CN103145528A - 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 a kind of high-speed countercurrent chromatography coupling high performance liquid chromatography of using and extract the method for separating the polyprenol lipoid cpd from plant leaf, belong to natural drug and separate and the Medicines and Health Product Application Areas.
Two, background technology
At present mostly adopt supercritical extraction technique, macroporous resin column chromatography technology, molecular distillation technique, membrane separation technique etc. for the isolation technique of natural product in the world, but the natural product very low for content and biological activity is high, face social progress and the mankind to the requirement of high-tech content purified product, extremely lack the separation and purification that can realize high purity substance, can realize again the new technology of suitable magnitude preparative capacibility.If there is no such new technology, just be difficult to realize research and development and the production of 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) is for separation and purification and the preparation of biological active constituents from natural medicines brought more excellent solution.
high-speed countercurrent chromatography (HSCCC), a kind ofly to be 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 following advantageous feature is arranged in the separation and purification of the high-purity monomer of some special component in the natural product complex mixture and preparation: 1. high separating efficiency: be separated in and carry out in rotatablely moving, two-phase solvent is all got rid of into small particle by the centrifuge field of high vibration, each component of sample can be distributed on the very big surface of two-phase particulate, and can effectively transmit in the environment of these particle vibrations and convection current.Realize thousands of inferior ground, high-efficiency and continuous solvent extraction process, reach the isolation and purification of high-resolution.Can be with more than one step of effective constituent in sample separating-purifying to 98%.2. hang down use cost: sepn process is not the process of absorption and drip washing, but the process that convection current penetrates, the expense of saving expensive filler, the consumption of saving solvent; In the preparation solvent, major part is water, and organic solvent consumption is few; Can realize the solvent recuperation recycling in scale operation.Follow-up input during operation is used is very low.3. easily technique is amplified: process repeatability 100%.Technique is groped large-scale instrument and is amplified to produce easily and realize from the miniature instrument.4. high-recovery: exempted supporter shared void volume in post, without the impacts such as irreversible adsorption, pollution, sex change, inactivation, sample theoretical recovery 100%.5. clean environmental protection: whole experiment production process sealing is carried out, and avoids solvent evaporates cause environmental pollution and operator are caused actual bodily harm.
Polyprenol (polyprenols) extensively is present in the plants such as green needle, ginkgo and mulberry leaf, be the lipoid cpd single take the C5 isopentene group as structure, can divide on structure: the n-OH of 3-(cis) n-OH of 2-(cis) n-OH of betulin type ω-(trans), the pure type ω of luxuriant and rich with fragrance card-(trans) and alltrans Salanesol type ω-(trans).As shown in Figure 1.
The polyprenol lipoid is nontoxic to human body, 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 mobility, stability and the perviousness of film, thus but the fusion of reinforcing membrane.But exogenous polyprenol lipoid metabolism in vivo is dolichol, with the deficiency of dolichol in added body, to improving immunologic function, liver cell regeneration, anticancer shifts and assisting Radiotherapy chemotherapy etc. to have obvious effect, also can be used as the diagnostic reagent of cancer patient.State's approveds such as Latvia are used for protective foods production and drug development, have prepared " ROPREN " medicine series.Both at home and abroad for the existing bibliographical information of the separation and purification of Plant Polyprenols, main purifying route is by silica gel column chromatography several times, polyprenol crude extract purity to be brought up to more than 90%.In addition, also have some other purification process, as adopting polarizable medium resin (aluminum oxide or polymeric amide etc.) absorption, solvent extraction and crystallization obtain purity and are 70%～75% polyprenol product, yield 1.0% left and right.Carry out again purification by silica gel column chromatography after adopting the freezing removing impurities of petroleum ether-ethyl acetate, preparation purity 92.7% polyprenol.But still contain a large amount of yellow pigment such as 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 the polyprenol loss is large
The HSCCC separation efficiency is high, simple to operate, and sample does not need strict pre-treatment; Can be from extremely complicated rough sample one step separate and obtain highly purified component, can separating flavone, the micromolecular compounds such as alkaloid, quinones, lipoid, also can isolated polypeptide, the macromolecular compounds such as polysaccharide, protein, circulation ratio is good.Also do not have at present article and patent report to adopt high-speed countercurrent chromatography to separate polyprenol.
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, preparation content 5～20% polyprenol class fat ointments, adopt again high-speed countercurrent chromatography, preparation 60%～90% polyprenol lipoid, the exploitation for preparing in batches with pharmaceuticals for the high purity polyprenol provides technology.
Three, summary of the invention
For achieving the above object, invented a kind of method that high speed adverse current chromatogram and high performance liquid chromatography coupling prepare high purity polyprenol lipoid, formed by following steps:
The first step, the 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 100 following powder of order, add any solvent in anhydrous propanone or C1～C3 alcohol, leaf and solvent are 1: 8～30, preferred 1: 8～15 in mass ratio, and ℃ immersion is 10～72 hours in room temperature～80, as 25～50 ℃, 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 with respect to plant leaf powder yield 4～10%, polyprenol lipid levels 5～20%;
Second step, high speed adverse current chromatogram separates
1) select by volume normal hexane: acetonitrile: acetone=3～4: 0.5～1: 0.8～1.5 solution is as high-speed counter-current extraction solvent system, three kinds of solvent are fully rear standing, separate by the up and down two-phase, get to be mutually stationary phase, 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, the high-speed counter-current chromatograph separation condition is under 500～1000r/min rotating speed, flow velocity with 1～5mL/min injects moving phase, UV-detector at wavelength 210～230nm detects, when obviously having moving phase to flow out, beginning test sample solution sample introduction, whole centrifugal process first adopts the positive centrifugal rotation, time is 180～240min, adopt again anti-phase centrifugal rotation, time is 30～60min, collects distillate with automatic fraction collector;
In the 3rd step, high performance liquid chromatography detects
The second step automatic fraction collector is collected distillate, detect polyprenol lipoid position collection with high performance liquid chromatography and 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 leaf of this patent collection be in Ginkgo Leaf, needle and mulberry leaf any, Ginkgo Leaf is the fresh leaf that gathers during August to December, the Ginkgo Leaf of 3～10 years age of trees gathered 8～October, more than 10 years, age of tree Ginkgo Leaf gathered in 10～December, and containing the isopentene group unit number is 15～23 polyprenol lipoids.Pine needle is the fresh leaf of any collection from Pinus massoniana Lamb, slash pine, black pine, cdear, seashore pine and Chinese pine during February～August, and being rich in the isopentene group unit number is 13～19 polyprenol lipoids.Mulberry leaf are the fresh leaf that gathers during 10 months Augusts, and containing the isopentene group unit number is 10～12 polyprenol lipoids.
Traditional extraction process adopts sherwood oil, normal hexane, chlorine alkane, ethyl acetate, acetone etc. to extract the Plant Polyprenols lipoid cpd as the extraction agent of nonpolar or small and weak polarity, extraction yield general 3～6%, these public affairs profit adopts the larger absolute alcohol solvent of polarity as extraction agent first, as disclosed C1～C3 ethanol-extracted agent be in methyl alcohol, ethanol, n-propyl alcohol any, adopt simultaneously anhydrous propanone as extraction agent, enlarge polyprenol and extracted polarity, also obviously improved polyprenol extraction yield and yield.This patent is disclosed in room temperature～80 ℃ immersion 10～72 hours, as 25～50 ℃, 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 with respect to plant leaf powder yield 4～10%, polyprenol lipid levels 5～20%, relative with traditional method, extraction effect obviously improves.
Traditional separation method is brought up to polyprenol crude extract purity more than 90% by silica gel column chromatography repeatedly, not only the time long, and solvent and silica gel many, especially silica gel can not regeneration, cost is high; Adopt polarizable medium resin (aluminum oxide or polymeric amide etc.) absorption, solvent extraction and crystallization can only obtain purity and be 70%～75% polyprenol product, can not satisfy in new drug development the requirement to polyprenol high-content (＞90%).Compare traditional separation method, this patent adopts high speed adverse current chromatogram preparation high purity polyprenol lipoid from the polyprenol class fat ointment that Ginkgo Leaf, pine needle and mulberry leaf extract first, especially whole centrifugal process adopts positive centrifugal rotation and the anti-phase centrifugal rotation method that combines first, whole process time is 210～300min, can prepare in batches, the sample size of every batch of polyprenol class fat ointment is 10～500mg.This patent single stage method, step is few, simple possible, the polyprenol purity of preparation obviously improves, and can satisfy 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, high performance liquid chromatography detect parameters ODS C18column first
Granularity 2.5～10 μ m of filler, 20～30 ℃ of column temperatures, 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, detect wavelength: 210nm.The yellow polyprenol lipoid oily matter of high speed adverse current chromatogram preparation, 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 first absolute alcohol as extraction agent, adopt microwave extraction or ultrasonic extraction polyprenol lipoid, not only the polyprenol extraction yield obviously improves, and alcohol is more safer than non-polar solvents such as sherwood oil, normal hexanes, loses still less in leaching process, and cost is low.
2. adopt first positive centrifugal rotation and the anti-phase centrifugal rotation method that combines, adopt first high speed adverse current chromatogram preparation high purity polyprenol lipoid from the polyprenol class fat ointment that Ginkgo Leaf, pine needle and mulberry leaf extract.
3. adopt first high speed adverse current chromatogram and high performance liquid chromatography coupling to prepare high purity polyprenol lipoid, polyprenol lipid levels 60%～90%.
Description of drawings
Fig. 1 polyprenol lipoid structure
The high speed adverse current chromatogram figure of Fig. 2 standard substance
HPLC after the high speed adverse current chromatogram of Fig. 3 standard substance separates
The high speed adverse current chromatogram figure of Fig. 4 polyprenol lipoid
The HPLC after high speed adverse current chromatogram separates of Fig. 5 polyprenol lipoid
Five, embodiment
Following examples are more of the present invention giving an example, and should not be seen as limitation of the invention.
Embodiment 1 high speed adverse current chromatogram standard model separates
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; OptiChromeA half countercurrent chromatography instrument (Jiangyin Countercurrent Technology Co., Ltd.); Spiral tube separator column (revolution radius: 80cm; Value scope: 0.50～0.80; The 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; Take 80% methanol solution as solvent, prepare respectively 200mg/mL pyrocatechol solution, 200mg/mL Resorcinol solution and 100mg/mL Resorcinol solution, fully after dissolving, respectively get 1mL, 80% methanol solvate that adds 7mL, mixing is made biased sample to be separated.
Solvent system: normal hexane: ethyl acetate: methyl alcohol: water=3: 5: 1: 5.With 300mL normal hexane, 500mL ethyl acetate, 100mL methyl alcohol, with after 500mL distilled water fully mixes, the separating funnel phase-splitting.
4. experiment parameter:
The mixture of pyrocatechol, Resorcinol and Resorcinol separates, and separation condition is: solvent system: normal 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 ℃; Rotating speed: 1800rpm; Detect wavelength: 254nm.Sampling volume: 1mL; Sample introduction concentration: Catechol 2 mg/mL+ Resorcinol 2mg/mL+ Resorcinol 1mg/mL (biased sample is dissolved in moving phase at 1: 10)
Preparation type instrument separation condition: twin columns separate, column volume 360mL; Flow velocity: 5mL/min; Room temperature: 22 ℃; Rotating speed: 1200rpm; Detect wavelength: 254nm, sampling volume: 20mL; Sample concentration: Catechol 2 mg/mL+ Resorcinol 2mg/mL+ Resorcinol 1mg/mL (biased sample is dissolved in moving phase at 1: 10).
5. experimental procedure:
5.1. stationary phase injects: open constant flow pump, with Peak Flow Rate, stationary phase is injected separator column by entrance, be full of the separator column pipeline, after flowing out to exit end, continue pump termination of pumping afterwards in 1 minute.Define enough stationary phase and be used for filling with separator column (analytical column should be prepared more than 50mL, preparative column should more than 400mL), carefully do not advance air or moving phase in this process.
5.2. the connection external power, the power switch in the instrument postnotum lower left corner that closes arranges the centrifugal rotation parameter; Constant flow pump is set separates flow velocity (analysis mode 1mL/min, preparation type 5mL/min)
5.3. system balancing: confirm that parameter of noncentricity is correct, and filled with 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 simultaneously " Run " button green above centrifugal control panel, start centrifugal.
5.4. the solvent systems balance, namely the outflow solution of outlet be all limpid moving phase, perhaps the on-line detector signal steady after, record the withdrawal volume of stationary phase, with calculating stationary phase retention rate;
Sf=(stationary phase volume of pipeline cumulative volume-discharge)/separator column volume * 100%
This experiment pumps into moving phase with the full stationary phase of 50mL/min speed pump with 5mL/min speed, starts OptiChrome
TMThe A-300 forward is centrifugal, observes UV3000 output and reaches system balancing, calculates stationary phase retention rate 65%.
5.5. sample preparation: (analysis mode: 0.1mL is diluted in 0.9mL moving phase with the moving phase that is diluted in solvent system at 1: 10 to get 50mg/mL phenols sample mix liquid (Catechol 2 0mg/mL+ Resorcinol 20mg/mL+ Resorcinol 10mg/mL); Preparation type: 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 as is connected with the sampling aperture of syringe adapter, observe loading ring and sample feeding pipe, inject emitter to discharge the air in sample feeding pipe, make to be full of the moving phase that flows out in sample feeding pipe from the loading ring.Then sample feeding pipe is inserted the sample tube (attention is inserted into the bottom and avoids the air inlet bubble) for preparing, the suction syringe sucks the loading ring with sample.Sample can be filled with the loading ring, also can partly be full of the loading ring, but note to have air to enter the loading ring.Then with the vertical zero position of loading valve cycle, sample will enter separator column with the moving phase that pumps into.
5.7. the detach Spline of biased sample is recorded in " data gathering " in the click chromatographic working station.
5.8. when separate finishing, stop centrifugally by " stopping " button on centrifugal control panel, and constant flow pump stopped.Click " stopping gathering " key in chromatographic working station, and preserve collection of illustrative plates.
5.9. can select to blow out solution last in separator column and the component of reservation with nitrogen or pressurized air; Perhaps select with stationary phase, the component of solution in post and reservation to be released at a high speed.
5.10. pipeline-cleaning: pump into the approximately methyl alcohol of 1/3 column volume with constant flow pump, then blow out with nitrogen; Triplicate.After cleaning for the last time and blowing out washed with methanol liquid, when continuing to blow, press " turning to " key on centrifugal control panel, display screen " running " item will show " oppositely centrifugal stopping ", then rotating speed is made as 400rpm, starts centrifugally, be conducive to emptying of remaining solvent in pipeline.
6. separating resulting:
Pyrocatechol, Resorcinol and Resorcinol have been realized good separation, as Fig. 2 and Fig. 3.
Embodiment 2 high speed adverse current chromatograms and high performance liquid chromatography method for combined use
The first step, the 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 100 following powder of order, add any solvent in anhydrous propanone or C1～C3 alcohol, preferred anhydrous propanone or ethanol, leaf powder and solvent are 1: 8～30 in mass ratio, preferred 1: 8～15, ℃ immersion is 10～72 hours in room temperature～80, as 25～50 ℃, 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 with respect to plant leaf powder yield 4～10%, polyprenol lipid levels 5～20%;
Second step, high speed adverse current chromatogram separates
(1) select by volume normal hexane: acetonitrile: acetone=3～4: 0.5～1: 0.8～1.5 solution is as high-speed counter-current extraction solvent system, three kinds of solvent are fully rear standing, separate by the up and down two-phase, get to be mutually stationary phase, 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, preferred 1: 30～60;
(3) stationary phase is full of the multilayer coil separator column of high-speed counter-current chromatograph, the high-speed counter-current chromatograph separation condition is under 500～1000r/min rotating speed, flow velocity with 1～5mL/min injects moving phase, UV-detector at wavelength 210～230nm detects, when obviously having moving phase to flow out, beginning test sample solution sample introduction, whole centrifugal process first adopts the positive centrifugal rotation, time is 180～240min, adopt again anti-phase centrifugal rotation, time is 30～60min, collects distillate with automatic fraction collector;
In the 3rd step, high performance liquid chromatography detects
The second step automatic fraction collector is collected distillate, detect polyprenol lipoid position collection with high performance liquid chromatography and 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, the fresh plant leaf is any in Ginkgo Leaf, pine needle and mulberry leaf, Ginkgo Leaf is the fresh leaf that gathers during August to December, the Ginkgo Leaf of 3～10 years age of trees gathered 8～October, more than 10 years, age of tree Ginkgo Leaf gathered in 10～December, and containing the isopentene group unit number is 15～23 polyprenol lipoids; Pine needle is the fresh leaf of any collection from Pinus massoniana Lamb, slash pine, black pine, cdear, seashore pine and Chinese pine during February～August, being rich in the isopentene group unit number is 13～19 polyprenol lipoids, mulberry leaf are the fresh leaf that gathers during 10 months Augusts, and containing the isopentene group unit number is 10～12 polyprenol lipoids.
In the present embodiment, plant material separates with high speed adverse current chromatogram through anhydrous propanone or ethanol microwave extraction, whole centrifugal process adopts positive centrifugal rotation and the anti-phase centrifugal rotation method that combines, whole process time is 210～300min, can prepare in batches, the sample size of every batch of polyprenol class fat ointment is 10～500mg.
The present embodiment detects high speed adverse current chromatogram with high performance liquid chromatography and separates polyprenol lipoid extraction position and content thereof, and the high performance liquid chromatography detect parameters is chromatographic column adopting ODS C18column
Granularity 2.5～10 μ m of filler, 20～30 ℃ of column temperatures, 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, detect wavelength: 210nm.Analyze the polyprenol lipid levels 60%～90% that high speed adverse current chromatogram separates through HPLC.
Embodiment 3 high speed adverse current chromatogram polyprenol separating experiments
1. solvent is prepared: normal hexane: acetonitrile: acetone=by volume prepare 2000mL at 3: 1: 1, above is stationary phase mutually, below is moving phase mutually.
2. preparation of samples: get polyprenol class fat ointment 50mg in embodiment 1, be 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
TMThe A-300 forward is centrifugal, observes UV3000 output and reaches system balancing, calculates the stationary phase retention rate: 60%
5. loading: sample is injected OptiChrome by the loading valve
TMIn A-300.Forward is centrifugal: with OptiChrome
TMA-300 is set as the forward centrifugal rotation, sets.
6. oppositely centrifugal: will separate flow velocity is 5mL/min, centrifugal through the forward of 180 minutes.OptiChrome
TMA-300 is set as reverse centrifugal rotation, and set separating flow velocity is 10mL/min, oppositely centrifugal through 30 minutes.
7. in above-mentioned steps (5) and (6), sample device after testing obtains collection of illustrative plates, as accompanying drawing 4.
8.HPLC analyze: get Fig. 3 detector and absorb the isolated liquid of maximum and be placed in HPLC and detect, compare by the collection of illustrative plates with the polyprenol sample of standard, obtain polyprenol lipoid collection of illustrative plates.Can see clearly OptiChrome from the HPLC collection of illustrative plates of Fig. 4 as accompanying drawing 5.
TMThe A-300 success with part mixture separation in the polyprenol mixture out.
Embodiment 4 high speed adverse current chromatograms prepare the Polyprenols From Ginkgo Biloba L lipoid
1. raw material is collected, and Ginkgo Leaf is the fresh leaf that gathers during 8～December, and wherein the Ginkgo Leaf of 3～10 years age of trees gathered 8～October, and 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 100 following powder of order, and is standby.
2. the 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 to extract twice, united extraction liquid, vacuum reclaims organic solvent, enriched material is polyprenol class fat ointment, is total to 5g, and it is 8% that HPLC analyses purity;
3. getting 100mg adopts high-speed counter-current chromatograph to separate, get volume ratio and be normal hexane, acetonitrile and the acetone of 3: 0.5: 0.8 as the high-speed counter-current solvent system, standing after mixing fully, by the up and down 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, the setting high-speed counter current chromatograph, under the 800r/min rotating speed, flow velocity with 2mL/min injects moving phase, UV-detector at wavelength 210nm detects, when obviously having moving phase to flow out, begin to get trial-product as the sample solution sample introduction, begin simultaneously to collect distillate with automatic fraction collector, whole centrifugal process first adopts the positive centrifugal rotation, time is 180～240min, then to adopt anti-phase centrifugal rotation, time be 30～60min.The distillate that collection obtains 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%, and containing the isopentene group unit number is 15～23.
Embodiment 5 high speed adverse current chromatograms prepare needle polyprenol lipoid
1. raw material is collected, and pine needle is the fresh leaf of any collection from Pinus massoniana Lamb, slash pine, black pine, cdear, seashore pine and Chinese pine during February～August, and room temperature is dried in the shade, and is crushed to the 100 following powder of order, and is standby.
2. the 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 to extract twice, united extraction liquid, vacuum reclaims organic solvent, enriched material is polyprenol class fat ointment, is total to 5.8g, and it is 15% that HPLC analyses purity;
3. get 100mg polyprenol class fat ointment, adopt high-speed counter-current chromatograph to separate, get volume ratio and be normal hexane, acetonitrile and the acetone of 4: 1: 1.5 as the high-speed counter-current solvent system, mixing is fully rear standing, separates by the up and down two-phase, gets to be mutually stationary phase, 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, the setting high-speed counter current chromatograph, under the 1000r/min rotating speed, flow velocity with 2mL/min injects moving phase, UV-detector at wavelength 210nm detects, when obviously having moving phase to flow out, begin to get trial-product as the sample solution sample introduction, begin simultaneously to collect distillate with automatic fraction collector, whole centrifugal process first adopts the positive centrifugal rotation, time is 180～240min, then to adopt anti-phase centrifugal rotation, time be 30～60min.The distillate that collection obtains adopts rotary evaporation to remove solvent, obtains sample 20.5mg.
4. high performance liquid chromatography detects, and needle polyprenol purity is 86%, and containing the isopentene group unit number is 13～19.
Embodiment 6 high speed adverse current chromatograms prepare mulberry leaf polyprenol lipoid
1. raw material is collected, and mulberry leaf are the fresh leaf that gathers during 10 months Augusts, and room temperature is dried in the shade, and is crushed to the 100 following powder of order, and is standby.
2. the polyprenol lipoid extracts: get mulberry leaves powder 1kg, add anhydrous methanol, leaf and solvent are 1: 15 in mass ratio, 50 ℃ of microwave extraction 60 minutes, repeat to extract twice, united extraction liquid, vacuum reclaims organic solvent, enriched material is polyprenol class fat ointment, is total to 3.8g, and it is 18% that HPLC analyses purity;
3. getting 500mg adopts high-speed counter-current chromatograph to separate, get volume ratio and be normal hexane, acetonitrile and the acetone of 4: 1: 1.5 as the high-speed counter-current solvent system, standing after mixing fully, by the up and down 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, the setting high-speed counter current chromatograph, under the 1000r/min rotating speed, flow velocity with 5mL/min injects moving phase, UV-detector at wavelength 210nm detects, when obviously having moving phase to flow out, begin to get trial-product as the sample solution sample introduction, begin simultaneously to collect distillate with automatic fraction collector, whole centrifugal process first adopts the positive centrifugal rotation, time is 180～240min, then to adopt anti-phase centrifugal rotation, time be 30～60min.The distillate that collection obtains adopts rotary evaporation to remove solvent, obtains sample 172.3mg.
4. high performance liquid chromatography detects, and mulberry leaf polyprenol lipoid purity is 73%, and containing the isopentene group unit number is 10～12.
Embodiment 7 high speed adverse current chromatograms prepare the Polyprenols From Ginkgo Biloba L lipoid
1. raw material is collected, and Ginkgo Leaf is the fresh leaf that gathers during August to December, and wherein the Ginkgo Leaf of 3～10 years age of trees gathered 8～October, and 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 100 following powder of order, and is standby.
2. the polyprenol lipoid extracts: get Ginkgo Leaf 1kg, add anhydrous methanol, leaf and solvent are 1: 10 in mass ratio, 60 ℃ of microwave extraction 60 minutes, repeat to extract twice, united extraction liquid, vacuum reclaims organic solvent, enriched material is polyprenol class fat ointment, is total to 4.5g, and it is 18% that HPLC analyses purity;
3. getting 100mg adopts high-speed counter-current chromatograph to separate, get volume ratio and be normal hexane, acetonitrile and the acetone of 4: 1: 1.5 as the high-speed counter-current solvent system, standing after mixing fully, by the up and down 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, the setting high-speed counter current chromatograph, under the 1000r/min rotating speed, flow velocity with 1mL/min injects moving phase, UV-detector at wavelength 210nm detects, when obviously having moving phase to flow out, begin to get trial-product as the sample solution sample introduction, begin simultaneously to collect distillate with automatic fraction collector, whole centrifugal process first adopts the positive centrifugal rotation, time is 180～240min, then to adopt anti-phase centrifugal rotation, time be 30～60min.The distillate that collection obtains adopts rotary evaporation to remove solvent, obtains sample 37mg.
4. high performance liquid chromatography detects, and the Polyprenols From Ginkgo Biloba L lipoid is 90%, and containing the isopentene group unit number is 15～23.
Embodiment 8 high speed adverse current chromatograms prepare needle polyprenol lipoid
1. pine needle is the fresh leaf of any collection from Pinus massoniana Lamb, black pine and cdear during February～August, and room temperature is dried in the shade, and is crushed to the 100 following powder of order, polyprenol lipid levels 1.75%.
2. the 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 to extract twice, united extraction liquid, vacuum reclaims organic solvent, enriched material is polyprenol class fat ointment, is total to 5.8g, and it is 25% that HPLC analyses purity;
3. getting 300mg adopts high-speed counter-current chromatograph to separate, get volume ratio and be normal hexane, acetonitrile and the acetone of 3: 1: 1.5 as the high-speed counter-current solvent system, standing after mixing fully, by the up and down 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, the setting high-speed counter current chromatograph, under the 600r/min rotating speed, flow velocity with 5mL/min injects moving phase, UV-detector at wavelength 230nm detects, when obviously having moving phase to flow out, begin to get trial-product as the sample solution sample introduction, begin simultaneously to collect distillate with automatic fraction collector, whole centrifugal process first adopts the positive centrifugal rotation, time is 180～240min, then to adopt anti-phase centrifugal rotation, time be 30～60min.The distillate that collection obtains adopts rotary evaporation to remove solvent, obtains sample 108.75mg.
4. high performance liquid chromatography detects, and needle polyprenol lipoid purity is 86%, and containing the isopentene group unit number is 13～19.
Claims (8)
1. a high speed adverse current chromatogram and high performance liquid chromatography coupling prepare the method for high purity polyprenol lipoid, it is characterized in that being comprised of following steps:
The first step, the 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 100 following powder of order, adds any solvent in anhydrous propanone or C1～C3 alcohol, leaf and solvent are 1: 8～30 in mass ratio, soaked 10～72 hours in room temperature～80 ℃, 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 separates
1) select by volume normal hexane: acetonitrile: acetone=3～4: 0.5～1: 0.8～1.5 solution is as high-speed counter-current extraction solvent system, three kinds of solvent are fully rear standing, separate by the up and down two-phase, get to be mutually stationary phase, 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, the high-speed counter-current chromatograph separation condition is under 500～1000r/min rotating speed, flow velocity with 1～5mL/min injects moving phase, UV-detector at wavelength 210～230nm detects, when obviously having moving phase to flow out, beginning test sample solution sample introduction, whole centrifugal process first adopts the positive centrifugal rotation, time is 180～240min, adopt again anti-phase centrifugal rotation, time is 30～60min, collects distillate with automatic fraction collector; In the 3rd step, high performance liquid chromatography detects
The second step automatic fraction collector is collected distillate, detect polyprenol lipoid position collection with high performance liquid chromatography and divide, merge polyprenol lipoid collection and divide, reclaim organic solvent, enriched material is yellow polyprenol lipoid oily matter, polyprenol lipid levels 60%～90%.
2. a kind of high speed adverse current chromatogram and high performance liquid chromatography coupling prepare the method for high purity polyprenol lipoid according to claim 1, it is characterized in that the fresh plant leaf of the first step polyprenol lipoid in extracting is any in Ginkgo Leaf, pine needle and mulberry leaf.
3. a kind of high speed adverse current chromatogram and high performance liquid chromatography coupling prepare the method for high purity polyprenol lipoid according to claim 1, it is characterized in that the first step polyprenol lipoid extract in C1～C3 alcohol in methyl alcohol, ethanol, n-propyl alcohol any.
4. a kind of high speed adverse current chromatogram and high performance liquid chromatography coupling prepare the method for high purity polyprenol lipoid according to claim 1, it is characterized in that the second step high speed adverse current chromatogram separate in the sample size of polyprenol class fat ointment be 10～500mg.
5. a kind of high speed adverse current chromatogram and high performance liquid chromatography coupling prepare the method for high purity polyprenol lipoid according to claim 1, it is characterized in that the 3rd step high performance liquid chromatography detect parameters ODS C18column
Granularity 2.5～10 μ m of filler, 20～30 ℃ of column temperatures, 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, detect wavelength: 210nm.
6. Ginkgo Leaf according to claim 2, it is characterized in that Ginkgo Leaf is the fresh leaf that gathers during August to December, the Ginkgo Leaf of 3～10 years age of trees of silver gathered 8～October, more than 10 years, age of tree Ginkgo Leaf gathered in 10～December, and containing the isopentene group unit number is 15～23 polyprenol lipoids.
7. pine needle according to claim 2, it is characterized in that pine needle is the fresh leaf of any collection from Pinus massoniana Lamb, slash pine, black pine, cdear, seashore pine and Chinese pine during February～August, being rich in the isopentene group unit number is 13～19 polyprenol lipoids.
8. mulberry leaf according to claim 2 is characterized in that mulberry leaf are the fresh leaf that gathers during 10 months Augusts, and containing the isopentene group unit number is 10～12 polyprenol lipoids.
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