CN101698634A - 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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- CN101698634A CN101698634A CN 200910207395 CN200910207395A CN101698634A CN 101698634 A CN101698634 A CN 101698634A CN 200910207395 CN200910207395 CN 200910207395 CN 200910207395 A CN200910207395 A CN 200910207395A CN 101698634 A CN101698634 A CN 101698634A
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- 235000021283 resveratrol Nutrition 0.000 title abstract description 16
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Images
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 giant knotweed 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 from giant knotweed the method for extraction separation trans-resveratrol.
Background technology
Trans-resveratrol (Resveratrol) is resveratrol again, 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 polygonaceae plant giant knotweed rhizome and root or Vitis vitaceae grape.Found trans-resveratrol in 1940 first, find to contain in the grape this material the seventies in 20th century 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 the anti-cardiovascular disorder of potential, 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, preparing resveratrol has chemical synthesis, culture plant cell method and natural product extraction method.
Existing synthesizing resveratrol need just can reach 98% content by above synthesis technique of 5 steps, and the synthetic technological condition harshness, the reagent costliness, and yield is low, is difficult to realize suitability for industrialized production;
Plant cell culture technology, be meant 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, from cell, extract the natural medicinal ingredients useful then to the mankind, this technology only limits to the cultivation of little experiment, and artificial 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 made full use of the natural resources of China's abundant, have the economically viable production advantage again.
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 giant knotweed rhizome organic solvent extraction, through ethyl acetate extraction, concentrate, column chromatography and recrystallization, obtain purity up to trans-resveratrol more than 97% and polidatin.But, this method is to use ethyl acetate extraction, emulsion is obvious, product loss is serious, thereby cause yield to reduce, and this method is from giant knotweed trans-resveratrol and polidatin extraction separation to be come out, polidatin is not converted into trans-resveratrol, this step is not implemented, and can influence 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, stifled easily post, and Polydatin is not converted into trans-resveratrol in the patent, technology is uneconomical.
Chinese patent CN 1513822A " giant knotweed extracts high-purity trans-resveratrol technology ", the present invention has simplified technology 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 equipment microwave extraction preferably; shorten the production cycle; but microwave extraction is used for the still old now certain degree of difficulty of large-scale production; technology is infeasible, only is confined to breadboard experiment.
Chinese patent CN 1384088A " a kind of preparing resveratrol of extracting from giant knotweed " and Chinese patent CN the 1926592A method of separation and purification polydatin and trans-resveratrol " a kind of from the Chinese medicine giant knotweed " adopt special-purpose high pressure chromatography column to separate trans-resveratrol with high-speed countercurrent chromatography respectively, the equipment cost height, only can reach feather weight production, productive rate is low, the production cost height, be confined to breadboard separation, inapplicable big production.
Chinese patent CN1090603C " a kind of technology of extracting trans-resveratrol from the Chinese medicine giant knotweed " 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 promptly.This patent technology 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 big, the shortcoming that production cost is high.
Summary of the invention
In order to solve the extraction separation trans-resveratrol method cost height from giant knotweed that exists in the background technology, polidatin is not converted into trans-resveratrol, the trans-resveratrol yield is low, and technology 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, trans-resveratrol yield height, technology is simple, the cycle is short, is applicable to the method for extraction separation trans-resveratrol from giant knotweed of industrialized production.
The technical solution adopted for the present invention to solve the technical problems is:
The method of extraction separation trans-resveratrol from giant knotweed 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; This lytic enzyme can be a kind of, two or more the mixed enzyme of cellulase, plant extract prozyme, 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, and this proportion is at 60～70 ℃, and normal pressure is measured down, adds the water that volume is 1～3 times of amount of concentrated solution volume, sedimentation then;
4) centrifugal
With step 3) make to add concentrated liquid centrifugal, filter, obtain precipitation;
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, dry, obtain mixing all product;
To add the sedimentary mass ratio that column packing and step 4) obtain be 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, 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.
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 a raw material weight, and the mass ratio of beta-glucan glycosides enzyme and polygalacturonase is 19: 1, and hydrolysis temperature is 37 ℃, the 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, ethanol/water, acetone 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 ℃, and normal pressure is measured down, and adding volume in 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; Using whizzer to filter in the step 6), 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:
The method of extraction separation trans-resveratrol from giant knotweed may further comprise the steps:
1) pulverizes
With the giant knotweed raw material pulverizing;
2) extract
Raw material after step 1) pulverized extracts with extracting solvent refluxing or cold soaking, and it is 1.10～1.30 that extracting solution is concentrated into proportion, and this proportion is at 60～70 ℃, and normal pressure is measured down, 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 extract prozyme, amylase, polygalacturonase or beta-glucan glycosides enzyme;
4) centrifugal
The enzymolysis solution that step 3) makes is centrifugal, filter, obtain precipitation;
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, and obtains lysate and precipitation, abandons precipitation, and lysate and column packing are mixed sample, dries, and obtains mixing all product;
To add the sedimentary mass ratio that column packing and step 4) obtain be 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, 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.
Above-mentioned steps 3) the optimum hydrolysis enzyme that adds in is the mixed enzyme of beta-glucan glycosides enzyme, plant extract prozyme and cellulase, consumption is 2 ‰ of a raw material weight, the mass ratio of beta-glucan glycosides enzyme, plant extract prozyme and cellulase is 12: 5: 3, hydrolysis temperature is 40 ℃, the 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, ethanol/water, acetone 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 ℃, and normal pressure is measured down, 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; Using whizzer to filter in the step 6), 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, makes the yield of product improve.Simultaneously, traditional processing method is extraction separation trans-resveratrol and a polidatin from giant knotweed, polidatin is not converted into trans-resveratrol, above-mentioned two kinds of schemes are by adding lytic enzyme before extraction step or behind the extraction step, make polidatin further be converted into trans-resveratrol, the control of each link in post separation and recrystallization technology again makes the yield of trans-resveratrol improve.
Advantage of the present invention:
1, this method adopts organic solvent extraction, adds depositing in water afterwards and falls, and uses pure sample dissolution again, avoids extracting the loss that brings, and improves product yield, reduces cost.
2, this method is converted into trans-resveratrol with polidatin, has improved the yield of trans-resveratrol.
3, this method cost is low.
4, this method technology 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 ℃, and normal pressure is measured down) 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 standby; Getting mass ratio is all product of 1: 3 mix and polyacrylic resin CG71 upper prop, separate, methanol eluent system with the different volumes ratio is carried out wash-out, be 3: 7 wash-out impurity with the methanol volume ratio earlier, be 5: 5 wash-out effective constituent with the methanol volume ratio again, collect the effective constituent section, concentrate and produce crystallization, crystallization is centrifugal, obtain coarse crystallization, 's 80% recrystallizing methanol with coarse crystallization with the volumetric concentration of 1.5 times of amounts of coarse crystallization weight, 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 |
Embodiment 2
Get 1000kg giant knotweed raw material pulverizing, add the water of 1500kg and plant extract prozyme 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 ℃, and normal pressure is measured down) 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 standby; Getting mass ratio is all product of 1: 5 mix and polyacrylic resin RPC40 upper prop, separate, acetone eluent system with the different volumes ratio is carried out wash-out, be 9: 1 wash-out impurity with the acetone volume ratio earlier, be 8: 1 wash-out effective constituent with the acetone volume ratio again, collect the effective constituent section, concentrate and produce crystallization, crystallization is filtered with whizzer, obtain coarse crystallization, it with the volumetric concentration of 2 times of amounts of coarse crystallization weight 95% recrystallizing methanol, 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 ℃, and normal pressure is measured down) 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 standby; 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 earlier, be 5: 1 wash-out effective constituent with the petrol ether/ethyl acetate volume ratio again, collect the effective constituent section, concentrate and produce crystallization, filter with whizzer, obtain coarse crystallization, it with the volumetric concentration of 2 times of amounts of coarse crystallization weight 95% recrystallizing methanol, 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 |
Embodiment 4
Get 1000kg giant knotweed raw material pulverizing, extract with ethyl acetate backflow, it is 1.10 (this proportion is at 60 ℃, and normal pressure is measured down) 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 extract prozyme 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 standby; 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 earlier, be 2: 1 wash-out effective constituent with the ethanol/water volume ratio again, collect the effective constituent section, concentrate and produce crystallization, crystallization is filtered with whizzer, obtain coarse crystallization, it with the volumetric concentration of 1.5 times of amounts of coarse crystallization weight 95% recrystallizing methanol, 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 (10)
1. the method for extraction separation trans-resveratrol from giant knotweed 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, plant extract prozyme, 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 ℃, normal pressure is measured down, adds the water that volume is 1～3 times of amount of concentrated solution volume then, places sedimentation;
4) centrifugal
With step 3) make to add concentrated liquid centrifugal, filter, obtain precipitation;
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, to add the sedimentary mass ratio that column packing and step 4) obtain be 1: 2; Described column packing 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;
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, 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, 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. according to claim 1 from giant knotweed the method for extraction separation trans-resveratrol, 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 a 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. according to claim 1 and 2 from giant knotweed the method for extraction separation trans-resveratrol, it is characterized in that: described extraction solvent is methyl alcohol, ethanol, ethyl acetate or acetone.
4. according to claim 3 from giant knotweed the method for extraction separation trans-resveratrol, it is characterized in that: described eluent system is methanol, ethanol/water, acetone or petrol ether/ethyl acetate system.
5. according to claim 4 from giant knotweed the method for extraction separation trans-resveratrol, 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 ℃, normal pressure is measured down, and adding volume in 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. the method for extraction separation trans-resveratrol from giant knotweed may further comprise the steps:
1) pulverizes
With the giant knotweed raw material pulverizing;
2) extract
Raw material after step 1) pulverized extracts with extracting solvent refluxing or cold soaking, and it is 1.10～1.30 that extracting solution is concentrated into proportion, and this proportion is at 60～70 ℃, and normal pressure is measured down, 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, plant extract prozyme, amylase, polygalacturonase or beta-glucan glycosides enzyme;
4) centrifugal
The enzymolysis solution that step 3) makes is centrifugal, filter, obtain precipitation;
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, to add the sedimentary mass ratio that column packing and step 4) obtain be 1: 2; Described column packing is polyacrylic resin CG71, polyacrylic resin RPC40, polystyrene resin CG161, polystyrene resin CG300, dextrane gel Sephadex G-25 or dextrane gel SephadexLH-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, 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, 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. according to claim 6 from giant knotweed the method for extraction separation trans-resveratrol, it is characterized in that: the lytic enzyme that adds in the described step 3) is the mixed enzyme of beta-glucan glycosides enzyme, plant extract prozyme, three kinds of enzymes of cellulase, consumption is 2 ‰ of a raw material weight, the mass ratio of beta-glucan glycosides enzyme, plant extract prozyme and cellulase is 12: 5: 3, hydrolysis temperature is 40 ℃, and enzymolysis time is 12h.
According to claim 6 or 7 described from giant knotweed the method for extraction separation trans-resveratrol, it is characterized in that: described extraction solvent is methyl alcohol, ethanol, ethyl acetate or acetone.
9. according to claim 8 from giant knotweed the method for extraction separation trans-resveratrol, it is characterized in that: described eluent system is methanol, ethanol/water, acetone or petrol ether/ethyl acetate system.
10. according to claim 9 from giant knotweed the method for extraction separation trans-resveratrol, 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 ℃, normal pressure is measured down, adds the water of 1 times of amount of concentrated solution volume in 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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Denomination of invention: Method for extracting and separating resveratrol from giant knotweed rhizome Effective date of registration: 20190408 Granted publication date: 20130327 Pledgee: Bank of Xi'an Limited by Share Ltd high tech branch Pledgor: Sanyuan Runhe Biological Technology Co. Ltd. Registration number: 2019610000061 |
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Date of cancellation: 20200616 Granted publication date: 20130327 Pledgee: Bank of Xi'an Limited by Share Ltd. high tech branch Pledgor: Sanyuan Runhe Biological Technology Co.,Ltd. Registration number: 2019610000061 |
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Denomination of invention: Extraction and separation of resveratrol from Polygonum cuspidatum Effective date of registration: 20200916 Granted publication date: 20130327 Pledgee: Bank of Xi'an Limited by Share Ltd. high tech branch Pledgor: Sanyuan Runhe Biological Technology Co.,Ltd. Registration number: Y2020610000149 |
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Date of cancellation: 20210531 Granted publication date: 20130327 Pledgee: Bank of Xi'an Limited by Share Ltd. high tech branch Pledgor: Sanyuan Runhe Biological Technology Co.,Ltd. Registration number: Y2020610000149 |