CN103788036B - A kind of method extracting high purity ε-grape element from grape branches and tendrils - Google Patents
A kind of method extracting high purity ε-grape element from grape branches and tendrils Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
- C07D307/77—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D307/78—Benzo [b] furans; Hydrogenated benzo [b] furans
- C07D307/79—Benzo [b] furans; Hydrogenated benzo [b] furans with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to carbon atoms of the hetero ring
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Abstract
The present invention relates to a kind of method extracting high purity ε-grape element from plant, particularly relate to a kind of method extracting high purity ε-grape element from grape branches and tendrils.The present invention is complicated and confused for raw material with discarded grape, after extraction, purifying, carry out column chromatography, after wash-out, obtain the elutriant containing ε-grape element crude product, 40-50 DEG C it is concentrated, dry, obtain ε-grape element crude product, loaded in hydroxypropyl sephadex column after ε-grape element crude product is dissolved in methyl alcohol again, chromatography, obtains the elutriant containing ε-grape element, then through dilution, crystallization, centrifugal, ε-grape element monomer of purity >=98% is obtained.The present invention for raw material with discarded grape branches and tendrils, has successfully extracted highly purified ε-grape element, has played utilization of waste material, economize on resources, the effect of protection of the environment; Byproduct of the present invention is trans-resveratrol, is also a kind of medicine of preciousness, chemical feedstocks, and this just further improves the utility value of grape branches and tendrils.Present invention process is simple, easy and simple to handle, is easy to industrialization and produces.
Description
Technical field
The present invention relates to a kind of method extracting high purity ε-grape element from plant, particularly relate to a kind of method extracting high purity ε-grape element from grape branches and tendrils.
Background technology
Domestic and international research in recent years finds, containing abundant ε-grape element in grape branches and tendrils.ε-grape element is a kind of natural polyphenol that in grape and grape wine, critical function composition trans-resveratrol is polymerized.ε-grape element is the same with trans-resveratrol has the effects such as anticancer, anti-oxidant, preventing cardiovascular disease, antibacterial, anti-inflammatory, and its activity and stability are all higher than trans-resveratrol.Thus receive much concern in plant disease-resistant, the active field of Human Physiology.Grape need prune several times in the course of cultivation every year, and conservative estimation every mu is produced 100-200kg per year and pruned useless branch.For a long time, useless branch is directly burnt by peasant household mostly, serious environment pollution.Therefore, from the useless branch of pruning, develop natural product, not only can economize on resources, and safe natural product can be obtained.
In recent years, more to the functionally active research of ε-grape element abroad, existing relevant grape vine extract product listing, stilbene U content 30-50% in commercialized product, wherein ε-grape cellulose content was 10-20%.
The domestic research to ε-grape element is less, " a kind of improve method (application number is 200810097510.x) containing grape cellulose content in grape extract plant by fermentation " of the patent Jin You Guilin Laiyin Biotechnology Co., Ltd. that can find and " a kind of stilbene group compounds and contain its mattae extract and extracting method (application number is 200710302004.5) ".What mainly study in the application number patent that is 200810097510.x is how to improve grape cellulose content by fermentation; At application number be in the patent of 200710302004.5 main research be the plain and/or tripolymer stilbene compound of grape by extractions, purifying, the refining main products that obtains; But because grape element and tripolymer stilbene compound have various structures formula, so its product should be the mixture of multiple grape element and the mixture of multiple tripolymer stilbene compound.As for how obtaining high purity (purity >98%) ε-grape element, also there is not the correlative study report of relevant preparation and/or extracting method so far both at home and abroad.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, provide a kind of method extracting high purity ε-grape element from grape branches and tendrils, purity >=98% of ε-grape element in products obtained therefrom.
A kind of method extracting high purity ε-grape element from grape branches and tendrils of the present invention, comprises the steps:
Step one is separated trans-resveratrol, ε-grape element
After the solid mixture of trans-resveratrol and ε-grape element is dissolved in methyl chloride, carries out column chromatography, during chromatography, adopt the mixing solutions be made up of methyl chloride and methyl alcohol to carry out gradient elution, obtain the elutriant containing ε-grape element crude product;
Step 2 is refined
Elutriant step one gained being contained ε-grape element crude product obtains ε-grape element crude product being less than 60 DEG C of concentrated, dryings, loading hydroxypropyl sephadex column is adopted after ε-grape element crude product is dissolved in methyl alcohol, carry out column chromatography, during chromatography, adopt methyl alcohol to be that elutriant carries out isocratic elution, obtain the elutriant containing ε-grape element, leave standstill, crystallization, centrifugal, obtain ε-grape element monomer.
A kind of method extracting high purity ε-grape element from grape branches and tendrils of the present invention, in step one, when carrying out column chromatography, chromatography column used is alumina chromatographic column or silica gel column chromatography, is preferably silica gel column chromatography; In described silica gel column chromatography, the size of silica gel particle is 200-300 order.
A kind of method extracting high purity ε-grape element from grape branches and tendrils of the present invention, methyl chloride described in step one is methylene dichloride or trichloromethane.
A kind of method extracting high purity ε-grape element from grape branches and tendrils of the present invention, in step, during chromatography dress post, adopt wet method loading, during wash-out, first use 2-4BV's, carry out wash-out for the first time by methyl chloride and the methyl alcohol mixing solutions that 19:1 forms by volume and also collect the elutriant of first time wash-out, use 2-4BV's again, carry out second time wash-out by methyl chloride and the methyl alcohol mixing solutions that 16:1 forms by volume and collect the elutriant of second time wash-out, then use 2-4BV's, carry out wash-out for the third time by methyl chloride and the methyl alcohol mixing solutions that 13:1 forms by volume and also collect the elutriant of third time wash-out, finally use 2-4BV's, carried out the 4th wash-out by methyl chloride and the methyl alcohol mixing solutions that 10:1 forms by volume and collected the 4th elutriant, during wash-out, control the equal 0.5-1.5BV/h of flow velocity of elutriant, described first time wash-out elutriant and the elutriant of second time wash-out be elutriant containing trans-resveratrol, described third time wash-out elutriant and the elutriant of the 4th wash-out be elutriant containing ε-grape element.
A kind of method extracting high purity ε-grape element from grape branches and tendrils of the present invention, when in step one, wash-out layer inhales post, each wash-out elutriant used is preferably 3BV, and during each wash-out, the flow velocity of elutriant is preferably 1BV/h.
A kind of method extracting high purity ε-grape element from grape branches and tendrils of the present invention, generally adopts following proposal time refining:
Elutriant step one gained being contained ε-grape element crude product concentrates at 40-50 DEG C, drying obtains ε-grape element crude product, after ε-grape element crude product is dissolved in methyl alcohol, adopt the mode of wet method loading to be loaded in hydroxypropyl sephadex column, carry out column chromatography, during chromatography, methyl alcohol is adopted to be that elutriant carries out isocratic elution, obtain the elutriant containing ε-grape element, with described in equal-volume distilled water diluting containing ε-grape element elutriant after, be placed in the stink cupboard of room temperature, volatilization dilution after containing ε-grape element elutriant in methyl alcohol to when starting to occur crystallization, be cooled to 2-6 DEG C, be preferably 4 DEG C, leave standstill 20-28 hour, be preferably 24 hours, centrifugal, obtain the ε-grape element monomer of purity >=98%, the model of described hydroxypropyl sephadex column is LH-20, during chromatography, adopt methyl alcohol to be that elutriant carries out isocratic elution, the flow velocity of elutriant is 1-2BV/h.
A kind of method extracting high purity ε-grape element from grape branches and tendrils of the present invention, the described solid mixture containing trans-resveratrol and ε-grape element is prepared by following step:
Step a extracts
Be the ratio of 4-6mL/g in liquid-solid ratio, add in extraction agent after the plant containing ε-grape element is crushed to-20 orders, extract 0.5-1h 60 DEG C below after, the concentrated organic solvent removed in extraction agent, obtains extracting solution; Described extraction agent is water by water and organic solvent by volume: organic solvent=3-5:15-17 forms, and described organic solvent is selected from the one in ethanol, methyl alcohol, ethyl acetate;
Step b purifying
With the centrifugal speed of 3000-5000r/min, centrifugal treating is carried out to step a gained extracting solution, centrifugal treating gained supernatant liquor macroporous resin carries out chromatography purification, first wash with water during chromatography, then ethanolic soln wash-out is used, collect ethanol eluate, at 50-60 DEG C, concentrated, drying ethanol elutriant obtains the solid mixture containing trans-resveratrol and ε-grape element; Or
Add isopyknic extraction agent after organic solvent below 60 DEG C in removing step a gained extracting solution, 40-60 DEG C of concentrated, dry organic phase after extracting, obtain the solid mixture containing trans-resveratrol and ε-grape element; The selection system of extraction agent is: when the organic solvent of extraction agent be ethanol or methyl alcohol time, extraction agent used is ethyl acetate; When the organic solvent of extraction agent is ethyl acetate, extraction agent used is chloroform or methylene dichloride.
A kind of method extracting high purity ε-grape element from grape branches and tendrils of the present invention, the described plant containing ε-grape element is grape branches and tendrils; Described grape branches and tendrils are through naturally drying in the shade the 1-3 month.
A kind of method extracting high purity ε-grape element from grape branches and tendrils of the present invention, described in be extracted as supersound extraction or refluxing extraction; Be preferably supersound extraction; Extracting temperature is preferably room temperature-30 DEG C.
A kind of method extracting high purity ε-grape element from grape branches and tendrils of the present invention, macroporous resin described in step b is selected from the one in D101 type macroporous resin, AB-8 type macroporous resin, D102 type macroporous resin.A kind of method extracting high purity ε-grape element from grape branches and tendrils of the present invention, in step b, during chromatography purification, controlling loading speed is 2-3BV/h; After completion of the sample, first carry out wash-out with the water of 1-3BV, then carry out wash-out with the ethanol of 2-6BV and collect ethanol eluate; Ethanol eluate carries out vacuum-drying after being evaporated to medicinal extract shape at 50-60 DEG C, obtains the solid mixture containing trans-resveratrol and ε-grape element; The concentration expressed in percentage by volume of described ethanol is 70-90%.
A kind of method extracting high purity ε-grape element from grape branches and tendrils of the present invention, in step 4, model is that the hydroxypropyl sephadex column (SephadexLH-20) of LH-20 can be distinguished according to the molecular weight of trans-resveratrol and polymkeric substance thereof and constitutional features, effectively they are separated, thus obtain high purity ε-grape element.
Technique effect
The present invention is using discarded grape branches and tendrils as raw material, and carry out the extraction of ε-grape element, it compared with prior art has following difference and advantage:
1, the polarity due to the isomers of ε-grape element is close, is difficult to be separated by common method obtain highly purified ε-grape element; Contriver is after attempting in a large number, and the initiative hydroxypropyl sephadex column (SephadexLH-20) that utilizes carrys out the isomers of separate epsilon-grape element, and achieves beyond thought effect, obtains the ε-grape element of purity >=98%;
2, less to the Separation Research of trans-resveratrol and ε-grape element in prior art, the present invention makes full use of the polarity difference of trans-resveratrol and ε-grape element, adopts the method for column chromatography well to achieve being separated of trans-resveratrol and grape element; By reclaiming the elutriant of different elution stage, effectively achieve being separated of trans-resveratrol and ε-grape element, for obtaining trans-resveratrol and high purity ε-grape element provides prerequisite;
3, because ε-grape element is comparatively responsive to temperature, temperature is very easily degraded higher than when 60 DEG C, so the present invention strictly controls the temperature (≤60 DEG C) of each step; Especially, in leaching process, preferred room temperature supersound extraction, so both achieved good extraction effect, shortened extraction time again, thus greatly improved production efficiency;
In sum, the present invention is using discarded grape branches and tendrils as raw material, temperature≤60 DEG C of strict rate-determining steps in whole preparation process, adopt and reasonably extract, purification technique obtains the solid mixture containing trans-resveratrol and ε-grape element, selecting and the control realization high efficiency separation of trans-resveratrol and ε-grape element of parameter by rational isolation technique, at ε-grape element with the sepn process of its isomers, by selecting hydroxypropyl sephadex column (SephadexLH-20) as chromatography column, obtain the ε-grape element of purity >=98% unexpectedly, make a general survey of whole technique, present invention process is simple, easy and simple to handle, and be easy to industrialization and produce, products therefrom purity is high, has very high industry, medical value, and gained by product is trans-resveratrol, and it also has very high industry, medical value.The present invention is using discarded grape branches and tendrils as raw material in a word, is achieved the recovery of efficient, the high value of grape branches and tendrils by rational technique; Obtain the ε-grape element of purity >=98%.
Accompanying drawing explanation
The purity of accompanying drawing 1 prepared by embodiment 1 is high-efficient liquid phase chromatogram spectrum (HPLC collection of illustrative plates) of the ε-grape element of 98.5%;
The Structural Identification figure of accompanying drawing 2 ε prepared by embodiment 1-grape element.
As can be seen from Figure 1, unimodal about 43min appearance one, contrast with ε-grape element standard specimen, be separated ε-grape element purity is greater than 98%.
As can be seen from Figure 2, the light yellow unformed powder prepared by embodiment 1 is ε-grape element.
Embodiment
Below in conjunction with figure, method of the present invention is described in further detail: the above-mentioned embodiment of embodiment is used for illustrative purposes only, and be not limitation of the present invention, the those of ordinary skill of relevant technical field, without departing from the spirit and scope of the present invention, can also make various change and modification, therefore all equivalent technical schemes also should belong to category of the present invention.
Embodiment 1
(1) pre-treatment: the branch that given up by the grape of pruning dries in the shade 2 months naturally, then cross 20 mesh sieves after pulverizing.
(2) extract: by pretreated raw material, drop in extractor, add 75% supersound extraction 2 times, extract 1h at every turn, during extracted twice, the solid-to-liquid ratio of solvent and raw material is respectively 6mL/g and 4mL/g.Merge extracted twice liquid, proceeded to by extracting solution in thickener, be evaporated to without alcohol taste, temperature controls at 60 DEG C.
(3) purifying: by step (2) gained extracting solution 3000r/min centrifugal treating, obtain supernatant liquor, through macroporous resin D101 chromatography purification, loading speed is 2BV/h.After completion of the sample, first wash 2BV with water, then directly use 80% ethanol elution 4BV.Proceed in thickener by 80% ethanol eluate, be evaporated to medicinal extract shape, then proceed to dry (temperature controls at 60 DEG C) in vacuum drying oven, after dry, in crude extract, ε-grape cellulose content is 11.5%.
(4) trans-resveratrol, ε-grape element is separated: by wet method loading after as far as possible few methylene dichloride dissolving step (3) gained crude extract, then be separated trans-resveratrol, ε-grape element through normal pressure silica gel (200-300 order) chromatography column, wash-out obtains the elutriant containing trans-resveratrol crude product and the elutriant containing ε-grape element crude product respectively, during chromatographic elution, first use 2BV's, carry out wash-out for the first time by methyl chloride and the methyl alcohol mixing solutions that 19:1 forms by volume and also collect the elutriant of first time wash-out, use 4BV's again, carry out second time wash-out by methyl chloride and the methyl alcohol mixing solutions that 16:1 forms by volume and collect the elutriant of second time wash-out, then use 3BV's, carry out wash-out for the third time by methyl chloride and the methyl alcohol mixing solutions that 13:1 forms by volume and also collect the elutriant of third time wash-out, finally use 3BV's, carried out the 4th wash-out by methyl chloride and the methyl alcohol mixing solutions that 10:1 forms by volume and collected the 4th elutriant, during wash-out, control the equal 0.5BV/h of flow velocity of elutriant, the elutriant of wash-out and the elutriant of second time wash-out are the elutriant containing trans-resveratrol for the first time, the elutriant of wash-out and the elutriant of the 4th wash-out are the elutriant containing ε-grape element for the third time.
Elutriant gained being contained ε-grape element crude product obtains ε-grape element crude product at 40 DEG C of concentrate dryings, and ε-grape cellulose content is 79.8%.
(5) finished product preparation: by step (4) gained ε-as far as possible few dissolve with methanol wet method loading of grape element crude product, through hydroxypropyl sephadex column (SephadexLH-20) chromatography, pure methanol-eluted fractions, the flow velocity of elutriant is 1BV/h, obtain the elutriant containing ε-grape element, isopyknic distilled water is added again to containing in the elutriant of ε-grape element, be placed in ventilating kitchen, volatilization organic phase is to starting to occur crystallization, then go to 4 DEG C of refrigerators and place 24 hours, 5000r/min centrifugal treating, centrifugal gained throw out is through recrystallization, after lyophilize, obtain light yellow unformed powder, adopt high performance liquid chromatography to detect and show that ε in product-grape cellulose content is 98.5%, its detection figure is shown in Fig. 1.
Employing electronics bombards
ion source-MS (EI-MS) detects products therefrom, records m/z (%): 454 [M]
+(100), 360,347,107.UVλmaxnm:232(sh),328(MeOH)；
Adopt hydrogen nuclear magnetic resonance to detect products therefrom, record:
1hNMR (400MHz, DMSO-d
6, ppm) δ: 6.70 (2H, d, J=7.2Hz, H-2, 6), 7.18 (2H, d, J=7.2Hz, H-3, 5), 6.62 (1H, d, J=16.8Hz, H-7), 6.87 (1H, d, J=16.8Hz, H-8), 6.29 (1H, H-12), 6.60 (1H, H-14), 6.52 (2H, d, J=7.2Hz, H-2 ', 6 '), 6.93 (2H, d, J=7.2Hz, H-3 ', 5 '), 5.78 (1H, d, J=7.6Hz, H-7 '), 4.62 (1H, d, J=7.6Hz, H-8 '), 5.64 (2H, H-10 ', 14 '), 5.78 (1H, H-12 '), 8.77 (2H, s, C11 '-OH, C
13 '-OH), 9.17(1H, s, C
1 '-OH), 9.36 (1H, s, C
13-OH), 9.53 (1H, s, C
1-OH),
Connexus modal data and hydrocarbon nuclear magnetic resonance data, identifying this compound is ε-grape element, and its structure as shown in Figure 2.
Embodiment 2
(1) pre-treatment: the branch that given up by the grape of pruning dries in the shade March naturally, then cross 40 mesh sieves after pulverizing.
(2) extract: by pretreated raw material, drop in extractor, add 80% ethanol ultrasonic extraction 2 times, extract 0.5h at every turn, during extracted twice, the solid-to-liquid ratio of solvent and raw material is respectively 6mL/g and 4mL/g.Merge extracted twice liquid, extracting solution is proceeded in thickener.Be evaporated to without alcohol taste, temperature controls at 55 DEG C.
(3) purifying: step (2) gained extracting solution is added equal-volume extraction into ethyl acetate, organic phase after extraction is proceeded in thickener, be evaporated to medicinal extract shape, then proceed to dry (temperature controls at 50 DEG C) in vacuum drying oven, after dry, in crude extract, ε-grape cellulose content is 9.6%.
(4) trans-resveratrol, ε-grape element is separated: by wet method loading after as far as possible few methylene dichloride dissolving step (3) gained crude extract, then be separated trans-resveratrol, ε-grape element through normal pressure silica gel (200-300 order) chromatography column, wash-out obtains the elutriant containing trans-resveratrol crude product and the elutriant containing ε-grape element crude product respectively, during chromatographic elution, first use 4BV's, carry out wash-out for the first time by methyl chloride and the methyl alcohol mixing solutions that 19:1 forms by volume and also collect the elutriant of first time wash-out, use 3BV's again, carry out second time wash-out by methyl chloride and the methyl alcohol mixing solutions that 16:1 forms by volume and collect the elutriant of second time wash-out, then use 3BV's, carry out wash-out for the third time by methyl chloride and the methyl alcohol mixing solutions that 13:1 forms by volume and also collect the elutriant of third time wash-out, finally use 3BV's, carried out the 4th wash-out by methyl chloride and the methyl alcohol mixing solutions that 10:1 forms by volume and collected the 4th elutriant, during wash-out, control the equal 1BV/h of flow velocity of elutriant, the elutriant of wash-out and the elutriant of second time wash-out are the elutriant containing trans-resveratrol for the first time, the elutriant of wash-out and the elutriant of the 4th wash-out are the elutriant containing ε-grape element for the third time.
Elutriant gained being contained ε-grape element crude product obtains ε-grape element crude product 40 DEG C of concentrating under reduced pressure dryings, and ε-grape cellulose content is 76.5%.
(5) finished product preparation: by step (4) gained ε-grape element crude product wet method loading after as far as possible few dissolve with methanol, through hydroxypropyl sephadex column (SephadexLH-20) chromatography, pure methanol-eluted fractions, the flow velocity of elutriant is 1.5BV/h, obtain the elutriant containing ε-grape element, isopyknic distilled water is added again to containing in the elutriant of ε-grape element, be placed in ventilating kitchen, volatilization organic phase is to starting to occur crystallization, then go to 4 DEG C of refrigerators and place 24 hours, 5000r/min centrifugal treating, centrifugal gained throw out is through recrystallization, after lyophilize, ε in product-grape cellulose content is 98.1%.
Embodiment 3
(1) pre-treatment: the branch that given up by the grape of pruning dries in the shade January naturally, then cross 20 mesh sieves after pulverizing.
(2) extract: by pretreated raw material, drop in extractor, add 85% ethanol ultrasonic extraction 2 times, extract 0.7h at every turn, during extracted twice, the solid-to-liquid ratio of solvent and raw material is respectively 6mL/g and 4mL/g.Merge extracted twice liquid, proceeded to by extracting solution in thickener, be evaporated to without alcohol taste, temperature controls at 50 DEG C.
(3) purifying: by step (2) gained extracting solution 5000r/min centrifugal treating, obtain supernatant liquor, through macroporous resin D101 chromatography purification, loading speed is 3BV/h.After completion of the sample, first wash 2BV with water, then directly use 80% ethanol elution 4BV.Proceed in thickener by 80% ethanol eluate, be evaporated to medicinal extract shape, then proceed to dry (temperature controls at 55 DEG C) in vacuum drying oven, after dry, in crude extract, ε-grape cellulose content is 10.3%.。
(4) trans-resveratrol, ε-grape element is separated: by wet method loading after as far as possible few trichloromethane dissolving step (3) gained crude extract, then be separated trans-resveratrol, ε-grape element through normal pressure silica gel (200-300 order) chromatography column, wash-out obtains the elutriant containing trans-resveratrol crude product and the elutriant containing ε-grape element crude product respectively, during chromatographic elution, first use 3BV's, carry out wash-out for the first time by methyl chloride and the methyl alcohol mixing solutions that 19:1 forms by volume and also collect the elutriant of first time wash-out, use 3BV's again, carry out second time wash-out by methyl chloride and the methyl alcohol mixing solutions that 16:1 forms by volume and collect the elutriant of second time wash-out, then use 3BV's, carry out wash-out for the third time by methyl chloride and the methyl alcohol mixing solutions that 13:1 forms by volume and also collect the elutriant of third time wash-out, finally use 3BV's, carried out the 4th wash-out by methyl chloride and the methyl alcohol mixing solutions that 10:1 forms by volume and collected the 4th elutriant, during wash-out, the flow velocity controlling elutriant is 1.5BV/h, the elutriant of wash-out and the elutriant of second time wash-out are the elutriant containing trans-resveratrol for the first time, the elutriant of wash-out and the elutriant of the 4th wash-out are the elutriant containing ε-grape element for the third time.
Elutriant gained being contained ε-grape element crude product obtains ε-grape element crude product 50 DEG C of concentrating under reduced pressure dryings, and ε-grape cellulose content is 78.1%.
(5) finished product preparation: by step (4) gained ε-grape element crude product wet method loading after as far as possible few dissolve with methanol, through hydroxypropyl sephadex column (SephadexLH-20) chromatography, pure methanol-eluted fractions, the flow velocity of elutriant is 2BV/h, obtain the elutriant containing ε-grape element, isopyknic distilled water is added again to containing in the elutriant of ε-grape element, be placed in ventilating kitchen, volatilization organic phase is to starting to occur crystallization, then go to 4 DEG C of refrigerators and place 24 hours, 5000r/min centrifugal treating, centrifugal gained throw out is through recrystallization, after lyophilize, ε in product-grape cellulose content is 98.3%.
Claims (5)
1. from grape branches and tendrils, extract a method for high purity ε-grape element, it is characterized in that comprising the steps:
Step one is separated trans-resveratrol, ε-grape element
After the solid mixture of trans-resveratrol and ε-grape element is dissolved in methyl chloride, carry out column chromatography, obtain the elutriant containing ε-grape element crude product;
During chromatography dress post, adopt wet method loading; During wash-out, elutriant is made up of methyl chloride and methyl alcohol, adopts gradient elution mode, is that 19:1,16:1,13:1,10:1 carry out wash-out successively by the volume ratio of methyl chloride in elutriant and methyl alcohol; When the volume ratio of methyl chloride and methyl alcohol is 19:1,16:1 in elutriant, collect the elutriant obtained containing trans-resveratrol; When the volume ratio of methyl chloride and methyl alcohol is 13:1,10:1 in elutriant, collect the elutriant for containing ε-grape element obtained; During each wash-out, the consumption of elutriant is 2-4BV; During wash-out, control the equal 0.5-1.5BV/h of flow velocity of elutriant;
Described methyl chloride is methylene dichloride or trichloromethane;
Chromatography column used is alumina chromatographic column or silica gel column chromatography; In described silica gel column chromatography, the size of silica gel particle is 200-300 order;
The described solid mixture containing trans-resveratrol and ε-grape element is prepared by following step:
Step a extracts
Be the ratio of 4-6mL/g in liquid-solid ratio, add in extraction agent after the plant containing ε-grape element is crushed to-20 orders, after extracting 0.5-1h at 20-60 DEG C, obtain extracting solution; Described extraction agent is water by water and organic solvent by volume: organic solvent=3-5:15-17 forms, and described organic solvent is selected from the one in ethanol, methyl alcohol, ethyl acetate;
Step b purifying
Carry out centrifugation with the centrifugal speed of 3000-5000r/min to step a gained extracting solution, the supernatant liquor macroporous resin obtained carries out chromatography purification, first washes with water during chromatography, then uses ethanolic soln wash-out; Collect ethanol eluate, in 50-60 DEG C, concentrated, drying obtains the solid mixture containing trans-resveratrol and ε-grape element; Or
By step a gained extracting solution, below 60 DEG C after removing organic solvent wherein, add isopyknic extraction agent wherein and extract, after extraction terminates, get organic phase concentrated, dry in 40-60 DEG C, obtain the solid mixture containing trans-resveratrol and ε-grape element; The selection system of extraction agent is: when organic solvent in extraction agent be ethanol or methyl alcohol time, extraction agent used is ethyl acetate; When organic solvent in extraction agent is ethyl acetate, extraction agent used is chloroform or methylene dichloride;
Step 2 is refined
Elutriant step one gained being contained ε-grape element crude product concentrates, drying temperature is 40-50 DEG C, after the ε obtained-grape element crude product is dissolved in methyl alcohol, the mode of wet method loading is adopted to load in hydroxypropyl sephadex column, carry out column chromatography, the elutriant containing ε-grape element obtained, after equal-volume distilled water diluting, the methyl alcohol volatilized under room temperature in elutriant is to when starting to occur crystallization, be cooled to 2-6 DEG C, after leaving standstill, completing crystallization, centrifugation, obtains the ε-grape element monomer of purity >=98%; The model of described hydroxypropyl sephadex column is LH-20; , the flow velocity of elutriant is 1-2BV/h.
2. a kind of method extracting high purity ε-grape element from grape branches and tendrils according to claim 1, is characterized in that: the described plant containing ε-grape element is grape branches and tendrils; Described grape branches and tendrils are through naturally drying in the shade the 1-3 month.
3. a kind of method extracting high purity ε-grape element from grape branches and tendrils according to claim 1, is characterized in that: described in be extracted as supersound extraction or refluxing extraction.
4. a kind of method extracting high purity ε-grape element from grape branches and tendrils according to claim 1, is characterized in that: macroporous resin described in step b is selected from the one in D101 type macroporous resin, AB-8 type macroporous resin, D102 type macroporous resin.
5. a kind of method extracting high purity ε-grape element from grape branches and tendrils according to claim 1, is characterized in that: in step b, and during chromatography purification, controlling loading speed is 2-3BV/h; After completion of the sample, first carry out wash-out with the water of 1-3BV, then carry out wash-out with the ethanol of 2-6BV and collect ethanol eluate; Ethanol eluate carries out vacuum-drying after being evaporated to medicinal extract shape at 50-60 DEG C, obtains the solid mixture containing trans-resveratrol and ε-grape element; The concentration expressed in percentage by volume of described ethanol is 70-90%.
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