CN103145528B - Method of preparing high-purity polyprenol lipid by means of both high-speed countercurrent chromatography and high performance liquid chromatography - Google Patents
Method of preparing high-purity polyprenol lipid by means of both high-speed countercurrent chromatography and high performance liquid chromatography Download PDFInfo
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Abstract
The invention discloses a method of preparing high-purity polyprenol lipid by means of both a high-speed countercurrent chromatography and a high performance liquid chromatography. Ginkgo leaves, pine needles and mulberry leaves are used as raw materials, alcohol of anhydrous acetone or anhydrous carbon 1 - carbon 3 is used as extraction solvent, and extraction is carried out through low temperature microwaves or ultrasonic waves to prepare polyprenol paste. Solvents, such as normal hexane, acetonitrile and acetone, are proportionally mixed to form a high-speed countercurrent solvent system, the high-speed countercurrent solvent system is kept still after sufficiently mixed, an upper phase is a fixed phase, a low phase is a flowing phase, the polyprenol paste is dissolved in the lower phase to serve as a product to be tested, the fixed phase is filled in a separation column of a high-speed countercurrent chromatographic instrument, the rotation speed is set as 500-1000 rounds/min, the fixed phase flows to the flowing phase with the flowing speed 1-5mL/min and is detected in an ultraviolet detecting device with wavelength 210-230mm, in the centrifuge process, normal phase centrifugal rotation is adopted for 180-240min at first, then opposite phase centrifugal rotation is adopted for 30-60 min, when the flowing phase starts to flow out of a chromatographic column, distillate is collected, different constituents are collected in a classified mode, and after high performance liquid chromatography (HPLC) detection, the polyprenol lipid with the purity 60-90% is prepared.
Description
One, technical field
The present invention relates to and a kind ofly apply the method for high-speed countercurrent chromatography coupling high performance liquid chromatography from plant leaf extraction and isolation polyprenol lipoid cpd, belong to natural drug and be separated and Medicines and Health Product Application Areas.
Two, background technology
In the world supercritical extraction technique, macroporous resin column chromatography technology, molecular distillation technique, membrane separation technique etc. are adopted mostly for the isolation technique of natural product at present, but the very low and natural product that biological activity is high for content, in the face of social progress and the mankind are to the requirement of high-tech content purified product, extremely lack the separation and purification that can realize high purity substance, the new technology of suitable magnitude preparative capacibility can be realized again.If there is no such new technology, be just difficult to the research and development and the production that realize product of new generation, just can not realize the preparation of high purity reference material and the foundation of high precision quality arbitration and control method.Therefore, in the face of this technical bottleneck of isolation technique, the appearance of high-speed countercurrent chromatography (HSCCC), for the separation and purification of biological active constituents from natural medicines brings more excellent solution with preparation.
High-speed countercurrent chromatography (HSCCC), that one is assigned as ultimate principle with liquid liquid, do not adopt any solid-state supporter (as column packing, sorbent material, affinity agent, plank bed, sieve membrane, Deng), therefore, for the high-purity monomer of some special component in natural product complex mixture separation and purification with preparation in have following advantageous feature: 1. high separating efficiency: be separated in rotary motion and carry out, two-phase solvent is all got rid of into small particle by the centrifuge field of high vibration, the each component of sample can be distributed on the very big surface of two-phase particulate, and effectively can transmit in the vibration of these particles with the environment of convection current.Realize thousands of ground, high-efficiency and continuous solvent extraction processes, reach the isolation and purification of high-resolution.Can by more than effective constituent in sample one step separating-purifying to 98%.2. low use cost: sepn process is not the process of absorption and drip washing, but the process that convection current penetrates, save the expense of expensive filler, save the consumption of solvent; In preparation solvent, major part is aqueous phase, and organic solvent consumption is few; Solvent recuperation recycling can be realized in scale operation.The follow-up input run in using is very low.3. easily technique is amplified: process repeatability 100%.From miniature instrument, technique is groped large-scale instrument amplification and is produced easily realization.4. high-recovery: eliminate supporter shared void volume in post, without impacts such as irreversible adsorption, pollution, sex change, inactivations, sample theoretical recovery 100%.5. clean environmental protection: whole experiment production process is closed and carried out, and avoids solvent evaporates to cause environmental pollution and causes actual bodily harm to operator.
Polyprenol (polyprenols) is extensively present in the plants such as green needles, ginkgo and mulberry leaf, be with C5 isopentene group for the single lipoid cpd of structure, can divide structure: birch alcohol type ω-(trans) 2-(cis) n-OH, luxuriant and rich with fragrance card alcohol type ω-(trans) 3-(cis) n-OH and alltrans Salanesol type ω-(trans) n-OH.As shown in Figure 1.
Polyprenol lipoid is to human non-toxic, without mutagenesis, without teratogenesis with without carcinogenesis, have obvious physiology, pharmacological action.Physiologic Studies shows, the structure and function of polyprenol lipoid cell membrane has regulating effect, can improve the mobility of film, stability and perviousness, thus can the fusion of reinforcing membrane.Exogenous polyprenol lipoid can metabolism be dolichol in vivo, with the deficiency of dolichol in added body, shifts and assists Radiotherapy chemotherapy etc. to have obvious effect, also can be used as the diagnostic reagent of cancer patient to raising immunologic function, liver cell regeneration, anticancer.State's approveds such as Latvia are used for protective foods and produce and drug development, have prepared " ROPREN " medicine series.Separation and purification both at home and abroad for Plant Polyprenols has bibliographical information, and main purification route is, by silica gel column chromatography several times, polyprenol crude extract purity is brought up to more than 90%.In addition, also have some other purification process, as adopted polarizable medium resin (aluminum oxide or polymeric amide etc.) absorption, solvent extraction and crystallization, obtain the polyprenol product that purity is 70% ~ 75%, yield about 1.0%.Purification by silica gel column chromatography is carried out again, preparation purity 92.7% polyprenol after adopting the freezing removing impurities of petroleum ether-ethyl acetate.But still containing yellow pigment such as a large amount of carotenoid in product, Tetsuo Takigawa etc. once adopted a kind of discoloring agent of gac to the decolouring of polyprenol crude extract, but activated carbon dosage is large, and decolorizing effect is poor, and polyprenol loss is large
HSCCC separation efficiency is high, simple to operate, and sample does not need strict pre-treatment; Can one step be separated and obtain highly purified component from extremely complicated Raw samples, can the micromolecular compound such as separating flavone, alkaloid, quinones, lipoid, also can the macromolecular compound such as isolated polypeptide, polysaccharide, protein, circulation ratio is good.Article and patent report is not also had to adopt high-speed countercurrent chromatography to be separated polyprenol at present.
The present invention will be rich in the fresh plant leaf of polyprenol lipoid, pass through soaking at room temperature, or through microwave extraction or ultrasonic extraction, prepare content 5 ~ 20% polyprenol class fat ointment, adopt high-speed countercurrent chromatography again, preparation 60% ~ 90% polyprenol lipoid, for the exploitation of the preparation of high purity polyprenol batch and pharmaceuticals provides technology.
Three, summary of the invention
For achieving the above object, invented a kind of method that high purity polyprenol lipoid is prepared in high speed adverse current chromatogram and high performance liquid chromatography coupling, be made up of following steps:
The first step, polyprenol lipoid extracts
The fresh plant leaf of polyprenol lipoid will be rich in, room temperature is dried in the shade, be crushed to the following powder of 100 order, add any one solvent in anhydrous propanone or C1 ~ C3 alcohol, leaf and solvent are 1: 8 ~ 30 in mass ratio, preferably 1: 8 ~ 15, DEG C to soak 10 ~ 72 hours in room temperature ~ 80, as 25 ~ 50 DEG C, quiet bubble 30 ~ 50 hours; Or through microwave extraction or ultrasonic extraction 10 ~ 60 minutes, extract power 100W ~ 1000W, twice, united extraction liquid, vacuum reclaims organic solvent, and enriched material is polyprenol class fat ointment, ointment relative to plant leaf powder yield 4 ~ 10%, polyprenol lipid levels 5 ~ 20%;
Second step, high speed adverse current chromatogram is separated
1) normal hexane is selected by volume: acetonitrile: acetone=3 ~ 4: 0.5 ~ 1: 0.8 ~ 1.5 solution are as high-speed counter-current extraction solvent system, three kinds of solvent leave standstill fully afterwards, and by upper and lower two-phase separately, getting is stationary phase mutually, lower is moving phase mutually, ultrasonic degas;
2) get polyprenol class fat ointment be dissolved in lower mutually in as test sample, the ointment of described polyprenol is 1: 20 ~ 80 with the mass volume ratio (g/mL) of lower phase;
3) stationary phase is full of the multilayer coil separator column of high-speed counter-current chromatograph, high-speed counter-current chromatograph separation condition is under 500 ~ 1000r/min rotating speed, moving phase is injected with the flow velocity of 1 ~ 5mL/min, detect in the UV-detector of wavelength 210 ~ 230nm, when obviously there being moving phase to flow out, start test sample solution sample introduction, whole centrifugal process first adopts positive centrifugal rotation, time is 180 ~ 240min, adopt anti-phase centrifugal rotation again, time is 30 ~ 60min, collects distillate with automatic fraction collector;
3rd step, high performance liquid chromatography detects
Second step automatic fraction collector is collected distillate, carry out detection polyprenol lipoid position collection with high performance liquid chromatography to divide, merge polyprenol lipoid collection and divide, reclaim organic solvent, enriched material is yellow polyprenol lipoid oily matter, polyprenol lipid levels 60% ~ 90%.
The Fresh leaves of this patent collection is any one in Ginkgo Leaf, needle and mulberry leaf, the Fresh leaves that Ginkgo Leaf gathers during being August to December, the Ginkgo Leaf of 3 ~ 10 years age of trees gathered 8 ~ October, within more than 10 years, age of tree Ginkgo Leaf gathered in 10 ~ December, was 15 ~ 23 polyprenol lipoids containing isopentene group unit number.Pine needle be February ~ August during from Pinus massoniana Lamb, slash pine, black pine, cdear, seashore pine and Chinese pine any one gather Fresh leaves, being rich in isopentene group unit number is 13 ~ 19 polyprenol lipoids.Mulberry leaf are the Fresh leaves gathered period 10 months Augusts, are 10 ~ 12 polyprenol lipoids containing isopentene group unit number.
Traditional extraction process adopts sherwood oil, normal hexane, chlorine alkane, ethyl acetate, acetone etc. to extract Plant Polyprenols lipoid cpd as the extraction agent of nonpolar or small and weak polarity, extraction yield general 3 ~ 6%, these public affairs profit absolute alcohol solvent that employing polarity is larger is first as extraction agent, if disclosed C1 ~ C3 ethanol-extracted agent is any one in methyl alcohol, ethanol, n-propyl alcohol, adopt anhydrous propanone as extraction agent simultaneously, expand polyprenol and extract polarity, also significantly improve polyprenol extraction yield and yield.This patent is disclosed in room temperature ~ 80 DEG C and soaks 10 ~ 72 hours, as 25 ~ 50 DEG C, and quiet bubble 30 ~ 50 hours; Or through microwave extraction or ultrasonic extraction 10 ~ 60 minutes, extract power 100W ~ 1000W, twice, united extraction liquid, vacuum reclaims organic solvent, and enriched material is polyprenol class fat ointment, ointment is relative to plant leaf powder yield 4 ~ 10%, polyprenol lipid levels 5 ~ 20%, relative with traditional method, extraction effect significantly improves.
Polyprenol crude extract purity is brought up to more than 90% by repeatedly silica gel column chromatography by traditional separation method, and not only the time is long, and solvent and silica gel many, especially silica gel can not regeneration, and cost is high; Adopt polarizable medium resin (aluminum oxide or polymeric amide etc.) absorption, solvent extraction and crystallization, the polyprenol product that purity is 70% ~ 75% can only be obtained, the requirement to polyprenol high-content (> 90%) in new drug development can not be met.Compare traditional separation method, this patent adopts high speed adverse current chromatogram to prepare high purity polyprenol lipoid first from the polyprenol class fat ointment that Ginkgo Leaf, pine needle and mulberry leaf extract, especially whole centrifugal process adopts positive centrifugal rotation and anti-phase centrifugal rotation to combine method first, whole process time is 210 ~ 300min, can prepare in batches, the sample size often criticizing polyprenol class fat ointment is 10 ~ 500mg.This patent single stage method, step is few, simple possible, and the polyprenol purity of preparation significantly improves, and can meet the development requirement of new drug.
This patent adopts high speed adverse current chromatogram and high performance liquid chromatography coupling to prepare the method for high purity polyprenol lipoid first, high performance liquid chromatography detect parameters ODS C18column
the granularity of filler 2.5 ~ 10 μm, column temperature 20 ~ 30 DEG C, moving phase: methyl alcohol: Virahol: normal hexane: water=0.18: 0.32: 0.04: 0.01v/v, flow velocity: 0.6 ~ 1.2mL/min
-1, determined wavelength: 210nm.Yellow polyprenol lipoid oily matter prepared by high speed adverse current chromatogram, through efficient liquid phase chromatographic analysis, polyprenol lipid levels 60% ~ 90%.
Beneficial effect of the present invention shows as:
1., for traditional extraction process, adopt absolute alcohol as extraction agent first, adopt microwave extraction or ultrasonic extraction polyprenol lipoid, not only polyprenol extraction yield significantly improves, and alcohol is more safer than the non-polar solvent such as sherwood oil, normal hexane, in leaching process, loss is less, and cost is low.
2. adopt positive centrifugal rotation and anti-phase centrifugal rotation to combine method first, adopt high speed adverse current chromatogram to prepare high purity polyprenol lipoid first from the polyprenol class fat ointment that Ginkgo Leaf, pine needle and mulberry leaf extract.
3. adopt high speed adverse current chromatogram and high performance liquid chromatography coupling to prepare high purity polyprenol lipoid, polyprenol lipid levels 60% ~ 90% first.
Accompanying drawing explanation
Fig. 1 polyprenol lipid structure
The high speed adverse current chromatogram figure of Fig. 2 standard substance
The high speed adverse current chromatogram of Fig. 3 standard substance is separated rear HPLC
The high speed adverse current chromatogram figure of Fig. 4 polyprenol lipoid
The HPLC after high speed adverse current chromatogram is separated of Fig. 5 polyprenol lipoid
Five, embodiment
Following examples are citings more of the present invention, should not be seen as limitation of the invention.
Embodiment 1 high speed adverse current chromatogram standard model is separated
1. laboratory apparatus:
FastChrome analysis mode high-speed counter-current chromatograph (Jiangyin Countercurrent Technology Co., Ltd.); Separator column volume: 25mL; Value scope: 0.56 ~ 0.91; The semi-preparative high-speed counter-current chromatograph of OptiChromeA (Jiangyin Countercurrent Technology Co., Ltd.); Spiral tube separator column (revolution-radius: 80cm; Value scope: 0.50 ~ 0.80; Spiral tube internal diameter: 1.59mm), single-column volume 180mL, twin columns volume 360mL.
2. experiment material:
Analytical pure isomers phenols sample pyrocatechol, Resorcinol, Resorcinol; Normal hexane, ethyl acetate, methyl alcohol, distilled water
3. prepare solvent and sample:
The methanol mixed solution of preparation phenols sample, biased sample concentration: Catechol 2 0mg/mL+ Resorcinol 20mg/mL+ Resorcinol 10mg/mL; With 80% methanol solution for solvent, prepare 200mg/mL pyrocatechol solution, 200mg/mL resorcinol solution and 100mg/mL quinol solution respectively, after fully dissolving, respectively get 1mL, add 80% methanol solvate of 7mL, mixing, makes biased sample to be separated.
Solvent system: normal hexane: ethyl acetate: methyl alcohol: water=3: 5: 1: 5.By 300mL normal hexane, 500mL ethyl acetate, 100mL methyl alcohol, fully mix with 500mL distilled water after, separating funnel phase-splitting.
4. experiment parameter:
Pyrocatechol, Resorcinol are separated with the mixture of Resorcinol, and separation condition is: solvent system: n-hexane-ethyl acetate-methanol-water (3: 5: 1: 5); Stationary phase: upper phase; Moving phase: lower phase.
Analysis mode instrument separation condition: column volume: 34mL; Flow velocity: 1mL/min; Room temperature: 22 DEG C; Rotating speed: 1800rpm; Determined wavelength: 254nm.Sampling volume: 1mL; Sample introduction concentration: Catechol 2 mg/mL+ Resorcinol 2mg/mL+ Resorcinol 1mg/mL (biased sample 1: 10 is dissolved in moving phase)
Preparative instrument separation condition: twin columns are separated, column volume 360mL; Flow velocity: 5mL/min; Room temperature: 22 DEG C; Rotating speed: 1200rpm; Determined wavelength: 254nm, sampling volume: 20mL; Sample concentration: Catechol 2 mg/mL+ Resorcinol 2mg/mL+ Resorcinol 1mg/mL (biased sample 1: 10 is dissolved in moving phase).
5. experimental procedure:
5.1. stationary phase injection: open constant flow pump, injects separator column by stationary phase by entrance with Peak Flow Rate, is full of separator column pipeline, after flowing out, continues pump termination of pumping afterwards in 1 minute to exit end.Defining enough stationary phase for filling separator column (analytical column should prepare more than 50mL, preparative column should more than 400mL), in this process, carefully not entering air or moving phase.
5.2. connect external power, the power switch in the instrument postnotum lower left corner of closing, arranges centrifugal rotation parameter; Constant flow pump is set and is separated flow velocity (analysis mode 1mL/min, preparative 5mL/min)
5.3. system balancing: confirm that parameter of noncentricity is correct, and filled liquid (stationary phase) in separator column.The filter head of constant flow pump import is inserted moving phase.Start constant flow pump according to preset flow rate, press " Run " button green above centrifugal control panel simultaneously, start centrifugal.
5.4. solvent systems balance, the outflow solution namely exported be all limpid moving phase, or on-line detector signal steadily after, record the withdrawal volume of stationary phase, to calculate stationary phase retention rate;
Sf=(stationary phase volume of pipeline cumulative volume-discharge)/separator column volume * 100%
This experiment, with the full stationary phase of 50mL/min speed pump, pumps into moving phase with 5mL/min speed, starts OptiChrome
tMa-300 forward is centrifugal, observes UV3000 output and reaches system balancing, calculate stationary phase retention rate 65%.
5.5. sample preparation: get 50mg/mL phenols sample mix liquid (Catechol 2 0mg/mL+ Resorcinol 20mg/mL+ Resorcinol 10mg/mL) and be diluted in the moving phase of solvent system with 1: 10 (analysis mode: 0.1mL is diluted in 0.9mL moving phase; Preparative: 2mL is diluted in 18mL moving phase), mixing;
5.6. loading: the loading valve of corresponding separator column is threaded to " loading " position, syringe is inserted the sampling aperture as being connected with syringe adapter, observe loading ring and sample feeding pipe, inject emitter to discharge the air in sample feeding pipe, make in sample feeding pipe, to be full of the moving phase flowed out from loading ring.Then sample feeding pipe is inserted the sample tube (attention is inserted into bottom and avoids air inlet to steep) prepared, sample is sucked loading ring by aspirating syringe.Sample can be filled loading ring, also can partly be full of loading ring, but note having air to enter loading ring.Then by the vertical zero position of loading valve cycle, sample enters separator column by with the moving phase pumped into.
5.7. " data gathering " in chromatographic working station is clicked, the detach Spline of record biased sample.
5.8., at the end of being separated, stopping centrifugal by " stopping " button on centrifugal control panel, and constant flow pump is stopped.Click " stopping gathering " key in chromatographic working station, and preserve collection of illustrative plates.
5.9. the component with remaining solution and reservation in nitrogen or pressurized air blowout separator column can be selected; Or the component of solution in post and reservation is released at a high speed by selection stationary phase.
5.10. pipeline-cleaning: the methyl alcohol pumping into about 1/3 column volume with constant flow pump, then blows out with nitrogen; In triplicate.Last cleaning also, after blowing out washed with methanol liquid, while continuing to blow, presses " turning to " key on centrifugal control panel, display screen " running " item will show " reverse centrifugal stopping ", then rotating speed is set to 400rpm, starts centrifugal, be conducive to emptying of residual solvents in pipeline.
6. separating resulting:
Pyrocatechol, Resorcinol and Resorcinol achieve good separation, as Fig. 2 and Fig. 3.
Embodiment 2 high speed adverse current chromatogram and high performance liquid chromatography method for combined use
The first step, polyprenol lipoid extracts
The fresh plant leaf of polyprenol lipoid will be rich in, room temperature is dried in the shade, be crushed to the following powder of 100 order, add any one solvent in anhydrous propanone or C1 ~ C3 alcohol, preferred anhydrous propanone or ethanol, leaf powder and solvent are 1: 8 ~ 30 in mass ratio, preferably 1: 8 ~ 15, DEG C immersion 10 ~ 72 hours in room temperature ~ 80, as 25 ~ 50 DEG C, quiet bubble 30 ~ 50 hours; Or through microwave extraction or ultrasonic extraction 10 ~ 60 minutes, extract power 100W ~ 1000W, twice, united extraction liquid, vacuum reclaims organic solvent, and enriched material is polyprenol class fat ointment, ointment relative to plant leaf powder yield 4 ~ 10%, polyprenol lipid levels 5 ~ 20%;
Second step, high speed adverse current chromatogram is separated
(1) normal hexane is selected by volume: acetonitrile: acetone=3 ~ 4: 0.5 ~ 1: 0.8 ~ 1.5 solution are as high-speed counter-current extraction solvent system, three kinds of solvent leave standstill fully afterwards, and by upper and lower two-phase separately, getting is stationary phase mutually, lower is moving phase mutually, ultrasonic degas;
(2) get polyprenol class fat ointment be dissolved in lower mutually in as test sample, the ointment of described polyprenol is 1: 20 ~ 80 with the mass volume ratio (g/mL) of lower phase, preferably 1: 30 ~ 60;
(3) stationary phase is full of the multilayer coil separator column of high-speed counter-current chromatograph, high-speed counter-current chromatograph separation condition is under 500 ~ 1000r/min rotating speed, moving phase is injected with the flow velocity of 1 ~ 5mL/min, detect in the UV-detector of wavelength 210 ~ 230nm, when obviously there being moving phase to flow out, start test sample solution sample introduction, whole centrifugal process first adopts positive centrifugal rotation, time is 180 ~ 240min, adopt anti-phase centrifugal rotation again, time is 30 ~ 60min, collects distillate with automatic fraction collector;
3rd step, high performance liquid chromatography detects
Second step automatic fraction collector is collected distillate, carry out detection polyprenol lipoid position collection with high performance liquid chromatography to divide, merge polyprenol lipoid collection and divide, reclaim organic solvent, enriched material is yellow polyprenol lipoid oily matter, polyprenol lipid levels 60% ~ 90%.
In the present embodiment, fresh plant leaf is any one in Ginkgo Leaf, pine needle and mulberry leaf, the Fresh leaves that Ginkgo Leaf gathers during being August to December, the Ginkgo Leaf of 3 ~ 10 years age of trees gathered 8 ~ October, within more than 10 years, age of tree Ginkgo Leaf gathered in 10 ~ December, was 15 ~ 23 polyprenol lipoids containing isopentene group unit number; Pine needle be February ~ August during from Pinus massoniana Lamb, slash pine, black pine, cdear, seashore pine and Chinese pine any one gather Fresh leaves, being rich in isopentene group unit number is 13 ~ 19 polyprenol lipoids, mulberry leaf are the Fresh leaves gathered period 10 months Augusts, are 10 ~ 12 polyprenol lipoids containing isopentene group unit number.
In the present embodiment, plant material is separated with high speed adverse current chromatogram through anhydrous propanone or ethanol microwave extraction, whole centrifugal process adopts positive centrifugal rotation and anti-phase centrifugal rotation to combine method, whole process time is 210 ~ 300min, can prepare in batches, the sample size often criticizing polyprenol class fat ointment is 10 ~ 500mg.
The present embodiment detects high speed adverse current chromatogram with high performance liquid chromatography and is separated polyprenol lipid extraction position and content thereof, and high performance liquid chromatography detect parameters is that chromatographic column adopts ODS C18column
the granularity of filler 2.5 ~ 10 μm, column temperature 20 ~ 30 DEG C, moving phase: methyl alcohol: Virahol: normal hexane: water=0.18: 0.32: 0.04: 0.01v/v, flow velocity: 0.6 ~ 1.2mL/min
-1, determined wavelength: 210nm.Analyze through HPLC, the polyprenol lipid levels 60% ~ 90% that high speed adverse current chromatogram is separated.
Embodiment 3 high speed adverse current chromatogram polyprenol separating experiment
1. solvent prepares: normal hexane: acetonitrile: acetone=3: by volume prepare 2000mL at 1: 1, above is stationary phase mutually, is moving phase below mutually.
2. preparation of samples: polyprenol class fat ointment 50mg in Example 1, is dissolved in moving phase 50mL.
3. system balancing: with the full stationary phase of 50mL/min speed pump, pump into moving phase with 5mL/min speed, start
4.OptiChrome
tMa-300 forward is centrifugal, observes UV3000 output and reaches system balancing, calculate stationary phase retention rate: 60%
5. loading: sample is injected OptiChrome by loading valve
tMin A-300.Forward is centrifugal: by OptiChrome
tMa-300 is set as forward centrifugal rotation, setting.
6. oppositely centrifugal: to be 5mL/min by separation flow velocity, centrifugal through the forward of 180 minutes.OptiChrome
tMa-300 is set as reverse centrifugal rotation, and it is 10mL/min that setting is separated flow velocity, oppositely centrifugal through 30 minutes.
7., in above-mentioned steps (5) and (6), sample after testing device obtains collection of illustrative plates, as accompanying drawing 4.
8.HPLC analyzes: get Fig. 3 detector and absorb maximum isolated liquid and be placed in HPLC and detect, contrasted, obtain polyprenol lipoid collection of illustrative plates by the collection of illustrative plates of the polyprenol sample with standard.As the HPLC collection of illustrative plates of accompanying drawing 5. from Fig. 4 can be seen clearly, OptiChrome
tMa-300 successfully by part mixture separation in polyprenol mixture out.
Embodiment 4 high speed adverse current chromatogram prepares Polyprenols From Ginkgo Biloba L lipoid
1. material collection, the Fresh leaves that Ginkgo Leaf gathers during being 8 ~ December, wherein the Ginkgo Leaf of 3 ~ 10 years age of trees gathered 8 ~ October, and within more than 10 years, age of tree Ginkgo Leaf gathered in 10 ~ December, and room temperature is dried in the shade, and is crushed to the following powder of 100 order, for subsequent use.
2. polyprenol lipoid extracts: get Ginkgo Leaf 1kg, add anhydrous propanone, leaf and solvent are 1: 25 in mass ratio, soaking at room temperature 60 hours, repeat extraction twice, united extraction liquid, vacuum reclaims organic solvent, enriched material is polyprenol class fat ointment, and 5g, HPLC analyse purity is altogether 8%;
3. getting 100mg adopts high-speed counter-current chromatograph to be separated, get volume ratio be 3: 0.5: 0.8 normal hexane, acetonitrile and acetone is as high-speed counter-current solvent system, leave standstill after mixing fully, by upper and lower two-phase separately, getting is stationary phase mutually, and lower is moving phase mutually, ultrasonic degas, get polyprenol class fat ointment be dissolved in lower mutually in as trial-product, the ointment of described polyprenol is 1g: 50mL with the mass volume ratio of lower phase; Stationary phase is full of the multilayer coil separator column of high-speed counter-current chromatograph, setting high-speed counter current chromatograph, under 800r/min rotating speed, moving phase is injected with the flow velocity of 2mL/min, detect in the UV-detector of wavelength 210nm, when obviously there being moving phase to flow out, start to get trial-product as sample solution sample introduction, start to collect distillate with automatic fraction collector simultaneously, whole centrifugal process first adopts positive centrifugal rotation, time is 180 ~ 240min, then adopts anti-phase centrifugal rotation, and the time is 30 ~ 60min.Collecting the distillate obtained adopts rotary evaporation to remove solvent, obtains sample 29.8mg.
4. high performance liquid chromatography detects, and Polyprenols From Ginkgo Biloba L lipoid purity is 75%, is 15 ~ 23 containing isopentene group unit number.
Embodiment 5 high speed adverse current chromatogram prepares needle polyprenol lipoid
1. material collection, pine needle be February ~ August during from Pinus massoniana Lamb, slash pine, black pine, cdear, seashore pine and Chinese pine any one gather Fresh leaves, room temperature is dried in the shade, and is crushed to the following powder of 100 order, for subsequent use.
2. polyprenol lipoid extracts: get pine needle powder 1kg, add dehydrated alcohol, leaf and solvent are 1: 20 in mass ratio, ultrasonic extraction 50 minutes, repeat extraction twice, united extraction liquid, vacuum reclaims organic solvent, enriched material is polyprenol class fat ointment, and 5.8g, HPLC analyse purity is altogether 15%;
3. get 100mg polyprenol class fat ointment, high-speed counter-current chromatograph is adopted to be separated, get volume ratio be 4: 1: 1.5 normal hexane, acetonitrile and acetone is as high-speed counter-current solvent system, leave standstill after mixing fully, by upper and lower two-phase separately, getting is stationary phase mutually, lower is moving phase mutually, ultrasonic degas, get polyprenol class fat ointment be dissolved in lower mutually in as trial-product, the ointment of described polyprenol is 1g: 50mL with the mass volume ratio of lower phase; Stationary phase is full of the multilayer coil separator column of high-speed counter-current chromatograph, setting high-speed counter current chromatograph, under 1000r/min rotating speed, moving phase is injected with the flow velocity of 2mL/min, detect in the UV-detector of wavelength 210nm, when obviously there being moving phase to flow out, start to get trial-product as sample solution sample introduction, start to collect distillate with automatic fraction collector simultaneously, whole centrifugal process first adopts positive centrifugal rotation, time is 180 ~ 240min, then adopts anti-phase centrifugal rotation, and the time is 30 ~ 60min.Collecting the distillate obtained adopts rotary evaporation to remove solvent, obtains sample 20.5mg.
4. high performance liquid chromatography detects, and needle polyprenol purity is 86%, is 13 ~ 19 containing isopentene group unit number.
Embodiment 6 high speed adverse current chromatogram prepares mulberry leaf polyprenol lipoid
1. material collection, mulberry leaf are the Fresh leaves gathered period 10 months Augusts, and room temperature is dried in the shade, and is crushed to the following powder of 100 order, for subsequent use.
2. polyprenol lipoid extracts: get mulberry leaves powder 1kg, add anhydrous methanol, leaf and solvent are 1: 15 in mass ratio, 50 DEG C of microwave extraction 60 minutes, repeat extraction twice, united extraction liquid, vacuum reclaims organic solvent, enriched material is polyprenol class fat ointment, and 3.8g, HPLC analyse purity is altogether 18%;
3. getting 500mg adopts high-speed counter-current chromatograph to be separated, get volume ratio be 4: 1: 1.5 normal hexane, acetonitrile and acetone is as high-speed counter-current solvent system, leave standstill after mixing fully, by upper and lower two-phase separately, getting is stationary phase mutually, and lower is moving phase mutually, ultrasonic degas, get polyprenol class fat ointment be dissolved in lower mutually in as trial-product, the ointment of described polyprenol is 1g: 50mL with the mass volume ratio of lower phase; Stationary phase is full of the multilayer coil separator column of high-speed counter-current chromatograph, setting high-speed counter current chromatograph, under 1000r/min rotating speed, moving phase is injected with the flow velocity of 5mL/min, detect in the UV-detector of wavelength 210nm, when obviously there being moving phase to flow out, start to get trial-product as sample solution sample introduction, start to collect distillate with automatic fraction collector simultaneously, whole centrifugal process first adopts positive centrifugal rotation, time is 180 ~ 240min, then adopts anti-phase centrifugal rotation, and the time is 30 ~ 60min.Collecting the distillate obtained adopts rotary evaporation to remove solvent, obtains sample 172.3mg.
4. high performance liquid chromatography detects, and mulberry leaf polyprenol lipoid purity is 73%, is 10 ~ 12 containing isopentene group unit number.
Embodiment 7 high speed adverse current chromatogram prepares Polyprenols From Ginkgo Biloba L lipoid
1. material collection, the Fresh leaves that Ginkgo Leaf gathers during being August to December, wherein the Ginkgo Leaf of 3 ~ 10 years age of trees gathered 8 ~ October, and within more than 10 years, age of tree Ginkgo Leaf gathered in 10 ~ December, and room temperature is dried in the shade, and is crushed to the following powder of 100 order, for subsequent use.
2. polyprenol lipoid extracts: get Ginkgo Leaf 1kg, add anhydrous methanol, leaf and solvent are 1: 10 in mass ratio, 60 DEG C of microwave extraction 60 minutes, repeat extraction twice, united extraction liquid, vacuum reclaims organic solvent, enriched material is polyprenol class fat ointment, and 4.5g, HPLC analyse purity is altogether 18%;
3. getting 100mg adopts high-speed counter-current chromatograph to be separated, get volume ratio be 4: 1: 1.5 normal hexane, acetonitrile and acetone is as high-speed counter-current solvent system, leave standstill after mixing fully, by upper and lower two-phase separately, getting is stationary phase mutually, and lower is moving phase mutually, ultrasonic degas, get polyprenol class fat ointment be dissolved in lower mutually in as trial-product, the ointment of described polyprenol is 1g: 50mL with the mass volume ratio of lower phase; Stationary phase is full of the multilayer coil separator column of high-speed counter-current chromatograph, setting high-speed counter current chromatograph, under 1000r/min rotating speed, moving phase is injected with the flow velocity of 1mL/min, detect in the UV-detector of wavelength 210nm, when obviously there being moving phase to flow out, start to get trial-product as sample solution sample introduction, start to collect distillate with automatic fraction collector simultaneously, whole centrifugal process first adopts positive centrifugal rotation, time is 180 ~ 240min, then adopts anti-phase centrifugal rotation, and the time is 30 ~ 60min.Collecting the distillate obtained adopts rotary evaporation to remove solvent, obtains sample 37mg.
4. high performance liquid chromatography detects, and Polyprenols From Ginkgo Biloba L lipoid is 90%, is 15 ~ 23 containing isopentene group unit number.
Embodiment 8 high speed adverse current chromatogram prepares needle polyprenol lipoid
1. pine needle be February ~ August during from Pinus massoniana Lamb, black pine and cdear any one gather Fresh leaves, room temperature is dried in the shade, and is crushed to the following powder of 100 order, polyprenol lipid levels 1.75%.
2. polyprenol lipoid extracts: get pine needle powder 1kg, add anhydrous propanone, leaf and solvent are 1: 25 in mass ratio, soaking at room temperature 60 hours, repeat extraction twice, united extraction liquid, vacuum reclaims organic solvent, enriched material is polyprenol class fat ointment, and 5.8g, HPLC analyse purity is altogether 25%;
3. getting 300mg adopts high-speed counter-current chromatograph to be separated, get volume ratio be 3: 1: 1.5 normal hexane, acetonitrile and acetone is as high-speed counter-current solvent system, leave standstill after mixing fully, by upper and lower two-phase separately, getting is stationary phase mutually, and lower is moving phase mutually, ultrasonic degas, get polyprenol class fat ointment be dissolved in lower mutually in as trial-product, the ointment of described polyprenol is 1g: 50mL with the mass volume ratio of lower phase; Stationary phase is full of the multilayer coil separator column of high-speed counter-current chromatograph, setting high-speed counter current chromatograph, under 600r/min rotating speed, moving phase is injected with the flow velocity of 5mL/min, detect in the UV-detector of wavelength 230nm, when obviously there being moving phase to flow out, start to get trial-product as sample solution sample introduction, start to collect distillate with automatic fraction collector simultaneously, whole centrifugal process first adopts positive centrifugal rotation, time is 180 ~ 240min, then adopts anti-phase centrifugal rotation, and the time is 30 ~ 60min.Collecting the distillate obtained adopts rotary evaporation to remove solvent, obtains sample 108.75mg.
4. high performance liquid chromatography detects, and needle polyprenol lipoid purity is 86%, is 13 ~ 19 containing isopentene group unit number.
Claims (7)
1. a method for high purity polyprenol lipoid is prepared in high speed adverse current chromatogram and high performance liquid chromatography coupling, it is characterized in that being made up of following steps:
The first step, polyprenol lipoid extracts
The fresh plant leaf of polyprenol lipoid will be rich in, room temperature is dried in the shade, and is crushed to the following powder of 100 order, adds any one solvent in anhydrous propanone or C1 ~ C3 alcohol, leaf and solvent are 1: 8 ~ 30 in mass ratio, soak 10 ~ 72 hours in room temperature ~ 80 DEG C, or through microwave extraction or ultrasonic extraction 10 ~ 60 minutes, extract twice, united extraction liquid, vacuum reclaims organic solvent, and enriched material is polyprenol class fat ointment, polyprenol lipid levels 5 ~ 20%;
Second step, high speed adverse current chromatogram is separated
1) normal hexane is selected by volume: acetonitrile: acetone=3 ~ 4: 0.5 ~ 1: 0.8 ~ 1.5 solution are as high-speed counter-current extraction solvent system, three kinds of solvent leave standstill fully afterwards, and by upper and lower two-phase separately, getting is stationary phase mutually, lower is moving phase mutually, ultrasonic degas;
2) get polyprenol class fat ointment be dissolved in lower mutually in as test sample, the ointment of described polyprenol is 1g: 20mL ~ 80mL with the mass volume ratio of lower phase;
3) stationary phase is full of the multilayer coil separator column of high-speed counter-current chromatograph, high-speed counter-current chromatograph separation condition is under 500 ~ 1000r/min rotating speed, moving phase is injected with the flow velocity of 1 ~ 5mL/min, detect in the UV-detector of wavelength 210 ~ 230nm, when obviously there being moving phase to flow out, start test sample solution sample introduction, whole centrifugal process first adopts positive centrifugal rotation, time is 180 ~ 240min, adopt anti-phase centrifugal rotation again, time is 30 ~ 60min, collects distillate with automatic fraction collector; 3rd step, high performance liquid chromatography detects
Second step automatic fraction collector is collected distillate, carry out detection polyprenol lipoid position collection with high performance liquid chromatography to divide, merge polyprenol lipoid collection and divide, reclaim organic solvent, enriched material is yellow polyprenol lipoid oily matter, polyprenol lipid levels 60% ~ 90%.
2. the method for high purity polyprenol lipoid is prepared in a kind of high speed adverse current chromatogram and high performance liquid chromatography coupling according to claim 1, it is characterized in that the first step polyprenol lipoid extract in fresh plant leaf be any one in Ginkgo Leaf, pine needle and mulberry leaf.
3. the method for high purity polyprenol lipoid is prepared in a kind of high speed adverse current chromatogram and high performance liquid chromatography coupling according to claim 1, it is characterized in that in the extraction of the first step polyprenol lipoid, C1 ~ C3 alcohol is any one in methyl alcohol, ethanol, n-propyl alcohol.
4. the method for high purity polyprenol lipoid is prepared in a kind of high speed adverse current chromatogram and high performance liquid chromatography coupling according to claim 1, it is characterized in that the sample size of polyprenol class fat ointment in the separation of second step high speed adverse current chromatogram is 10 ~ 500mg.
5. the method for high purity polyprenol lipoid is prepared in a kind of high speed adverse current chromatogram and high performance liquid chromatography coupling according to claim 1, it is characterized in that the 3rd step high performance liquid chromatography detect parameters adopts chromatographic column
for the ODSC18column of 4.6mm × 250mm, the granularity of filler 2.5 ~ 10 μm, column temperature 20 ~ 30 DEG C, moving phase: methyl alcohol: Virahol: normal hexane: water=0.18: 0.32: 0.04: 0.01v/v, flow velocity: 0.6 ~ 1.2mL/min
-1, determined wavelength: 210nm.
6. the method for high purity polyprenol lipoid is prepared in a kind of high speed adverse current chromatogram according to claim 2 and high performance liquid chromatography coupling, it is characterized in that the Fresh leaves gathered during Ginkgo Leaf is August to December, the Ginkgo Leaf of silver 3 ~ 10 years age of trees gathered 8 ~ October, within more than 10 years, age of tree Ginkgo Leaf gathered in 10 ~ December, was 15 ~ 23 polyprenol lipoids containing isopentene group unit number.
7. the method for high purity polyprenol lipoid is prepared in a kind of high speed adverse current chromatogram according to claim 2 and high performance liquid chromatography coupling, it is characterized in that pine needle be February ~ August during from Pinus massoniana Lamb, slash pine, black pine, cdear, seashore pine and Chinese pine any one gather Fresh leaves, being rich in isopentene group unit number is 13 ~ 19 polyprenol lipoids.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20030083664A (en) * | 2003-10-08 | 2003-10-30 | (주)바이오플란텍 | Extracting method of polyprenol and cultivating method of plant with it |
| CN101654398A (en) * | 2009-09-09 | 2010-02-24 | 中国林业科学研究院林产化学工业研究所 | Method for extracting high purity polyprenol from plant needle leaf raw material |
| CN101967083A (en) * | 2010-09-20 | 2011-02-09 | 中国科学院山西煤炭化学研究所 | Method for separating and refining polyprenol in ginkgo biloba extract |
| CN102219645A (en) * | 2011-04-29 | 2011-10-19 | 浙江省林业科学研究院 | Ultrasonic countercurrent extraction method of polyprenol |
| CN102649803A (en) * | 2011-02-25 | 2012-08-29 | 苏州宝泽堂医药科技有限公司 | Preparation process for extracting and separating high-purity Cyasterone |
| CN102771496A (en) * | 2012-07-26 | 2012-11-14 | 中国林业科学研究院林产化学工业研究所 | Plant growth regulator composition containing polyprenols and preparation method thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS58201736A (en) * | 1982-05-19 | 1983-11-24 | Kuraray Co Ltd | Preparation of polyprenol |
-
2013
- 2013-01-24 CN CN201310028136.9A patent/CN103145528B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20030083664A (en) * | 2003-10-08 | 2003-10-30 | (주)바이오플란텍 | Extracting method of polyprenol and cultivating method of plant with it |
| CN101654398A (en) * | 2009-09-09 | 2010-02-24 | 中国林业科学研究院林产化学工业研究所 | Method for extracting high purity polyprenol from plant needle leaf raw material |
| CN101967083A (en) * | 2010-09-20 | 2011-02-09 | 中国科学院山西煤炭化学研究所 | Method for separating and refining polyprenol in ginkgo biloba extract |
| CN102649803A (en) * | 2011-02-25 | 2012-08-29 | 苏州宝泽堂医药科技有限公司 | Preparation process for extracting and separating high-purity Cyasterone |
| CN102219645A (en) * | 2011-04-29 | 2011-10-19 | 浙江省林业科学研究院 | Ultrasonic countercurrent extraction method of polyprenol |
| CN102771496A (en) * | 2012-07-26 | 2012-11-14 | 中国林业科学研究院林产化学工业研究所 | Plant growth regulator composition containing polyprenols and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| 王成章,等.五种针叶中聚戊烯醇的化学结构.《林产化学与工业》.1994,第14卷(第3期), * |
| 王成章,等.银杏叶中聚戊烯醇含量及其季节性变化.《植物资源与环境学报》.2000,第9卷(第2期), * |
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