CN100497307C - Preparation of raphanin sulfane - Google Patents
Preparation of raphanin sulfane Download PDFInfo
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- CN100497307C CN100497307C CNB2005101304679A CN200510130467A CN100497307C CN 100497307 C CN100497307 C CN 100497307C CN B2005101304679 A CNB2005101304679 A CN B2005101304679A CN 200510130467 A CN200510130467 A CN 200510130467A CN 100497307 C CN100497307 C CN 100497307C
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- sulforaphane
- ethyl acetate
- silica gel
- sherwood oil
- virahol
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Abstract
Production of raphsulfane with brassin vegetables as raw materials is carried out by crushing for seeds, flowers, stems and leaves, hydrolyzing at specific pH value by myrosase, extracting by acetic ether, adsorbing by silica gel, removing impurities by acetic ether and eluting by mixed solution of isopropyl alcohol, alcohol and petroleum ether. It's cheap and simple and can be used for large-scale industrial production.
Description
Technical field:
The present invention relates to a kind of is the method for feedstock production sulforaphane with the brassica vegetable.
Background technology:
Sulforaphane (sulforaphane, 1-isothiocyano-4R-[methyl sulfinyl] butane) extensively be present in Btassica in the cress (Brassica) vegetables.The daily edible vegetables of people all belong to this class vegetables as cabbage, brussels sprouts, leafy cabbage, kohlrabi, broccoli, leaf mustard and horseradish etc.Glucose sulforaphane (glucoraphanin) is the precursor of sulforaphane, and the glucose sulforaphane can generate sulforaphane through mustard seed sulphur thuja acid enzyme (myrosinase) hydrolysis.
The physiological function of sulforaphane:
1, sulforaphane does not have any cytotoxicity;
2, suppress the formation of tumour;
Experimental results demonstrate that sulforaphane all has good prevention effect to esophagus cancer, lung cancer, colorectal carcinoma, mammary cancer, liver cancer and large bowel cancer etc.;
3, sulforaphane improves the glutathione level of tissue;
4, sulforaphane can be separated toxenzyme by the synthetic II type of inducing cell, as Thiadiazolidine isomerase, NAD (P) H-quinone reductase, heme oxidase, makes cell avoid the attack of active oxygen;
5, suppress and kill helicobacter pylori.
In recent years, the physiological function of the brilliance of recognizing sulforaphane gradually and being had along with people, the preparation of sulforaphane also receives much attention.The preparation method of sulforaphane mainly contains chemosynthesis and natural product extraction at present.
(1) chemical synthesis
Though it is simple that initial chemosynthesis prepares the method reaction process of sulforaphane, the sulforaphane that is synthesized is racemic modification (mixture of the sulforaphane of D type and L type), and natural sulforaphane is the L type.Adopt the preparation of stereochemistry chirality synthetic method (Schenk, W.A. now mostly; Durr, M.; Steinmetz, B.; Adam, W.; Saha-Moller, C.R.Enantioselective oxidation of thioethers using ruthenium complexesas chiral auxiliaries.Proceedings of the Symposium, 3rd, Wuerzburg, Germany, Sept.17-19,1997, Meeting Date1997,245-246.).This method is a raw material with 1-oxygen-R-methyl sulfinyl-2-Santosol 360; be to react with the reactive metal molecular agents under the condition of solvent at room temperature, tetrahydrofuran (THF); the intermediate product that generates is reacting with hydrofluoric acid; generate 1-hydroxyl-4R-[methyl sulfinyl] butane, finally generating the sulforaphane of L type with sodiumazide, dithiocarbonic anhydride back flow reaction.Though the chirality synthesis method is the sulforaphane of synthetic L type only, has the reaction mechanism complexity, reactions steps is many, and productive rate is not high, and by product is many, and material toxicity is bigger, and shortcomings such as separation and Extraction difficulty are added the material cost height, are difficult to large-scale industrial production.
(2) natural product extraction
This method is after the mustard seed sulphur thuja acid enzymic hydrolysis glucose sulforaphane that utilizes the mustard seed sulphur thuja acid enzyme of plant self or add external source generates sulforaphane, prepares sulforaphane adopting organic solvent extraction and chromatography etc. to separate purifying process.Adopt the mixture of several hydrolysates often that obtain behind the extraction process treating water hydrolysis products, must carry out further separation and purification.The main at present hydrolyzate that adopts the dichloromethane extraction broccoli utilizes the anti-phase C of preparation type again
18Chromatographic separation and purification sulforaphane (Nathan V.Matusheski, Mathew A.Wallin, et al.PreparationHPLC Methodfor the purification of sulforaphane Nitrile from Brassicaoleracea.J.Agric.Chem, 2001,49,1867-1872.).Though this method can obtain highly purified product, the solvent that extraction is adopted is the bigger methylene dichloride of toxicity, be difficult to avoid residuing in the final product, thus the use of limits product; The anti-phase C of preparation type that separation and purification is selected
18The performance liquid chromatographic column price is very expensive, and moving phase is the acetonitrile solution of lower concentration, be difficult to recycle and reuse, thereby this method production cost is very high, is only limited to experimental size and prepares few products, is difficult to scale operation.
Publication number is that the Chinese patent application of CN 1149986A discloses the preparation method that a kind of preparation contains the brassica vegetable compressing tablet of sulforaphane.This method is added external myrosinase glucosinolate is transformed into sulforaphane by endogenous myrosinase in the steam heating passivation brassica vegetable.This method is raw-material, and to choose scope very limited, is example with the broccoli, and this method only is applicable to flower with broccoli as raw material, organizes then and can only throw aside for the seed of broccoli, stem, leaf etc.In addition, this method must be added the mustard seed sulphur thuja acid enzyme of external source, and this need set up the production line of external source mustard seed sulphur thuja acid enzyme or give a long price for its zymin, more than these factors cause this method in production application, to have limitation, and production cost is very high.
Summary of the invention:
The object of the present invention is to provide a kind of seed with brassica vegetable, flower, stem, leaf is the method for feedstock production sulforaphane.Raw material after crushed, utilize mustard seed sulphur thuja acid enzyme hydrolysis under specific pH value of plant self, adopt ethyl acetate extraction, adopt cheap silica gel adsorption sulforaphane, the ethyl acetate removal of impurities, the mixing solutions wash-out sulforaphane of Virahol, ethanol, Virahol and sherwood oil or ethanol and sherwood oil, thus the content of sulforaphane improved effectively, reduce cost simultaneously.
The invention main points:
Raw material of the present invention adopts brassica vegetable, comprise, but be not limited only to, cabbage, brussels sprouts, leafy cabbage, kohlrabi, broccoli, leaf mustard or horseradish, present method is applicable to that tissue such as seed, flower, stem or the leaf with brassica vegetable is as raw material.For the preferred brassica vegetable of present method is broccoli (another name: broccoli, Caulis et Folium Brassicae capitatae), and most preferred raw material is the flower or the seed of broccoli.
Concrete grammar and step are as follows:
A. hydrolysis generates sulforaphane
Seed, flower, stem or the leaf of brassica vegetable are pulverized, and adding quality is 1-3 deionized water doubly, and regulating the pH value is 2-7.5, stirs hydrolysis 2-8 hour under the room temperature;
The pH value can adopt pH value buffered soln or adding lime carbonate or hydrochloric acid to regulate and control;
Wherein pH value buffered soln can be Na
2HPO
4-citrate buffer solution, citric acid-sodium citrate damping fluid, acetate buffer solution, KH
2PO
4-NaOH damping fluid or Tutofusin tris-HCl damping fluid.
B. ethyl acetate extraction
Sulforaphane hydrolyzed solution with steps A obtains adds ethyl acetate extraction 2-3 time, and the amount of adding does not have special requirement, 1-2 with sulforaphane hydrolyzed solution volume doubly gets final product, the sulforaphane acetic acid ethyl acetate extract that obtains, underpressure distillation, after reclaiming ethyl acetate, obtain the thick product of sulforaphane.
C. silica gel separation and purification
The thick product of sulforaphane that step B is obtained is dissolved in the sherwood oil and ethyl acetate mixture that the ethyl acetate volumn concentration is 10-40%, after silica gel adsorption, again with the low polar impurity that adsorbs on the ethyl acetate flush away silica gel, the consumption of ethyl acetate did not lose sulforaphane simultaneously with the low polar impurity of flush away and exceeded this moment; Use the mixing solutions wash-out sulforaphane of Virahol, ethanol, Virahol and sherwood oil or ethanol and sherwood oil at last, underpressure distillation obtains the sulforaphane product to remove organic solvent.
In the aforesaid method, the vegetable seed that steps A adopted can be degreasing or unskimmed seed.
The mixing solutions of Virahol that aforesaid method step C is used and sherwood oil, ethanol and sherwood oil, the sherwood oil volumn concentration in mixing solutions is no more than 50%.
In the aforesaid method, the silica gel that step C adopted is silica gel for chromatography, can be for amorphous or spherical, and the silica gel particle diameter is preferably the 60-300 order, is preferably the 200-300 order.
The invention effect:
After the present invention pulverizes seed, flower, stem, the leaf of brassica vegetable, utilize mustard seed sulphur thuja acid enzyme hydrolysis under specific pH value of plant self, adopt ethyl acetate extraction, silica gel adsorption, the ethyl acetate removal of impurities, the mixing solutions wash-out sulforaphane of Virahol, ethanol, Virahol and sherwood oil or ethanol and sherwood oil, thus the content of sulforaphane improved effectively.This method raw material is applied widely, need not to add the mustard seed sulphur thuja acid enzyme of external source, and hydrolysis utilizes the mustard seed sulphur thuja acid enzymic hydrolysis glucosinolate of starting material self under specific pH value, reduced production cost, simplify extraction and purification process, be easy to automated operation, be fit to large-scale commercial production.
Embodiment:
Embodiment 1:
(1) hydrolysis generates sulforaphane
Take by weighing 100g broccoli seed, be ground into powder, add the 250ml deionized water, regulating pH with lime carbonate is 5-7, at room temperature stirs hydrolysis 5h;
(2) ethyl acetate extraction
Mixture after the hydrolysis adds the ethyl acetate of 500ml, and is centrifugal, collects ethyl acetate, repeats this step 3 time, the ethyl acetate that obtains for three times is mixed mutually, and the thick product of underpressure distillation simmer down to tawny buttery sulforaphane, content is 5.5%;
(3) silica gel separation and purification
The thick product of the above-mentioned sulforaphane that obtains is dissolved in sherwood oil and the ethyl acetate solution that the 600ml volume ratio is 8:2, after 30 gram 100-200 purpose chromatographies are with silica gel adsorption, use the removal of impurities of 100ml ethyl acetate again, use 300ml ethanol elution sulforaphane at last, ethanol is reclaimed in underpressure distillation, purity is 54.2%, and yield is 71.3%.
Embodiment 2:
(1) hydrolysis generates sulforaphane
Take by weighing through the 100g of petroleum ether degreasing broccoli seed powder, adding 300mL pH value is the Na of 5-7
2HPO
4-citric acid solution at room temperature stirs hydrolysis 3h;
(2) ethyl acetate extraction
Mixture after the hydrolysis adds the ethyl acetate of 500ml, and is centrifugal, collects ethyl acetate, repeats this step 3 time, the ethyl acetate that obtains for three times is mixed mutually, and the thick product of underpressure distillation simmer down to tawny buttery sulforaphane, content is 15.5%;
(3) silica gel separation and purification
The thick product of the above-mentioned sulforaphane that obtains is dissolved in sherwood oil and the ethyl acetate solution that the 600ml volume ratio is 6:4, after 50 gram 200-300 purpose chromatographies are with silica gel adsorption, use the removal of impurities of 100ml ethyl acetate again, be sherwood oil and the ethanolic soln wash-out sulforaphane of 4:6 at last with the 800ml volume ratio, collection contains the component of sulforaphane, sherwood oil and ethanol are reclaimed in underpressure distillation, and purity is 84.2%, and yield is 66.2%.
Embodiment 3:
(1) hydrolysis generates sulforaphane
Taking by weighing the fresh broccoli of 500g, add the 500ml deionized water, is 2-3 with the hydrochloric acid adjust pH, and homogenate was at room temperature stirred hydrolysis 7 hours;
(2) ethyl acetate extraction
Said hydrolyzed liquid adds the 500ml ethyl acetate, and extraction is centrifugal, collects the ethyl acetate phase, so triplicate. merge the ethyl acetate phase that obtains for three times, and the thick product of underpressure distillation simmer down to tawny buttery sulforaphane, content is 1.98%;
(3) silica gel separation and purification
The thick product of the above-mentioned sulforaphane that obtains is dissolved in sherwood oil and the ethyl acetate solution that the 100ml volume ratio is 7:3, after 30 gram 100-200 purpose chromatographies are with silica gel adsorption, use the removal of impurities of 20ml ethyl acetate again, use 1000ml Virahol wash-out sulforaphane at last, collection contains the component of sulforaphane, Virahol is reclaimed in underpressure distillation, and content is 8.37%, and product recovery rate is 82.6%.
Embodiment 4:
(1) hydrolysis generates sulforaphane
Take by weighing the fresh broccoli of 500g, the buffered soln adjust pH that adds 1000ml citric acid-Trisodium Citrate is 3-4, and homogenate was at room temperature stirred hydrolysis 8 hours;
(2) ethyl acetate extraction
Said hydrolyzed liquid adds the 1000ml ethyl acetate, and extraction is centrifugal, collects the ethyl acetate phase, so triplicate. merge the ethyl acetate phase that obtains for three times, and the thick product of underpressure distillation simmer down to tawny buttery sulforaphane, content is 0.85%;
(3) silica gel separation and purification
The thick product of the above-mentioned sulforaphane that obtains is dissolved in sherwood oil and the ethyl acetate solution that the 100ml volume ratio is 9:1, after 30 gram 100-200 purpose chromatographies are with silica gel adsorption, use the removal of impurities of 15ml ethyl acetate again, be sherwood oil and the aqueous isopropanol wash-out sulforaphane of 2:8 at last with the 1000ml volume ratio, collection contains the component of sulforaphane, Virahol is reclaimed in underpressure distillation, and content is 12.56%, and product recovery rate is 85.8%.
Claims (6)
1. the preparation method of a sulforaphane is a raw material with the brassica vegetable, in turn includes the following steps:
A. hydrolysis generates sulforaphane
Seed, flower, stem or the leaf of brassica vegetable are pulverized, and adding quality is 1-3 deionized water doubly, and regulating the pH value is 2-7.5, stirs hydrolysis 2-8 hour under the room temperature;
The pH value adopts pH value buffered soln or adding lime carbonate or hydrochloric acid to regulate and control;
B. ethyl acetate extraction
The sulforaphane hydrolyzed solution that steps A is obtained adds ethyl acetate extraction 2-3 time, obtains the acetic acid ethyl acetate extract of sulforaphane, and underpressure distillation obtains the thick product of sulforaphane;
C. silica gel separation and purification
The thick product of sulforaphane that step B is obtained is dissolved in the sherwood oil and ethyl acetate mixture that the ethyl acetate volumn concentration is 10-40%, after silica gel adsorption, again with the low polar impurity that adsorbs on the ethyl acetate flush away silica gel, use the mixing solutions wash-out sulforaphane of Virahol, ethanol, Virahol and sherwood oil or ethanol and sherwood oil at last, remove organic solvent through underpressure distillation, obtain the sulforaphane product.
2. method according to claim 1 is characterized in that: said pH value buffered soln is Na in the steps A
2HPO
4-citrate buffer solution, citric acid-sodium citrate damping fluid, acetate buffer solution, KH
2PO
4-NaOH damping fluid or Tutofusin tris-HCl damping fluid.
3. method according to claim 1 is characterized in that: adopting the flower or the seed of broccoli in the steps A is raw material.
4. method according to claim 1 is characterized in that: the used silica gel particle diameter of step C is the 60-300 order.
5. method according to claim 4 is characterized in that: the used silica gel particle diameter of step C is the 200-300 order.
6. method according to claim 1 is characterized in that: the mixing solutions PetroChina Company Limited. volumn concentration of ether in mixing solutions of Virahol that step C is used and sherwood oil or ethanol and sherwood oil is no more than 50%.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005101304679A CN100497307C (en) | 2005-12-13 | 2005-12-13 | Preparation of raphanin sulfane |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005101304679A CN100497307C (en) | 2005-12-13 | 2005-12-13 | Preparation of raphanin sulfane |
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| CN1982294A CN1982294A (en) | 2007-06-20 |
| CN100497307C true CN100497307C (en) | 2009-06-10 |
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| CNB2005101304679A Expired - Fee Related CN100497307C (en) | 2005-12-13 | 2005-12-13 | Preparation of raphanin sulfane |
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Families Citing this family (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101514174B (en) * | 2009-02-24 | 2011-11-02 | 黑龙江八一农垦大学 | Method for extracting multifunctional sulforaphane from broccoli sprouting vegetable |
| CN101831479B (en) * | 2010-04-23 | 2012-09-05 | 北京化工大学 | Method for preparing 4-methylsulfinylbutylthioglucoside by biotransformation |
| CN102492745B (en) * | 2011-11-28 | 2013-07-03 | 北京化工大学 | Method for preparing sulforaphane by using glucoerucin |
| CN102586352B (en) * | 2012-01-16 | 2014-02-26 | 南京农业大学 | Method for enriching and extracting sulforaphane from brassica vegetable seeds |
| CN102898341A (en) * | 2012-10-26 | 2013-01-30 | 贵州大学 | Extracting and purifying method of high-purity sulforaphane |
| CN104130171A (en) * | 2013-05-02 | 2014-11-05 | 西安瑞德生物科技有限公司 | Preparation method of sulforaphane |
| CN104086468A (en) * | 2014-05-22 | 2014-10-08 | 浙江大学苏州工业技术研究院 | Method for supercritical carbon dioxide extraction of sulforaphane |
| CN104086467B (en) * | 2014-07-08 | 2016-07-06 | 北京化工大学 | A kind of solvent extraction and molecularly distilled is utilized to combine the method preparing raphanin |
| KR101515573B1 (en) * | 2014-11-07 | 2015-04-28 | 주식회사 파미니티 | Functional composition containing extract of new green for detoxification |
| CN104498547B (en) * | 2014-11-23 | 2017-10-31 | 北京化工大学 | The sulforaphane for improving broccoli seed hydrolyzes the preprocess method of yield |
| CN104803900B (en) * | 2015-04-29 | 2016-08-24 | 重庆大学 | A kind of method preparing sulforaphen continuously |
| CN107496402B (en) * | 2017-08-24 | 2020-10-16 | 中国农业科学院蔬菜花卉研究所 | Sulforaphane with anti-influenza virus effect and preparation method and application thereof |
-
2005
- 2005-12-13 CN CNB2005101304679A patent/CN100497307C/en not_active Expired - Fee Related
Non-Patent Citations (2)
| Title |
|---|
| Separation by solid phase extraction and quantification byreverse phase HPLC of sulforaphane in broccoli. Bertelli，D.[J].Food chemistry，第63卷第3期. 1998 |
| Separation by solid phase extraction and quantification byreverse phase HPLC of sulforaphane in broccoli. Bertelli，D.[J].Food chemistry，第63卷第3期. 1998 * |
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