CN104087623A - Method for extracting resveratrol from giant knotweed by enzymolysis - Google Patents
Method for extracting resveratrol from giant knotweed by enzymolysis Download PDFInfo
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Abstract
The invention relates to a method for extracting resveratrol from a natural plant giant knotweed, particularly a method for extracting resveratrol from giant knotweed by enzymolysis. The method comprises the following steps: by using commercially available giant knotweed rhizome as the raw material, carrying out enzymolysis reaction, alcohol deep extraction and separation concentration, and drying to obtain the resveratrol crystal powder product. The method has the characteristics of sufficient enzymolysis, short time, low operating cost, high operation safety and the like, fully utilizes the raw materials and organic solvent, is beneficial to environmental protection since the waste liquid is mainly water, and can easily implement industrialized popularization and application. The resveratrol crystal powder product prepared by the method has high purity, and the maximum purity is 98.31%. The overall yield is high, and the maximum overall yield is 1.40%. The method can be widely used for producing the resveratrol crystal product. The product prepared by the method can be widely used in fine chemical engineering production of medical drugs for preventing cardiovascular diseases, protecting the liver and resisting cancers and skin-care beauty-treatment products.
Description
One, technical field
The invention belongs to the technical field of extracting trans-resveratrol from natural phant, be specifically related to prepare the method for trans-resveratrol from the giant knotweed of natural phant.
Two, background technology
Trans-resveratrol is a kind of non-flavonoid polyphenolic substance that contains stilbene class formation, chemistry Resveratrolk, white or light yellow crystalline powder by name.Trans-resveratrol can prophylaxis of tumours Cancerous disease, can prevent cardiovascular and cerebrovascular diseases again, have protect the liver, anti-oxidant, regulate blood fat balance, the health-care effect such as delay senility, be that human health is had to the significantly actively active substance of effect, so it applies very extensive aspect medicines and health protection.Because trans-resveratrol is the polyphenolic compound of stilbene class material, there is phenols and alkene class character, with Air exposing or illumination condition under, trans-resveratrol can be oxidized or isomeric transition occurs be generated irreversible material, the physiologically active loss that goes to pot, therefore in order to improve trans-resveratrol yield and to extend the shelf-time, the production of trans-resveratrol and to preserve range request stricter.Trans-resveratrol is prevalent in the natural fronds such as giant knotweed, peanut, mulberries, Vitaceae and is mainly to exist with metastable polydatin molecular form.Particularly the polydatin in giant knotweed and trans-resveratrol total content are higher than other natural phant, and suitability for industrialized production potential benefit is huge.Therefore, from giant knotweed, extract the emphasis that trans-resveratrol is current research.
The existing method of extracting trans-resveratrol from giant knotweed, as the publication No. of announcing on April 17th, 2013 " a kind of production technique of preparing trans-resveratrol from the giant knotweed " patent that is CN103044210A, disclosed method is: take giant knotweed as raw material, through enzymolysis dry, continuous countercurrent extracting tank acetone refluxing extraction, filtering and concentrating, ethyl acetate extraction, concentrated, silica gel column chromatography, condensing crystal makes trans-resveratrol crystal finished product.The main drawback of the method is: 1. the constant temperature enzyme digestion reaction time longer, long-time high temperature is unfavorable for the steady chemical structure of trans-resveratrol; 2. because trans-resveratrol reduces content exposing under illumination condition easily inactivation, so after enzymolysis, airing operation is also unfavorable for the steady chemical structure of trans-resveratrol; 3. because acetone boiling point is lower, volatile, there is certain toxicity, with the air mixed danger of easily blasting, therefore, it is during as countercurrent extraction solvent, the insulation of relevant device and relative the having relatively high expectations of sealing; 4. adopt ethyl acetate-sherwood oil as separated eluting solvent, wherein sherwood oil toxicity is larger and inflammable, and solvent recuperation process relates to larger potential safety hazard.
Three, summary of the invention
The object of the invention is the deficiency of extracting trans-resveratrol method for existing from giant knotweed, provide a kind of from giant knotweed the method for enzymolysis and extraction trans-resveratrol, have that enzymolysis is abundant and the time is short, production unit is simple, running cost is low, safety in operation is high, and environmental protection is easy to promote the features such as suitability for industrialized production.
Cardinal principle of the present invention is: Resveratrol in Rhizoma Polygoni Cuspidati is mainly with the existing of polydatin form, and its molecular formula is C
20h
22o
8, chemistry is by name 3,4 ', 5-trihydroxy-toluylene-3-β-D-Glucose glycosides, it has good solubleness in hot water is molten, is soluble in the organic solvents such as methyl alcohol, ethanol.Under suitable pH value certain temperature and agitation condition, progressively stripping from giant knotweed of polydatin, the enzyme in solution acts on β-D glycosidic link of polydatin simultaneously, makes its hydrolysis generate trans-resveratrol (C
14h
12o
3) and glucose.In addition, enzyme also can destroy giant knotweed cell walls, is conducive to stripping and the conversion of polydatin.
The technical scheme that realizes the object of the invention is: a kind of from giant knotweed the method for enzymolysis and extraction trans-resveratrol, take commercially available giant knotweed rhizome as raw material, through enzyme digestion reaction, alcohol depth extraction, make trans-resveratrol crystal powder product with separated concentrated, dry simple process.The concrete steps of described method are as follows:
(1) enzyme digestion reaction
First commercially available giant knotweed rhizome raw material is positioned in pulverizer and is pulverized, then carry out sub-sieve through 240～320 object sieves, collect the powder sieving, standby, unsifted particle returns in pulverizer and pulverizes again.Then, in the quality of Giant Knotweed Rhizome (g): the quality of cellulase (mg): the ratio of the volume of NaAc_HAc buffer solution (mL) is 1: (1～10): the ratio of (10～50), after first regulating the pH value of described buffered soln to be 4～6 with concentrated hydrochloric acid or sodium hydroxide solution, by the buffered soln after Giant Knotweed Rhizome and adjusting pH value, be positioned over successively in batch reactor, under the rotating speed of 30～60 ℃ of constant temperature and 80～200r/min, stir after 15～30min, add again cellulase, continuing constant temperature stirs, carry out enzyme digestion reaction 15～30h, just make the solidliquid mixture after enzyme digestion reaction.
(2) alcohol depth extraction
After (1) step completes, the solidliquid mixture after the enzyme digestion reaction first (1) step being made, is positioned in pressure filter and carries out solid-liquid separation, collects respectively filtrate (being the filtrate after enzymolysis and extraction) and solid residue.After according to the quality of solid residue (g): the ratio of the volume of the methanol solution that volume fraction is 50%～75% (mL) is 1: the ratio of (5～20), described solid residue and described methanol solution are positioned in extractor, in temperature, be under 35～65 ℃, the stirring velocity condition that is 80～200r/min, to carry out for the first time after alcohol extracting 15～60mm, and filter for the first time, collect respectively filtered liquid and the solid residue of alcohol extracting for the first time.Then, equal volume while getting alcohol extracting for the first time, the methanol solution of equal concentrations and the solid residue of collecting for the first time, put back to again in extractor, in temperature, be under 35～65 ℃, the stirring velocity condition that is 80～200r/min, to carry out for the second time after alcohol extracting 15～60min, and filter for the second time, collect respectively filtered liquid and the solid residue of alcohol extracting for the second time, so repeat 1～3 time, in order to solid residue is carried out to degree of depth alcohol extracting.Finally, merge the alcohol extracting filtered liquid of each collection, be positioned in evaporation concentrator, carry out evaporation concentration to without till alcohol taste, collect respectively the solvent of concentrated solution and evaporation.To the solvent (being methyl alcohol) of collecting, recycling.The concentrated solution that filtrate after merging enzymolysis and extraction and each alcohol extracting are collected, just makes trans-resveratrol crude product liquid, for next step separation and purification.
(3) prepare trans-resveratrol crystal powder product
1. the initial gross separation of macroporous resin and wash-out
After (2) step completes, according to macroporous resin quality (g): the ratio of the volume (mL) of the trans-resveratrol crude product liquid that (2) step makes is 1: the ratio of (6～10), first adopt conventional macroporous resin completion method, macroporous resin is packed in separator column, with constant flow pump, by volume, it is ethanolic soln 1～3BV (being the bed volume of 1～3 times of weighting material), that volume fraction is 10～20%, flow with 1～3BV/h passes in described separator column, carry out rinse, and the rinse liquid of collection outflow, through evaporation, reclaim alcohol solvent, recycling.Then, by described trans-resveratrol crude product liquid, with the flow of 1～3BV/h, pass into described separator column and adsorb, and collect the solution flowing out.To the solution-treated rear discharge up to standard of collecting.Then, the distilled water that is 2～5BV by volume, passes in described separator column and carries out wash-out for the first time with the flow of 1～3BV/h, in order to adsorption component (being mainly carbohydrate and plant proteins material) wash-out not, collect the elutriant flowing out for the first time, treated rear discharge up to standard.Again then, the ethanolic soln that is first 3～5BV by volume, volume fraction is 20～30%, passes in described separator column and carries out wash-out for the second time with the flow of 1～3BV/h, in order to by part pigment wash-out, collect the elutriant flowing out for the second time, through evaporation, reclaim alcohol solvent, recycling.After ethanolic soln that be 3～5BV by volume, that volume fraction is 30～50%, with the flow of 1～3BV/h, pass in described separator column and carry out wash-out for the third time, in order to by polydatin material wash-out, collect the elutriant flowing out for the third time, and carry out evaporation concentration by evaporation concentrator, collect respectively evaporated liquor (being alcohol solvent) and concentrated solution (being polydatin enrichment concentrated solution).The polydatin enrichment concentrated solution of collecting is stayed and used it for anything else.The ethanolic soln that is 4～10BV by volume again, volume fraction is 60～80%, with the flow of 1～3BV/h, pass in described separator column and carry out wash-out the 4th time again, in order to by trans-resveratrol material wash-out, collect the elutriant flowing out for the 4th time, and by evaporation concentrator evaporation concentration, collect respectively evaporated liquor (being alcohol solvent) and concentrated solution (being trans-resveratrol enrichment concentrated solution).Recycling to the evaporated liquor (being alcohol solvent) of collecting; The concentrated solution (being trans-resveratrol enrichment concentrated solution) of collecting is prepared to trans-resveratrol crystal product for next step.To the macroporous resin in described separator column, by conventional sodium hydroxide-salt acid system, carry out manipulation of regeneration, can be recycled.To described macroporous resin, be NKA-9 type or NKA-II type or AB-8 type or HPD950 type or HPD400 type etc.
2. prepare trans-resveratrol crystal powder product
(3)-after 1. having walked, in the quality of neutral alumina (g): the ratio of the volume (mL) of (3)-concentrated solution (being trans-resveratrol enrichment concentrated solution) that 1. step is collected is 1: the ratio of (10～20), after adopting conventional fill method that neutral alumina is packed in chromatography column, first use constant flow pump, the ethanolic soln that is 1～3BV by volume, volume fraction is 60～80%, with the flow of 1～3BV/h, pass in described chromatography column and carry out rinse, collect the rinse liquid flowing out, and by evaporation concentrator, alcohol solvent recycling is reclaimed in evaporation.Again respectively by described concentrated solution (being trans-resveratrol enrichment concentrated solution) and volume ethanolic soln that be 1～3BV, that volume fraction is 60～80%, with the flow of 1～3BV/h, pass in described chromatography column and decolour, collect the destainer (i.e. resveratrol solution after decolouring) flowing out.Then, to the destainer of collecting (i.e. resveratrol solution after decolouring), by evaporation concentrator evaporation concentration, collect respectively evaporated liquor (being alcohol solvent) and concentrated solution (being trans-resveratrol concentrated solution).Evaporated liquor (being alcohol solvent) to collecting, can reuse; To the concentrated solution (being trans-resveratrol concentrated solution) of collecting, the resveratrol solution after the purifying for the first time making exactly.Finally, in the quality of gac (g): the ratio of the volume of the resveratrol solution after purifying (mL) is 1 for the first time: the ratio of (50～100), by resveratrol solution and gac after purifying for the first time, be positioned in bleacher, stirring at normal temperature 1～5h carries out for the second time after purifying (by activated carbon decolorizing purifying), mixture after purifying is for the second time carried out to suction filtration, collect respectively suction filtration slag (being activated charcoal solid) and suction filtration liquid (being the resveratrol solution after purifying for the second time), the suction filtration slag of collecting is recycled to recycling.By suction filtration liquid (for the second time the resveratrol solution after purifying), pass through evaporation concentrator, to carry out evaporation concentration to the volume of concentrated solution be concentrated front volume till 1/5～1/30, collects respectively evaporated liquor (being alcohol solvent) and concentrated solution (for the second time the trans-resveratrol concentrated solution after purifying).The evaporated liquor (being alcohol solvent) of collecting, can reuse; To the concentrated solution of collecting (for the second time the trans-resveratrol concentrated solution after purifying), be positioned in freeze drying box, first at-10～-18 ℃ of pre-freeze 2～3h, at pressure, be that 20～60Pa, temperature are under-40～-60 ℃ of conditions again, lyophilize 24～30h, the trans-resveratrol crystal powder product that just make quality purity and be 98.07%～98.31%, productive rate is 1.12%～1.40%.
The present invention adopts after technique scheme, mainly contains following effect:
1. the present invention be take commercially available giant knotweed rhizome as raw material, after being crushed to certain order number, under the aqueous buffer solution action condition of cellulase and certain pH value, enzymolysis transforms and obtains trans-resveratrol, then through methanol solution, carry out degree of depth lixiviate, therefore trans-resveratrol product overall yield is high, and overall yield is up to 1.40%;
2. in macroporous resin separating treatment technique of the present invention, adopt the ethanolic soln of different volumes mark (20～30%, 30～50%, 60～80%), to absorption, there is the punching resin of trans-resveratrol crude product to carry out wash-out respectively, and last successively use the removal of impurities of further decolouring of aluminum oxide and gac, therefore the quality purity of trans-resveratrol product is high, and highest purity reaches 98.31%.
3. cellulase usage quantity of the present invention is few, and minimum enzyme amount is amounted to into butt Giant Knotweed Rhizome meter and is about 1 ‰, and enzymolysis is abundant and the time is short, and approximately 15～30 hours, greatly shorten the production cycle, reduce production costs, be easy to promote suitability for industrialized production;
4. methanol extraction of the present invention workshop section takes the mode of extracting after first solid-liquid separation, has greatly saved the usage quantity of methanol solvate solvent, has improved extraction efficiency and the trans-resveratrol productive rate of giant knotweed raw material;
5. in the present invention, be with an organic solvent mainly methyl alcohol and ethanol, solvent toxicity is low, and methyl alcohol and ethanol moderate boiling point are convenient to reclaim, and make full use of organic solvent and save energy, and main waste liquid is water, meets green production requirement and safety in operation high;
6. in the present invention, because foreign pigment is mainly Flavonoid substances and easy and Al
2o
3in conjunction with, therefore adopt Al
2o
3with the method that gac is successively used in conjunction with, trans-resveratrol is refined, efficiently removed the remaining foreign pigment in trans-resveratrol enrichment positions; In addition Al,
2o
3can reprocessing cycle use directly in 500～700 ℃ of calcining 1～2h, non-pollutant discharge, easy and simple to handle.Through Al
2o
3remove after most of pigment and impurity, remaining pigment only just can be removed with a small amount of gac, has reduced gac usage quantity, has further reduced production cost.
The present invention can be widely used in producing trans-resveratrol crystal product, and the product that adopts the inventive method to produce can be widely used in the medical drugs of preventing cardiovascular disease and liver-protecting and cancer-resisting and the fine chemical product of cosmetology.
Embodiment
Below in conjunction with embodiment, further the present invention.
Embodiment 1
A method for enzymolysis and extraction trans-resveratrol from giant knotweed, the concrete steps of described method are as follows:
(1) enzyme digestion reaction
First commercially available giant knotweed rhizome raw material is positioned in pulverizer and is pulverized, then carry out sub-sieve through 280 object sieves, collect the powder sieving, standby, unsifted particle returns in pulverizer and pulverizes again.Then, in the quality of Giant Knotweed Rhizome (g): the quality of cellulase (mg): the ratio that the ratio of the volume of NaAc_HAc buffer solution (mL) is 1: 2: 20, after first regulating the pH value of described buffered soln to be 4.7 with concentrated hydrochloric acid or sodium hydroxide solution, by the buffered soln after Giant Knotweed Rhizome and adjusting pH value, be positioned over successively in batch reactor, under the rotating speed of 45 ℃ of constant temperature and 200r/min, stir after 20min, add again cellulase, continuing constant temperature stirs, carry out enzyme digestion reaction 20h, just make the solidliquid mixture after enzyme digestion reaction.
(2) alcohol depth extraction
After (1) step completes, solidliquid mixture after the enzyme digestion reaction first (1) step being made, be positioned in pressure filter and carry out solid-liquid separation, collect respectively filtrate (being the filtrate after enzymolysis and extraction) and solid residue (being the Giant Knotweed Rhizome after enzyme digestion reaction).After according to the quality of solid residue (g): the ratio that the ratio of the volume of the methanol solution that volume fraction is 65% (mL) is 1: 10, described solid residue and described methanol solution are positioned in extractor, in temperature, be under 45 ℃, the stirring velocity condition that is 200r/min, to carry out for the first time after alcohol extracting 30min, and filter for the first time, collect respectively filtered liquid and the solid residue of alcohol extracting for the first time.Then, equal volume while getting alcohol extracting for the first time, the methanol solution of equal concentrations and the solid residue of collecting for the first time, put back to again in extractor, in temperature, be under 45 ℃, the stirring velocity condition that is 200r/min, to carry out for the second time after alcohol extracting 30min, and filter for the second time, collect respectively filtered liquid and the solid residue of alcohol extracting for the second time, so repeat 1 time, in order to solid residue is carried out to degree of depth alcohol extracting.Finally, merge the alcohol extracting filtered liquid of each collection, be positioned in evaporation concentrator, carry out evaporation concentration to without till alcohol taste, collect respectively the solvent of concentrated solution and evaporation.To the solvent (being methyl alcohol) of collecting, recycling.The concentrated solution that filtrate after merging enzymolysis and extraction and each alcohol extracting are collected, just makes trans-resveratrol crude product liquid, for next step separation and purification.
(3) prepare trans-resveratrol crystal powder product
1. the initial gross separation of macroporous resin and wash-out
After (2) step completes, according to macroporous resin quality (g): the ratio that the ratio of the volume (mL) of the trans-resveratrol crude product liquid that (2) step makes is 1: 6, first adopt conventional macroporous resin completion method, macroporous resin is packed in separator column, with constant flow pump, the ethanolic soln that is 2BV by volume, volume fraction is 20%, flow with 2BV/h passes in described separator column, carries out rinse, and collects the rinse liquid flowing out, through evaporation, reclaim alcohol solvent, recycling.Then, by described trans-resveratrol crude product liquid, with the flow of 1BV/h, pass into described separator column and adsorb, and collect the solution flowing out.To the solution-treated rear discharge up to standard of collecting.Then, the distilled water that is 3BV by volume, passes in described separator column and carries out wash-out for the first time with the flow of 1BV/h, in order to adsorption component (being mainly carbohydrate and plant proteins material) wash-out not, collect the elutriant flowing out for the first time, treated rear discharge up to standard.Again then, the ethanolic soln that is first 4BV by volume, volume fraction is 25%, passes in described separator column and carries out wash-out for the second time with the flow of 2BV/h, in order to by part pigment wash-out, collect the elutriant flowing out for the second time, through evaporation, reclaim alcohol solvent, recycling.After ethanolic soln that be 4BV by volume, that volume fraction is 45%, with the flow of 2BV/h, pass in described separator column and carry out wash-out for the third time, in order to by polydatin material wash-out, collect the elutriant flowing out for the third time, and carry out evaporation concentration by evaporation concentrator, collect respectively evaporated liquor (being alcohol solvent) and concentrated solution (being polydatin enrichment concentrated solution).The polydatin enrichment concentrated solution of collecting is stayed and used it for anything else.The ethanolic soln that is 8BV by volume again, volume fraction is 70%, with the flow of 2BV/h, pass in described separator column and carry out wash-out the 4th time again, in order to by trans-resveratrol material wash-out, collect the elutriant flowing out for the 4th time, and by evaporation concentrator evaporation concentration, collect respectively evaporated liquor (being alcohol solvent) and concentrated solution (being trans-resveratrol enrichment concentrated solution).Recycling to the evaporated liquor (being alcohol solvent) of collecting; The concentrated solution (being trans-resveratrol enrichment concentrated solution) of collecting is prepared to trans-resveratrol crystal product for next step.To the macroporous resin in described separator column, by conventional sodium hydroxide-salt acid system, carry out manipulation of regeneration, can be recycled.To described macroporous resin, it is NKA-9 type.
2. prepare trans-resveratrol crystal powder product
(3)-after 1. having walked, in the quality of neutral alumina (g): the ratio that the ratio of the volume (mL) of (3)-concentrated solution (being trans-resveratrol enrichment concentrated solution) that 1. step is collected is 1: 15, after adopting conventional fill method that neutral alumina is packed in chromatography column, first use constant flow pump, the ethanolic soln that is 2BV by volume, volume fraction is 70%, with the flow of 2BV/h, pass in described chromatography column and carry out rinse, collect the rinse liquid flowing out, and by evaporation concentrator, alcohol solvent recycling is reclaimed in evaporation.Again respectively by described concentrated solution (being trans-resveratrol enrichment concentrated solution) and volume ethanolic soln that be 2BV, that volume fraction is 70%, with the flow of 2BV/h, pass in described chromatography column and decolour, collect the destainer (i.e. resveratrol solution after decolouring) flowing out.Then, to the destainer of collecting (i.e. resveratrol solution after decolouring), by evaporation concentrator evaporation concentration, collect respectively evaporated liquor (being alcohol solvent) and concentrated solution (being trans-resveratrol concentrated solution).Evaporated liquor (being alcohol solvent) to collecting, can reuse; To the concentrated solution (being trans-resveratrol concentrated solution) of collecting, the resveratrol solution after the purifying for the first time making exactly.Finally, in the quality of gac (g): the ratio that the ratio of the volume of the resveratrol solution after purifying (mL) is 1: 75 for the first time, by resveratrol solution and gac after purifying for the first time, be positioned in bleacher, stirring at normal temperature 3h carries out for the second time after purifying (by activated carbon decolorizing purifying), mixture after purifying is for the second time carried out to suction filtration, obtain the resveratrol solution after purifying for the second time.Again by the resveratrol solution after purifying for the second time, pass through evaporation concentrator, to carry out evaporation concentration to the volume of concentrated solution be concentrated front volume till 1/5, collects respectively evaporated liquor (being alcohol solvent) and concentrated solution (for the second time the trans-resveratrol concentrated solution after purifying).The evaporated liquor (being alcohol solvent) of collecting, can reuse; To the concentrated solution of collecting (for the second time the trans-resveratrol concentrated solution after purifying), be positioned in freeze drying box, first at-18 ℃ of pre-freeze 2h, at pressure, be that 20Pa, temperature are under-50 ℃ of conditions again, lyophilize 27h, the trans-resveratrol crystal powder product that just make quality purity and be 98.31%, productive rate is 1.40%.
Embodiment 2
A method for enzymolysis and extraction trans-resveratrol from giant knotweed, with embodiment 1, wherein:
In (1) step, sub-sieve is 240 orders, the quality of Giant Knotweed Rhizome (g): the quality of cellulase (mg): the ratio of the volume of NaAc_HAc buffer solution (mL) is 1: 1: 10, regulate described pH value of buffer solution to 4.0, under the speed conditions that is 150r/min in 60 ℃ of constant temperature, stirring velocity, enzyme digestion reaction 30h.
In (2) step, the quality of solid residue (g): the ratio of the volume of the methanol solution that volume fraction is 50% (mL) is 1: 20, the volume fraction of methanol solution is 50%, in temperature, be under 35 ℃, the stirring velocity speed conditions that is 150r/min, carry out alcohol extracting 60min for the first time.In temperature, be under 35 ℃, the stirring velocity speed conditions that is 150r/min, carry out alcohol extracting 60min for the second time, so repeat 2 times.
(3)-in 1. walking, macroporous resin quality (g): the ratio of the volume (mL) of the trans-resveratrol crude product liquid that (2) step makes is 1: 8, during rinse, volumes of aqueous ethanol mark is 10%, volumes of aqueous ethanol is 1BV, and rinse flow is 1BV/h; The upper prop flow of trans-resveratrol crude product liquid is 2BV/h; In wash-out, distilled water elution volume is 2BV for the first time, and flow is 2BV/h; In wash-out, volumes of aqueous ethanol mark is 20% for the second time, and volumes of aqueous ethanol is 3BV, wash-out flow is 1BV/h; In wash-out, volumes of aqueous ethanol mark is 30% for the third time, and ethanolic soln body is 5BV, and wash-out flow is 1BV/h; In the 4th wash-out, the volume fraction of ethanolic soln is 60%, and volumes of aqueous ethanol is 10BV, wash-out flow is 3BV/h; To described macroporous resin, it is AB-8 type.
(3)-2. walking the quality of neutral alumina (g): the ratio of the volume (mL) of (3)-concentrated solution (being trans-resveratrol enrichment concentrated solution) that 1. step is collected is 1: 10; During rinse, volumes of aqueous ethanol mark is 60%, and volumes of aqueous ethanol is 3BV, and rinse flow is 1BV/h; During decolouring, volumes of aqueous ethanol mark is 60%, and volumes of aqueous ethanol is 1BV, and flow is 1BV/h; The quality of gac (g): the ratio of the volume of the resveratrol solution after purifying (mL) is 1: 50 for the first time, stirring at normal temperature 1h, the volume of evaporation concentration liquid is 1/15 of concentrated front volume; During lyophilize, first at-14 ℃ of pre-freeze 2.5h, then pressure be 40Pa, temperature under-60 ℃ of conditions, lyophilize 30h, the trans-resveratrol crystal powder product that make quality purity and be 98.07%, productive rate is 1.12%.
Embodiment 3
A method for enzymolysis and extraction trans-resveratrol from giant knotweed, with embodiment 1, wherein:
In (1) step, sub-sieve is 320 orders, the quality of Giant Knotweed Rhizome (g): the quality of cellulase (mg): the ratio of the volume of NaAc_HAc buffer solution (mL) is 1: 10: 50, regulate described pH value of buffer solution to 6.0, under the speed conditions that is 80r/min in 30 ℃ of constant temperature, stirring velocity, enzyme digestion reaction 15h.
In (2) step, the quality of solid residue (g): the ratio of the volume of the methanol solution that volume fraction is 50% (mL) is 1: 5, the volume fraction of methanol solution is 75%, in temperature, is under 65 ℃, the stirring velocity speed conditions that is 80r/min, carries out alcohol extracting 15min for the first time.In temperature, be under 65 ℃, the stirring velocity speed conditions that is 80r/min, carry out alcohol extracting 15min for the second time, so repeat 3 times.
(3)-in 1. walking, macroporous resin quality (g): the ratio of the volume (mL) of the trans-resveratrol crude product liquid that (2) step makes is 1: 10, during rinse, volumes of aqueous ethanol mark is 15%, volumes of aqueous ethanol is 3BV, and rinse flow is 3BV/h; The upper prop flow of trans-resveratrol crude product liquid is 3BV/h; In wash-out, distilled water elution volume is 5BV for the first time, and flow is 3BV/h; In wash-out, volumes of aqueous ethanol mark is 30% for the second time, and volumes of aqueous ethanol is 5BV, wash-out flow is 3BV/h; In wash-out, volumes of aqueous ethanol mark is 50% for the third time, and ethanolic soln body is 3BV, and wash-out flow is 3BV/h; In the 4th wash-out, the volume fraction of ethanolic soln is 80%, and volumes of aqueous ethanol is 4BV, wash-out flow is 1BV/h; To described macroporous resin, it is NKA-II type.
(3)-2. walking the quality of neutral alumina (g): the ratio of the volume (mL) of (3)-concentrated solution (being trans-resveratrol enrichment concentrated solution) that 1. step is collected is 1: 20; During rinse, volumes of aqueous ethanol mark is 80%, and volumes of aqueous ethanol is 1BV, and rinse flow is 3BV/h; During decolouring, volumes of aqueous ethanol mark is 80%, and volumes of aqueous ethanol is 3BV, and flow is 3BV/h; The quality of gac (g): the ratio of the volume of the resveratrol solution after purifying (mL) is 1: 100 for the first time, stirring at normal temperature 5h, the volume of evaporation concentration liquid is 1/30 of concentrated front volume; During lyophilize, first at-10 ℃ of pre-freeze 3h, then pressure be 60Pa, temperature under-40 ℃ of conditions, lyophilize 24h, the trans-resveratrol crystal powder product that make quality purity and be 98.15%, productive rate is 1.12%.
Claims (1)
1. a method for enzymolysis and extraction trans-resveratrol from giant knotweed, is characterized in that the concrete steps of described method are as follows:
(1) enzyme digestion reaction
First commercially available giant knotweed rhizome raw material is positioned in pulverizer and is pulverized, through 240～320 object sieves, carry out sub-sieve again, the powder that collection is sieved, unsifted particle returns in pulverizer and pulverizes again, then, in the quality of Giant Knotweed Rhizome: the quality of cellulase: the ratio of the volume of acetic acid one sodium acetate buffer solution is 1g: (1～10) mg: the ratio of (10～50) mL, after first regulating the pH value of described buffered soln to be 4～6 with concentrated hydrochloric acid or sodium hydroxide solution, by the buffered soln after Giant Knotweed Rhizome and adjusting pH value, be positioned over successively in batch reactor, under the rotating speed of 30～60 ℃ of constant temperature and 80～200r/min, stir after 15～30min, add again cellulase, continuing constant temperature stirs, carry out enzyme digestion reaction 15～30h, just make the solidliquid mixture after enzyme digestion reaction,
(2) alcohol depth extraction
After (1) step completes, solidliquid mixture after the enzyme digestion reaction first (1) step being made, be positioned over and in pressure filter, carry out solid-liquid separation, collect respectively filtrate, be filtrate and solid residue after enzymolysis and extraction, after according to the quality of solid residue: the ratio of the volume of the methanol solution that volume fraction is 50%～75% is 1g: the ratio of (5～20) mL, described solid residue and described methanol solution are positioned in extractor, in temperature, it is 35～65 ℃, stirring velocity is under the condition of 80～200r/min, to carry out for the first time after alcohol extracting 15～60min, and filter for the first time, collect respectively filtered liquid and the solid residue of alcohol extracting for the first time, then, equal volume while getting alcohol extracting for the first time, the methanol solution of equal concentrations and the solid residue of collecting for the first time, put back to again in extractor, in temperature, it is 35～65 ℃, stirring velocity is under the condition of 80～200r/min, to carry out for the second time after alcohol extracting 15～60min, and filter for the second time, collect respectively filtered liquid and the solid residue of alcohol extracting for the second time, so repeat 1～3 time, finally, the alcohol extracting filtered liquid that merges each collection, be positioned in evaporation concentrator, carry out evaporation concentration extremely without till alcohol taste, collect respectively the solvent of concentrated solution and evaporation, the concentrated solution that filtrate after merging enzymolysis and extraction and each alcohol extracting are collected, just make trans-resveratrol crude product liquid,
(3) prepare trans-resveratrol crystal powder product
1. the initial gross separation of macroporous resin and wash-out
After (2) step completes, according to macroporous resin quality: the ratio of the volume of the trans-resveratrol crude product liquid that (2) step makes is 1g: the ratio of (6～10) mL, first adopt conventional macroporous resin completion method, macroporous resin is packed in separator column, with constant flow pump, by volume, be 1～3BV, volume fraction is 10～20% ethanolic soln, flow with 1～3BV/h passes in described separator column, carry out rinse, and the rinse liquid of collection outflow, then, by described trans-resveratrol crude product liquid, with the flow of 1～3BV/h, passing into described separator column adsorbs, and the solution of collection outflow, then, the distilled water that is 2～5BV by volume, with the flow of 1～3BV/h, pass in described separator column and carry out wash-out for the first time, collect the elutriant flowing out for the first time, again then, by volume, be first 3～5BV, volume fraction is 20～30% ethanolic soln, with the flow of 1～3BV/h, pass in described separator column and carry out wash-out for the second time, collect the elutriant flowing out for the second time, after by volume, be 3～5BV, volume fraction is 30～50% ethanolic soln, with the flow of 1～3BV/h, pass in described separator column and carry out wash-out for the third time, collect the elutriant flowing out for the third time, and carry out evaporation concentration by evaporation concentrator, collect respectively evaporated liquor, be alcohol solvent and concentrated solution, it is polydatin enrichment concentrated solution, by volume, be 4～10BV again, volume fraction is 60～80% ethanolic soln, with the flow of 1～3BV/h, pass in described separator column and carry out wash-out the 4th time again, collect the elutriant flowing out for the 4th time, and by evaporation concentrator evaporation concentration, collect respectively evaporated liquor, be alcohol solvent and concentrated solution, it is trans-resveratrol enrichment concentrated solution, to described macroporous resin, be NKA-9 type or NKA-II type or AB-8 type or HPD950 type or HPD400 type,
2. prepare trans-resveratrol crystal powder product
(3)-after 1. having walked, press the quality of neutral alumina: (3)-concentrated solution that 1. step is collected, the ratio that is the volume of trans-resveratrol enrichment concentrated solution is 1g: the ratio of (10～20) mL, after adopting conventional fill method that neutral alumina is packed in chromatography column, first use constant flow pump, by volume, be 1～3BV, volume fraction is 60～80% ethanolic soln, with the flow of 1～3BV/h, pass in described chromatography column and carry out rinse, collect the rinse liquid flowing out, again respectively by described concentrated solution, be that trans-resveratrol enrichment concentrated solution and volume are 1～3BV's, volume fraction is 60～80% ethanolic soln, with the flow of 1～3BV/h, pass in described chromatography column and decolour, collect the destainer flowing out, the resveratrol solution after decolouring, then, to the destainer of collecting, the resveratrol solution after decolouring, by evaporation concentrator evaporation concentration, collect respectively evaporated liquor, be alcohol solvent and concentrated solution, it is trans-resveratrol concentrated solution, to the concentrated solution of collecting, be that trans-resveratrol concentrated solution is exactly the resveratrol solution after the purifying for the first time making, finally, in the quality of gac: the ratio of the volume of the resveratrol solution after purifying is 1g for the first time: the ratio of (50～100) mL, by resveratrol solution and gac after purifying for the first time, be positioned in bleacher, stirring at normal temperature 1～5h carries out for the second time after purifying, mixture after purifying is for the second time carried out to suction filtration, collect respectively suction filtration slag, be activated charcoal solid and suction filtration liquid, be the resveratrol solution after purifying for the second time, by suction filtration liquid, i.e. resveratrol solution after purifying for the second time, pass through evaporation concentrator, to carry out evaporation concentration to the volume of concentrated solution be concentrated front volume till 1/5～1/30, collect respectively evaporated liquor, be alcohol solvent and concentrated solution, i.e. trans-resveratrol concentrated solution after purifying for the second time, to the concentrated solution of collecting, i.e. trans-resveratrol concentrated solution after purifying for the second time, be positioned in freeze drying box, first at-10～-18 ℃ of pre-freeze 2～3h, at pressure, be 20～60Pa again, temperature is under-40～-60 ℃ of conditions, lyophilize 24～30h, just make trans-resveratrol crystal powder product.
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