle, remove the vinealeucoblastine(VLB) molecule with the washing of 10～40% Glacial acetic acid methyl alcohol, methanol solution successively then, vacuum-drying obtains molecular engram polymer of vinblastine.
5, the preparation method of a kind of molecular engram polymer of vinblastine according to claim 4, the described pore-creating agent of its feature is toluene or acetonitrile.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102432753A (en) * | 2011-09-02 | 2012-05-02 | 陕西科技大学 | Preparation method of epothilone B molecularly imprinted polymer |
| CN102504100A (en) * | 2011-11-04 | 2012-06-20 | 陕西科技大学 | Epothilone D molecular imprinted polymer and preparation method thereof |
| CN103319743A (en) * | 2013-06-03 | 2013-09-25 | 江南大学 | Molecularly imprinted polymer template molecule elution method |
| CN104072664A (en) * | 2014-07-17 | 2014-10-01 | 南方医科大学 | Catharanthus roseus alkaloidal drug carrier and preparation method |
| CN104693354A (en) * | 2015-03-30 | 2015-06-10 | 天津医科大学 | Preparation of molecular imprinting release controlled drug carrier through taking metal and organic gel as pore-foaming agent |
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2006
- 2006-10-30 CN CN200610123060A patent/CN100575399C/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102432753A (en) * | 2011-09-02 | 2012-05-02 | 陕西科技大学 | Preparation method of epothilone B molecularly imprinted polymer |
| CN102504100A (en) * | 2011-11-04 | 2012-06-20 | 陕西科技大学 | Epothilone D molecular imprinted polymer and preparation method thereof |
| CN102504100B (en) * | 2011-11-04 | 2013-12-25 | 陕西科技大学 | Epothilone D molecular imprinted polymer and preparation method thereof |
| CN103319743A (en) * | 2013-06-03 | 2013-09-25 | 江南大学 | Molecularly imprinted polymer template molecule elution method |
| CN104072664A (en) * | 2014-07-17 | 2014-10-01 | 南方医科大学 | Catharanthus roseus alkaloidal drug carrier and preparation method |
| CN104693354A (en) * | 2015-03-30 | 2015-06-10 | 天津医科大学 | Preparation of molecular imprinting release controlled drug carrier through taking metal and organic gel as pore-foaming agent |
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