CN101698634B - Method for extracting and separating resveratrol from giant knotweed rhizome - Google Patents
Method for extracting and separating resveratrol from giant knotweed rhizome Download PDFInfo
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- 235000021283 resveratrol Nutrition 0.000 title abstract description 15
- 229940016667 resveratrol Drugs 0.000 title abstract description 15
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Abstract
The invention relates to a method for extracting and separating resveratrol from giant knotweed rhizome. The method comprises the following steps of grinding, enzymolysis, extraction, centrifugation, column separation and re-crystallization or grinding, extraction, enzymolysis, centrifugation, column separation and re-crystallization. Due to the addition of hydrolytic enzymes at the enzymolysis step, and then separation of the column chromatography, wherein the filling material used in the column chromatography is one of polyacrylic resin CG71, polyacrylic resin RPC 40, polystyrene resin CG161, polystyrene resin CG300, sephadex Sephadex G-25 and sephadex Sephadex LH-20, the technical problems, existing in the conventional method for extracting and separating the resveratrol from the giantknotweed rhizome, of high cost, low product yield, incapacity of converting polydatin into resveratrol, complex process, long period and unsuitability for industrial large-scale production are solved. The method has the advantages of low cost, high yield, simple process, short production period and the suitability for mass industrialized production.
Description
Technical field
The present invention relates to a kind of extraction separation field of flavonoid compound, be specifically related to a kind of method of separating trans-resveratrol of from giant knotweed, extracting.
Background technology
Trans-resveratrol (Resveratrol) is again resveratrol, molecular formula: C
14H
12O
3, relative molecular mass is 228.24.Trans-resveratrol is tasteless, and canescence or white powder are insoluble in cold water, can be dissolved in hot water, ether, ethanol, methyl alcohol, ethyl acetate, acetone etc., and fusing point is 256～257 ℃.Trans-resveratrol is a kind of flavonoid compound that extracts from the pericarp of the rhizome of polygonaceae plant giant knotweed and root or Vitis vitaceae grape.Found first trans-resveratrol in 1940,20 century 70s find to contain in the grape this material first, it is found that afterwards in the plants such as giant knotweed, peanut, mulberry fruit and also contain this composition.Trans-resveratrol is a kind of important phytoalexin, it can stop the oxidation of low-density lipoprotein, thereby have potential anti-cardiovascular disorder, anti-cancer, an antiviral and immunoregulation effect, it is classified as one of " 100 kinds of effective anti-ageing materials the most popular " by U.S.'s monograph " anti-ageing canon " book, becomes the focus of whole world research.
At present, the preparation method of trans-resveratrol has chemical synthesis, culture plant cell method and natural product extraction method.
Existing synthesizing resveratrol need to just can reach 98% content by above synthesis technique of 5 steps, and synthetic technological condition is harsh, and reagent is expensive, and yield is low, is difficult to realize suitability for industrialized production;
Plant cell culture technology, refer under the prerequisite of the fundamental characteristics that does not change the natural plant attribute, originally should be in natural surroundings growing plants, place fully artificial controlled reaction environment, and utilize the cell proliferation condition of optimizing to impel vegetable cell to breed at a high speed, then from cell, extract the natural medicinal ingredients useful to the mankind, this technology only limits to the cultivation of little experiment, and manual control is strict in the culturing process, complicated operation, research and the discussion that also need go deep into for industrialized production.
From natural product, extract and separate trans-resveratrol, both taken full advantage of the natural resources of China's abundant, have again the economically viable production advantage.
Chinese patent CN 1116264C " trans-resveratrol and polidatin separation method and application thereof " and Chinese patent CN1724495A " method for preparing trans-resveratrol from the Chinese herbal medicine giant knotweed " mainly adopt the giant knotweed rhizome organic solvent extraction, through ethyl acetate extraction, concentrated, column chromatography and recrystallization, obtain purity up to the trans-resveratrol more than 97% and polidatin.But, the method is to use ethyl acetate extraction, emulsion is obvious, product loss is serious, thereby cause yield to reduce, and the method is trans-resveratrol and polidatin to be extracted to separate from giant knotweed, polidatin is not converted into trans-resveratrol, this step is not implemented, and can affect the yield of trans-resveratrol yet.
Chinese patent CN1269831C " new preparation process of Polydatin and trans-resveratrol " proposes the new preparation process of a kind of polygonin and trans-resveratrol, and it is with polymeric amide chromatography method separation and purification polygonin and trans-resveratrol.Separate with polyamide resin in this patent, polyamide resin order number is thinner, blocks up easily post, and Polydatin is not converted into trans-resveratrol in the patent, and technique is uneconomical.
Chinese patent CN 1513822A " Technology of extracting high purity resveratrol from polygonoum cuspidatum ", the present invention has simplified technique and schedule of operation, has shortened the production cycle, has reduced production cost, has improved product yield and purity.This technology utilization microbial transformation; with 40% ethanol and 4-methyl-2-amylalcohol mixture as extraction solvent; utilize preferably equipment microwave extraction; shorten the production cycle; but microwave extraction is used for the present still old certain difficulty of large-scale production; technique is infeasible, only is confined to the experiment in laboratory.
Chinese patent CN 1384088A " a kind of preparation method who extracts trans-resveratrol from giant knotweed " and Chinese patent CN the 1926592A method of separation and purification polydatin and trans-resveratrol " a kind of from Polygonum cuspidatum Sieb. et Zucc " adopt respectively the high pressure chromatography column of special use to separate trans-resveratrol with high-speed countercurrent chromatography, equipment cost is high, only can reach feather weight production, productive rate is low, production cost is high, be confined to the separation in laboratory, inapplicable large production.
Chinese patent CN1090603C " a kind of technique of extracting trans-resveratrol from Polygonum cuspidatum Sieb. et Zucc " the present invention relates to a kind of extraction process of medicinal raw material trans-resveratrol.Described extraction process is: add prozyme and carry out obtaining in enzyme digestion reaction 48-72 hour the enzymolysis raw material in Powdered giant knotweed raw material under constant temperature; Again with dissolution extraction, the concentrated work in-process that obtain to contain trans-resveratrol, again through making with extra care and get final product.This patent technique is simple, but needs periodic crystallisation just can obtain the elaboration of trans-resveratrol, and recrystallization can cause product yield low repeatedly, and loss is large, the shortcoming that production cost is high.
Summary of the invention
Separate trans-resveratrol method cost height in order to solve from giant knotweed, extracting of existing in the background technology, polidatin is not converted into trans-resveratrol, the trans-resveratrol yield is low, and technique is loaded down with trivial details, the cycle is long, be not suitable for the technical problem of industrialized production, the invention provides that a kind of cost is low, the trans-resveratrol yield is high, technique is simple, the cycle is short, is applicable to the method for extracting the separation trans-resveratrol from giant knotweed of industrialized production.
The technical solution adopted for the present invention to solve the technical problems is:
From giant knotweed, extract the method for separating trans-resveratrol, may further comprise the steps:
1) pulverizes
With the giant knotweed raw material pulverizing;
2) enzymolysis
With step 1) add the water of 1～2 times of amount of raw material weight and the lytic enzyme of raw material weight 1 ‰～10 ‰ in the raw material after pulverizing, mix thoroughly, enzymolysis 12～48h under 35～40 ℃ of conditions is with the raw material drying behind the enzymolysis; This lytic enzyme can be a kind of, two or more the mixed enzyme of cellulase, plant extraction complex enzymes, amylase, polygalacturonase or beta-glucan glycosides enzyme;
3) extract
With step 2) dried feed behind the enzymolysis, extract with extracting solvent refluxing or cold soaking, it is 1.10～1.30 that extracting solution is concentrated into proportion, this proportion is at 60～70 ℃, measures under the normal pressure, then adds the water that volume is 1～3 times of amount of concentrated solution volume, sedimentation;
4) centrifugal
With step 3) make to add concentrated liquid centrifugal, filter, be precipitated;
5) post separates
5.1) resolution of precipitate mixes sample
The volumetric concentration of getting precipitation weight 1 times of amount is that 60～80% methyl alcohol or ethanol add step 4) precipitation in dissolve, obtain lysate and precipitation, abandon precipitation, lysate and column packing are mixed sample, dry, obtain mixing all product;
Add column packing and step 4) mass ratio of the precipitation that obtains is 1: 2;
Column packing can be a kind of among polyacrylic resin CG71, polyacrylic resin RPC40, polystyrene resin CG161, polystyrene resin CG300, dextrane gel Sephadex G-25 or the dextrane gel Sephadex LH-20;
5.2) upper prop
Get step 5.1) make mix all product and column chromatography filler upper prop, carry out wash-out with eluent system again, by each stream of thin layer chromatography inspection part, collect stream part of containing trans-resveratrol, be concentrated into 1/10～1/3 of original volume, place and produce crystallization;
The mass ratio of mixing all product and column chromatography filler is 1: 3～1: 15;
The column chromatography filler can be polyacrylic resin CG71, polyacrylic resin RPC40, polystyrene resin CG161, polystyrene resin CG300, dextrane gel Sephadex G-25 or dextrane gel Sephadex LH-20;
6) recrystallization
With step 5.2) crystallization that produces filters with whizzer or plate filter, obtain coarse crystallization, be methyl alcohol or the ethyl alcohol recrystallization of 80%-95% with this coarse crystallization with the volumetric concentration of 1～4 times of amount of coarse crystallization weight, obtain content and be not less than 98% trans-resveratrol.
Above-mentioned steps 2) the optimum hydrolysis enzyme that adds in is the mixed enzyme of beta-glucan glycosides enzyme and polygalacturonase, and consumption is 4 ‰ of raw material weight, and the mass ratio of beta-glucan glycosides enzyme and polygalacturonase is 19: 1, and hydrolysis temperature is 37 ℃, and is best when enzymolysis time is 36h;
The said extracted solvent can be methyl alcohol, ethanol, ethyl acetate or acetone;
Above-mentioned eluent system can be methanol/water, ethanol/water, acetone/water or petrol ether/ethyl acetate system;
Above-mentioned steps 3) extracting solvent in is acetone, and cold soaking extracts, and it is 1.21 that extracting solution is concentrated into proportion, and this proportion is at 65 ℃, measures under the normal pressure, and adding volume in the concentrated solution is the water of 2 times of amounts of concentrated solution volume; The volumetric concentration of getting 1 times of amount of precipitation weight step 5.1) is 70% methyl alcohol, and column packing is polystyrene resin CG161; Step 5.2) the used mass ratio of mixing all product and column chromatography filler of upper prop is 1: 8 in; The column chromatography filler is polystyrene resin CG161; Eluent system is the petrol ether/ethyl acetate eluent system; Step 6) using whizzer to filter in, obtain coarse crystallization, is that 95% recrystallizing methanol is the best with coarse crystallization with the volumetric concentration of 2 times of amounts of coarse crystallization weight.
The present invention solves another technical scheme that its technical problem adopts:
From giant knotweed, extract the method for separating trans-resveratrol, may further comprise the steps:
1) pulverizes
With the giant knotweed raw material pulverizing;
2) extract
With step 1) raw material after pulverizing, extract with extracting solvent refluxing or cold soaking, it is 1.10～1.30 that extracting solution is concentrated into proportion, and this proportion is at 60～70 ℃, measures under the normal pressure, and adding volume is the water of 1～2 times of amount of concentrated solution volume;
3) enzymolysis
In step 2) add in the concentrated liquid lytic enzyme that adds raw material weight 1 ‰～10 ‰, stir, enzymolysis 6～36h under 35～40 ℃ of conditions obtains enzymolysis solution; This lytic enzyme can be a kind of, two or more the mixed enzyme of cellulase, plant extraction complex enzymes, amylase, polygalacturonase or beta-glucan glycosides enzyme;
4) centrifugal
With step 3) enzymolysis solution that makes is centrifugal, filters, be precipitated;
5) post separates
5.1) resolution of precipitate mixes sample
The volumetric concentration of getting precipitation weight 1 times of amount is that 60～80% methyl alcohol or ethanol add step 4) dissolve in the precipitation that makes, obtain lysate and precipitation, abandon precipitation, lysate and column packing are mixed sample, dry, obtain mixing all product;
Add column packing and step 4) mass ratio of the precipitation that obtains is 1: 2;
Column packing can be a kind of among polyacrylic resin CG71, polyacrylic resin RPC40, polystyrene resin CG161, polystyrene resin CG300, dextrane gel Sephadex G-25 or the dextrane gel Sephadex LH-20;
5.2) upper prop
Get step 5.1) make mix all product and column chromatography filler upper prop, carry out wash-out with eluent system again, by each stream of thin layer chromatography inspection part, collect stream part of containing trans-resveratrol, be concentrated into 1/10～1/3 of original volume, place and produce crystallization;
The described mass ratio of mixing all product and column chromatography filler is 1: 3～1: 15;
The column chromatography filler can be a kind of among polyacrylic resin CG71, polyacrylic resin RPC40, polystyrene resin CG161, polystyrene resin CG300, dextrane gel Sephadex G-25 or the dextrane gel Sephadex LH-20;
6) recrystallization
With step 5.2) crystallization that produces filters with whizzer or plate filter, obtain coarse crystallization, be methyl alcohol or the ethyl alcohol recrystallization of 80%-95% with this coarse crystallization with the volumetric concentration of 1～4 times of amount of coarse crystallization weight, obtain content and be not less than 98% trans-resveratrol.
Above-mentioned steps 3) the optimum hydrolysis enzyme that adds in is the mixed enzyme of beta-glucan glycosides enzyme, plant extraction complex enzymes and cellulase, consumption is 2 ‰ of raw material weight, the mass ratio of beta-glucan glycosides enzyme, plant extraction complex enzymes and cellulase is 12: 5: 3, hydrolysis temperature is 40 ℃, and is best when enzymolysis time is 12h;
The said extracted solvent can be methyl alcohol, ethanol, ethyl acetate or acetone;
Above-mentioned eluent system can be methanol/water, ethanol/water, acetone/water or petrol ether/ethyl acetate system;
Above-mentioned steps 2) extracting solvent in is ethyl acetate, refluxing extraction, and it is 1.10 that extracting solution is concentrated into proportion, and this proportion is at 60 ℃, measures under the normal pressure, and adding volume is the water of 1 times of amount of concentrated solution volume; The volumetric concentration of getting 1 times of amount of precipitation weight step 5.1) is 80% methyl alcohol, and column packing is dextrane gel Sephadex LH-20; Step 5.2) the used mass ratio of mixing all product and column chromatography filler of upper prop is 1: 8 in; The column chromatography filler is dextrane gel Sephadex LH-20; Eluent system is the ethanol/water eluent system; Step 6) using whizzer to filter in, obtain coarse crystallization, is that 95% recrystallizing methanol is the best with coarse crystallization with the volumetric concentration of 1.5 times of amounts of coarse crystallization weight.
It is a kind of among polyacrylic resin CG71, polyacrylic resin RPC40, polystyrene resin CG161, polystyrene resin CG300, dextrane gel Sephadex G-25 or the dextrane gel Sephadex LH-20 that above-mentioned two kinds of technical scheme center pillars separate the column packing of using, these fillers are owing to be polymer carrier all, dead absorption to the target isolate produces dead absorption less or not, so that the yield of product improves.Simultaneously, traditional processing method is to extract to separate trans-resveratrol and polidatin from giant knotweed, polidatin is not converted into trans-resveratrol, above-mentioned two schemes is by adding lytic enzyme before extraction step or behind the extraction step, make polidatin be further converted to trans-resveratrol, the again control of links in post separation and recrystallization technology is so that the yield of trans-resveratrol improves.
Advantage of the present invention:
1, the method adopts organic solvent extraction, adds afterwards depositing in water and falls, and uses pure dissolution sample again, avoids extracting the loss that brings, and improves product yield, reduces cost.
2, the method is converted into trans-resveratrol with polidatin, has improved the yield of trans-resveratrol.
3, the method cost is low.
4, the method technique is simple and direct, the cycle short, is applicable to industrialized production.
Description of drawings
Fig. 1 is the HPLC high performance liquid phase collection of illustrative plates of the Resveratrol content measurement result that obtains of embodiment 1.
Fig. 2 is the HPLC high performance liquid phase collection of illustrative plates of the Resveratrol content measurement result that obtains of embodiment 2.
Fig. 3 is the HPLC high performance liquid phase collection of illustrative plates of the Resveratrol content measurement result that obtains of embodiment 3.
Fig. 4 is the HPLC high performance liquid phase collection of illustrative plates of the Resveratrol content measurement result that obtains of embodiment 4.
Embodiment
Embodiment 1
Get 1000kg giant knotweed raw material pulverizing, add 1000kg water and 10kg cellulase, mix thoroughly, enzymolysis 40h under 35 ℃ of conditions carries out drying with the raw material behind the enzymolysis in the microwave drying mode; Getting dried raw material, is that 80% methanol eddy extracts with volumetric concentration, and it is 1.10 (this proportion is at 63 ℃, measures under the normal pressure) that extracting solution is concentrated into proportion, and adding volume is the water of 2 times of amounts of concentrated solution volume, and 12h is placed in sedimentation; Centrifugal with whizzer, filter, must precipitate 70kg; Get the 70kg volumetric concentration and be 80% methyl alcohol and add dissolving in the precipitation, obtain lysate and precipitation, abandon precipitation, lysate and 35kg polyacrylic resin CG71 are mixed sample, oven for drying is for subsequent use; Getting mass ratio is all product of 1: 3 mix and polyacrylic resin CG71 upper prop, separate, methanol/water eluent system with the different volumes ratio is carried out wash-out, be 3: 7 wash-out impurity with the methanol/water volume ratio first, be 5: 5 wash-out effective constituent with the methanol/water volume ratio again, collect the effective constituent section, concentrated generation crystallization, crystallization is centrifugal, obtain coarse crystallization, be 80% recrystallizing methanol with the volumetric concentration of 1.5 times of amounts of coarse crystallization weight with coarse crystallization, can obtain content and be 98.767% trans-resveratrol 12kg, the yield of trans-resveratrol is 1.2%, and table 1 is the detected result tabulation of this embodiment, and Fig. 1 is the HPLC high performance liquid phase collection of illustrative plates of the Resveratrol content measurement result that obtains of this embodiment.
Table 1
| Title | Retention time (min) | Area (microvolt * second) | Highly (microvolt) | Content % |
| Trans-resveratrol | 6.485 | 13398429 | 1313560 | 98.767 |
Get 1000kg giant knotweed raw material pulverizing, add the water of 1500kg and plant extraction complex enzymes and the 1kg cellulase of 7kg, mix enzymolysis 38h under 38 ℃ of conditions thoroughly; Raw material behind the enzymolysis is carried out drying in the forced air drying mode; Getting dried raw material, is 80% alcohol reflux with volumetric concentration, and it is 1.15 (this proportion is at 62 ℃, measures under the normal pressure) that extracting solution is concentrated into proportion, and adding volume is the water of 1 times of amount of concentrated solution volume, and 12h is placed in sedimentation; Centrifugal with whizzer, filter, must precipitate 68kg; Get the 68kg volumetric concentration and be 70% methyl alcohol with resolution of precipitate, obtain lysate and precipitation, abandon precipitation, lysate and 34kg polyacrylic resin RPC40 are mixed sample, oven for drying is for subsequent use; Getting mass ratio is all product of 1: 5 mix and polyacrylic resin RPC40 upper prop, separate, acetone/water eluent system with the different volumes ratio is carried out wash-out, be 9: 1 wash-out impurity with the acetone/water volume ratio first, be 8: 1 wash-out effective constituent with the acetone/water volume ratio again, collect the effective constituent section, concentrated generation crystallization, crystallization is filtered with whizzer, obtain coarse crystallization, it is 95% recrystallizing methanol with the volumetric concentration of 2 times of amounts of coarse crystallization weight, can obtain content and be 98.591% trans-resveratrol 11kg, the yield of trans-resveratrol is 1.1%, and table 2 is detected result tabulations of this embodiment, and Fig. 2 is the HPLC high performance liquid phase collection of illustrative plates of the Resveratrol content measurement result that obtains of this embodiment.
Table 2
| Title | Retention time (min) | Area (microvolt * second) | Highly (microvolt) | Content % |
| Trans-resveratrol | 6.481 | 13564213 | 1210271 | 98.591 |
Embodiment 3
Get giant knotweed 1000kg raw material pulverizing, add 1500kg water and 3.8kg beta-glucan glycosides enzyme and 0.2kg polygalacturonase, mix thoroughly, enzymolysis 36h under 37 ℃ of conditions is with the raw material seasoning behind the enzymolysis; Get dried raw material, extract with the acetone cold soaking, it is 1.21 (this proportion is at 65 ℃, measures under the normal pressure) that extracting solution is concentrated into proportion, adds the water of 2 times of amounts of concentrated solution volume, places 12h; With the centrifugal concentrated liquid that adds of whizzer, filter, must precipitate 72kg; Get the 72kg volumetric concentration and be 70% methyl alcohol and add dissolving in the precipitation, obtain lysate and precipitation, abandon precipitation, lysate and 36kg polystyrene resin CG161 are mixed sample, oven for drying is for subsequent use; Getting mass ratio is all product of 1: 8 mix and polystyrene resin CG161 upper prop, separate, petrol ether/ethyl acetate eluent system with the different volumes ratio is carried out wash-out, be 9: 1 wash-out impurity with the petrol ether/ethyl acetate volume ratio first, be 5: 1 wash-out effective constituent with the petrol ether/ethyl acetate volume ratio again, collect the effective constituent section, concentrated generation crystallization, filter with whizzer, obtain coarse crystallization, it is 95% recrystallizing methanol with the volumetric concentration of 2 times of amounts of coarse crystallization weight, can obtain content and be 99.258% trans-resveratrol 13kg, the yield of trans-resveratrol is 1.3%, and table 3 is detected result tabulations of this embodiment, and Fig. 3 is the HPLC high performance liquid phase collection of illustrative plates of the Resveratrol content measurement result that obtains of this embodiment.
Table 3
| Title | Retention time (min) | Area (microvolt * second) | Highly (microvolt) | Content % |
| Trans-resveratrol | 6.577 | 14207919 | 1259849 | 99.258 |
Get 1000kg giant knotweed raw material pulverizing, extract with ethyl acetate backflow, it is 1.10 (this proportion is at 60 ℃, measures under the normal pressure) that extracting solution is concentrated into proportion, the water that adds 1 times of amount of concentrated solution volume, add 1.2kg beta-glucan glycosides enzyme, 0.5kg plant extraction complex enzymes and 0.3kg cellulase, stir enzymolysis 12h under 40 ℃ condition, obtain enzymolysis solution, enzymolysis solution is centrifugal with whizzer, filter, must precipitate 73kg.Get the 73kg volumetric concentration and be 80% methyl alcohol and add dissolving in the precipitation, obtain lysate and precipitation, abandon precipitation, lysate and 36.5kg dextrane gel Sephadex LH-20 are mixed sample, oven for drying is for subsequent use; Getting mass ratio is all product of 1: 8 mix and dextrane gel Sephadex LH-20 upper prop, separate, ethanol/water eluent system with the different volumes ratio is carried out wash-out, be 1: 4 wash-out impurity with the ethanol/water volume ratio first, be 2: 1 wash-out effective constituent with the ethanol/water volume ratio again, collect the effective constituent section, concentrated generation crystallization, crystallization is filtered with whizzer, obtain coarse crystallization, it is 95% recrystallizing methanol with the volumetric concentration of 1.5 times of amounts of coarse crystallization weight, can obtain content and be 98.826% trans-resveratrol 12kg, the yield of trans-resveratrol is 1.2%, and table 4 is detected result tabulations of this embodiment, and Fig. 4 is the HPLC high performance liquid phase collection of illustrative plates of the Resveratrol content measurement result that obtains of this embodiment.
Table 4
| Title | Retention time (min) | Area (microvolt * second) | Highly (microvolt) | Content % |
| Trans-resveratrol | 6.525 | 11871085 | 1108212 | 98.826 |
Claims (9)
1. from giant knotweed, extract the method for separating trans-resveratrol, may further comprise the steps:
1) pulverizes
With the giant knotweed raw material pulverizing;
2) enzymolysis
With adding the water of 1～2 times of amount of raw material weight and the lytic enzyme of raw material weight 1 ‰～10 ‰ in the raw material after the step 1) pulverizing, mix thoroughly, enzymolysis 12～48h under 35～40 ℃ of conditions is with the raw material drying behind the enzymolysis; Described lytic enzyme is a kind of, two or more the mixed enzyme of cellulase, polygalacturonase or beta-glucan glycosides enzyme;
3) extract
With step 2) dried feed behind the enzymolysis, extract with extracting solvent refluxing or cold soaking, it is 1.10～1.30 that extracting solution is concentrated into proportion, this proportion is at 60～70 ℃, measure under the normal pressure, then add the water that volume is 1～3 times of amount of concentrated solution volume, place sedimentation;
4) centrifugal
With step 3) make to add concentrated liquid centrifugal, filter, be precipitated;
5) post separates
5.1) resolution of precipitate mixes sample
The volumetric concentration of getting precipitation weight 1 times of amount be 60～80% methyl alcohol or ethanol add step 4) precipitation in dissolve, obtain lysate and precipitation, abandon precipitation, lysate and column packing are mixed sample, oven dry obtains mixing all product, add the precipitation that column packing and step 4) obtain mass ratio be 1:2; Described column packing is a kind of among polyacrylic resin CG71, polystyrene resin CG161 or the dextrane gel Sephadex LH-20;
5.2) upper prop
Get step 5.1) make mix all product and column chromatography filler upper prop, carry out wash-out with eluent system again, by each stream of thin layer chromatography inspection part, collect stream part of containing trans-resveratrol, be concentrated into 1/10～1/3 of original volume, place and produce crystallization; The described mass ratio of mixing all product and column chromatography filler is 1:3～1:15, and described column chromatography filler is polyacrylic resin CG71, polystyrene resin CG161 or dextrane gel Sephadex LH-20;
6) recrystallization
With step 5.2) crystallization that produces filters with whizzer or plate filter, obtains coarse crystallization, is methyl alcohol or the ethyl alcohol recrystallization of 80%-95% with this coarse crystallization with the volumetric concentration of 1～4 times of amount of coarse crystallization weight, and obtain content and be not less than 98% trans-resveratrol.
2. method of from giant knotweed, extract separating trans-resveratrol according to claim 1, it is characterized in that: the lytic enzyme that adds described step 2) is the mixed enzyme of beta-glucan glycosides enzyme and polygalacturonase, consumption is 4 ‰ of raw material weight, the mass ratio of beta-glucan glycosides enzyme and polygalacturonase is 19:1, hydrolysis temperature is 37 ℃, and enzymolysis time is 36h.
3. method of extract separating trans-resveratrol from giant knotweed according to claim 1 and 2, it is characterized in that: described extraction solvent is methyl alcohol, ethanol, ethyl acetate or acetone.
4. method of extract separating trans-resveratrol from giant knotweed according to claim 3, it is characterized in that: described eluent system is methanol/water, ethanol/water, acetone/water or petrol ether/ethyl acetate system.
5. method of from giant knotweed, extract separating trans-resveratrol according to claim 4, it is characterized in that: extracting solvent in the described step 3) is acetone, cold soaking extracts, it is 1.21 that extracting solution is concentrated into proportion, this proportion is at 65 ℃, measure under the normal pressure, adding volume in the concentrated solution is the water of 2 times of amounts of concentrated solution volume; The volumetric concentration of getting 1 times of amount of precipitation weight described step 5.1) is 70% methyl alcohol, and column packing is polystyrene resin CG161; Described step 5.2) the used mass ratio of mixing all product and column chromatography filler of upper prop is 1:8 in; The column chromatography filler is polystyrene resin CG161; Eluent system is the petrol ether/ethyl acetate eluent system; Using whizzer to filter in the described step 6), obtain coarse crystallization, is 95% recrystallizing methanol with the volumetric concentration of 2 times of amounts of coarse crystallization weight with coarse crystallization.
6. from giant knotweed, extract the method for separating trans-resveratrol, may further comprise the steps:
1) pulverizes
With the giant knotweed raw material pulverizing;
2) extract
With the raw material after the step 1) pulverizing, extract with extracting solvent refluxing or cold soaking, it is 1.10～1.30 that extracting solution is concentrated into proportion, and this proportion is at 60～70 ℃, measures under the normal pressure, and adding volume is the water of 1～2 times of amount of concentrated solution volume;
3) enzymolysis
In step 2) add in the concentrated liquid lytic enzyme that adds raw material weight 1 ‰～10 ‰, stir, enzymolysis 6～36h under 35～40 ℃ of conditions obtains enzymolysis solution; Described lytic enzyme is a kind of, two or more the mixed enzyme of cellulase, polygalacturonase or beta-glucan glycosides enzyme;
4) centrifugal
The enzymolysis solution that step 3) makes is centrifugal, filter, be precipitated;
5) post separates
5.1) resolution of precipitate mixes sample
The volumetric concentration of getting precipitation weight 1 times of amount is that 60～80% methyl alcohol or ethanol add in the precipitation that step 4) makes and dissolves, obtain lysate and precipitation, abandon precipitation, lysate and column packing are mixed sample, oven dry, obtain mixing all product, add the precipitation that column packing and step 4) obtain mass ratio be 1:2; Described column packing is polyacrylic resin CG71, polystyrene resin CG161 or dextrane gel Sephadex LH-20;
5.2) upper prop
Get step 5.1) make mix all product and column chromatography filler upper prop, carry out wash-out with eluent system again, by each stream of thin layer chromatography inspection part, collect stream part of containing trans-resveratrol, be concentrated into 1/10～1/3 of original volume, place and produce crystallization; The described mass ratio of mixing all product and column chromatography filler is 1:3～1:15; Described column chromatography filler is polyacrylic resin CG71, polystyrene resin CG161 or dextrane gel Sephadex LH-20;
6) recrystallization
With step 5.2) crystallization that produces filters with whizzer or plate filter, obtains coarse crystallization, is methyl alcohol or the ethyl alcohol recrystallization of 80%-95% with this coarse crystallization with the volumetric concentration of 1～4 times of amount of coarse crystallization weight, and obtain content and be not less than 98% trans-resveratrol.
7. method of extract separating trans-resveratrol from giant knotweed according to claim 6, it is characterized in that: described extraction solvent is methyl alcohol, ethanol, ethyl acetate or acetone.
8. method of extract separating trans-resveratrol from giant knotweed according to claim 7, it is characterized in that: described eluent system is methanol/water, ethanol/water, acetone/water or petrol ether/ethyl acetate system.
9. method of from giant knotweed, extract separating trans-resveratrol according to claim 8, it is characterized in that: extracting solvent described step 2) is ethyl acetate, refluxing extraction, it is 1.10 that extracting solution is concentrated into proportion, this proportion is at 60 ℃, measure under the normal pressure, add the water of 1 times of amount of concentrated solution volume in the concentrated solution; The volumetric concentration of getting 1 times of amount of precipitation weight described step 5.1) is 80% methyl alcohol, and column packing is dextrane gel Sephadex LH-20; Described step 5.2) the used mass ratio of mixing all product and column chromatography filler of upper prop is 1:8 in; The column chromatography filler is dextrane gel Sephadex LH-20; Eluent system is the ethanol/water eluent system; Using whizzer to filter in the described step 6), obtain coarse crystallization, is 95% recrystallizing methanol with the volumetric concentration of 1.5 times of amounts of coarse crystallization weight with coarse crystallization.
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| CN102465158A (en) * | 2010-11-19 | 2012-05-23 | 苏州宝泽堂医药科技有限公司 | Method for preparing chrysin |
| CN102304028B (en) * | 2011-07-01 | 2013-10-30 | 中山大学 | Method for separating and purifying resveratrol in polygonum cuspidatum |
| CN102838455A (en) * | 2012-09-17 | 2012-12-26 | 湖南希尔天然药业有限公司 | Method for extracting high-content resveratrol from polygonum cuspidatum |
| CN102925497A (en) * | 2012-11-01 | 2013-02-13 | 恩施清江生物工程有限公司 | Method for preparing high-purity resveratrol from polygonum cuspidatum |
| CN104087623A (en) * | 2014-06-27 | 2014-10-08 | 湖南鑫利生物科技有限公司 | Method for extracting resveratrol from giant knotweed by enzymolysis |
| CN105063101A (en) * | 2015-07-21 | 2015-11-18 | 四川维尔仕生物科技有限公司 | Technology of preparing high-purity resveratrol from giant knot weed |
| CN104946689B (en) * | 2015-07-21 | 2018-06-26 | 四川维尔仕生物科技有限公司 | A kind of method that resveratrol is extracted from giant knotweed |
| CN107162888A (en) * | 2017-05-31 | 2017-09-15 | 湖北中鑫生物科技有限公司 | The production method of resveratrol and rheum emodin is prepared from giant knotweed |
| CN107353183A (en) * | 2017-07-21 | 2017-11-17 | 安徽龙津生物科技有限公司 | A kind of method that high-content of resveratrol is extracted from giant knotweed |
| CN107296280A (en) * | 2017-08-08 | 2017-10-27 | 合肥润雨农业科技有限公司 | A kind of preparation method of cherry functional component |
| CN107721825B (en) * | 2017-10-18 | 2020-12-22 | 洋县秦龙药业有限公司 | Method for extracting resveratrol and polydatin from giant knotweed rhizome |
| CN108048495B (en) * | 2017-12-17 | 2021-04-16 | 安徽济人药业有限公司 | Biological extraction method of resveratrol |
| CN109096054A (en) * | 2018-08-13 | 2018-12-28 | 安徽兆龙生物科技有限公司 | A kind of biological extraction process of resveratrol |
| CN115141084A (en) * | 2022-07-15 | 2022-10-04 | 东明格鲁斯生物科技有限公司 | Method for extracting high-purity resveratrol from giant knotweed rhizome |
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