CN102102113A - Method for extracting sulforaphane from leaves of cauliflowers - Google Patents
Method for extracting sulforaphane from leaves of cauliflowers Download PDFInfo
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- CN102102113A CN102102113A CN2009102013912A CN200910201391A CN102102113A CN 102102113 A CN102102113 A CN 102102113A CN 2009102013912 A CN2009102013912 A CN 2009102013912A CN 200910201391 A CN200910201391 A CN 200910201391A CN 102102113 A CN102102113 A CN 102102113A
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Abstract
The invention relates to a method for extracting sulforaphane from leaves of cauliflowers, which comprises the following steps of: after bulbs of the cauliflowers are harvested, immediately collecting waste leaves, cleaning, and performing freezing treatment in an ultralow temperature refrigerator; performing vacuum freeze drying, uniformly crushing into powder, taking 1 part of powder, adding exogenous myrosinase in an amount which is 6 to 10 times weight of the powder and deionized water in an amount which is 4 to 6 times weight of the powder, and fully mixing into uniform slurry; and performing enzymolysis reaction, solvent extraction, filtration, concentration, and vacuum freeze drying to obtain high-purity sulforaphane. The raw material sources are rich, the extraction method is simple, and the method is more suitable for industrial production.
Description
Technical field
The invention belongs to agricultural technology field, relate to the separation-extraction technology of activeconstituents in the plant materials.
Background technology
Sulforaphen, claim again " raphanin ", it is a kind of material of chemical functional efficiently, be glucose Semen Raphani glycosides (glucoraphanin, 4-methylsulfinyl-butyl glucosinolate is called for short RAA) through the isothiocyanate derivatives of myrosin (myrosinase) hydrolysis or acid hydrolysis generation, it is soluble in water, relative molecular weight 177.3, molecular formula C
6H
11S
2NO.Basic structure as shown in Figure 1.
A large amount of evidences, sulforaphen has good prevention effect to esophagus cancer, mammary cancer, lung cancer, colorectal carcinoma etc., and especially it can kill the spiral Helicobacter pylori that causes stomach ulcer and most cancer of the stomach.The YueshengZhang report; then can in each histoorgan of animal body, induce generation II type to separate toxenzyme by the little female mouse feeding sulforaphen of giving nascent 6d---glutathione transferring enzyme (GST) and quinone reductase (QR); and pass through above-mentioned immunoprotection mechanism, thereby eliminate the infringement of chemical carcinogen to body generation effect.Jie Defeiyi (1999) also studies show that, under laboratory environment, has successfully killed the spiral Helicobacter pylori from the chemical substance sulforaphen that Caulis et Folium Brassicae capitatae extracts.In addition; (1994) such as Yuesheng Zhang are also reported; with sulforaphen and its analogue outer-2-ethanoyl-outer-different thiocyanates norcamphane of 6-is fed foster by force to mouse in the harm time of carcinogenic substance being exposed to respectively; the incidence of mouse mammary cancer, recurrence rate and its cancer cells weight all significantly descend, the growth of cancer cells sluggishness.Because in recent years, the sickness rate of cancer has the trend that rises gradually, sulforaphen has stronger antitumous effect, this crude substance with very strong anti-curing cancers effect is used for the food and medicine industry, has positive effect for malignant tumour prevention and assisting therapy, and sulforaphen is active substance of plant, has wide researching value and market outlook.
Rape belongs to precursor substance---the glucose Semen Raphani glycosides that contains higher sulforaphen in the vegetables (Cauliflower, broccoli, wild cabbage etc.).The bouquet of Cauliflower all will produce a large amount of cauline leaf wastes after the utilization of gathering, these wastes do not obtain development and use always so far.And the tackling key problem of extracting preparation sulforaphen technology from Cauliflower will effectively utilize the cauline leaf waste, improve resource utilization and agricultural product added value, have good ecological benefits, social benefit and economic benefit.
Developed country such as Japan-US has obtained some aspect the method research that prepare sulforaphen and has made progress extensive the extraction, and the production technique of having explored mainly contains chemical synthesis and enzyme process.Existing great majority research all is directly to extract the sulforaphen precursor from brassicaceous vegetable, obtains sulforaphen through hydrolysis.Davide Bertelli (1998) etc. then obtain rough sulforaphen to the glucose Semen Raphani glycosides of Cauliflower (bouquet, leaf) through organic solvent extraction, utilize Solid-Phase Extraction to obtain refining sulforaphen then.Nathan etc. (2001) proposed a kind of from the defatted seed of Brigadier Cauliflower the method for the preparative liquid chromatography of separation and purification sulforaphen and sulforaphen nitrile simultaneously, all there is complex steps in described method, expensive big problem.
Summary of the invention
For improving existing is raw material extraction separation sulforaphen Technology with the Cauliflower, and at the extraction and separation technology defective of existing sulforaphen, the invention provides a kind of is the Technology that raw material extracts sulforaphen with the Cauliflower blade.
The present invention solves this technical problem the technical scheme that is adopted:
1. the cleaning of blade, freeze-drying, pulverizing
With fresh blade,, put into the Ultralow Temperature Freezer freezing treatment after adopting tap water to clean, for example temperature range :-65 ℃～-70 ℃ as the fresh blade of the Cauliflower of gathering year March in November to the second.Again through vacuum lyophilization to constant weight, take out then be ground into Powdered.
2. homogenate, enzyme digestion reaction
Take out a blade dry powder, (enzyme activity unit is 1.0units to add 6～10 times external source myrosin by weight, adopt 0.05mM, the preparation of the PBS damping fluid of pH=7.0) and 4～6 times deionized water or ultrapure water, be mixed into homogenate on the vortex instrument, 40～50 ℃ of water bath processing, fully enzyme digestion reaction 30～40min.
3. solvent extraction, filtration
After enzyme digestion reaction finishes,, in homogenate, add 8～10 times dichloromethane extraction, and be 5.0～8.0 (W according to the weight ratio of blade dry powder and anhydrous sodium sulphate according to the actual addition of exogenous enzyme and deionized water or ultrapure water
Blade dry powder: W
Anhydrous sodium sulphate=5.0～8.0) add the anhydrous sodium sulfate drying dehydration, medical absorbent cotton filters, and collects filtrate.
4. concentrated, vacuum lyophilization
Filtrate utilizes Rotary Evaporators (30～40 ℃ water-bath, 100～120mba vacuum tightness) to carry out underpressure distillation, and (35～-40 ℃, 4.0～5.0mbar) to constant weight, collects the freeze-drying sample, can obtain sulforaphen through vacuum lyophilization again after concentrating.
Because the beneficial effect that has adopted technique scheme, the present invention to obtain is:
Provided by the invention is a whole set of Technology that raw material extracts sulforaphen with the Cauliflower blade, and its raw material sources are abundant, and extracting method is simple, is more suitable in suitability for industrialized production; So reduced production cost, this has positive effect for applying of sulforaphen.This method can not only be simplified extraction step loaded down with trivial details in the prior art, and can separate and obtain the higher sulforaphen of purity.
Description of drawings
Fig. 1 is the chemical structural drawing of sulforaphen.
Fig. 2 is a method flow synoptic diagram of the present invention.
Embodiment
To describe the technology contents of the technology of the present invention below in detail:
1, cleaning, freeze-drying, pulverizing
The raw material that Cauliflower extracts sulforaphen mainly is taken from the Cauliflower bouquet later discarded blade (the fresh blade of the Cauliflower that gather year March in November to the second) of gathering, because Cauliflower mainly is open-air cultivating and growing, so the fresh blade that needs to gather thoroughly cleans up, can adopt tap water cleaning, cold wind to dry up, remove vein, put into Ultralow Temperature Freezer (65 ℃～-70 ℃) freezing treatment immediately.Take out vacuum lyophilization then to constant weight, utilize pulverizer that blade is broken into the sealing bag of packing into immediately after Powdered ,-40 ℃ of cryodrying conditions are preserved.
2, homogenate, collection filtrate
The a blade dry powder of weighing, (enzyme activity unit is 1.0units to add 6～10 times external source myrosin by weight, adopt 0.05mM, the preparation of the PBS damping fluid of pH=7.0) and 4～6 times deionized water or ultrapure water (pH7.0～8.0), thorough mixing becomes homogenate on the vortex instrument, 40～50 ℃ of water bath processing 30～40min carry out enzyme digestion reaction.
3, solvent extraction, filtration
After enzyme digestion reaction finishes, actual addition according to exogenous enzyme and deionized water or ultrapure water, (3～5min) methylene dichloride that add 8～10 times (are not limited to methylene dichloride fast in homogenate, so long as utilize the similar principle that mixes, the extraction sulforaphen get final product) mixing shake up, extraction 15～20min, and be 5.0～8.0 (W according to the weight ratio of blade dry powder and anhydrous sodium sulphate
Blade dry powder: W
Anhydrous sodium sulphate=5.0～8.0) add anhydrous sodium sulphate (being not limited to anhydrous sodium sulphate) drying and dehydrating, medical absorbent cotton filtration, collection filtrate as long as use this material can not introduce other metal ion, influence the transformation efficiency of sulforaphen.
4, concentrated, vacuum lyophilization
Filtrate adopts Rotary Evaporators (30 ℃ water-bath, 110mba vacuum tightness) to carry out underpressure distillation, and (35～-40 ℃, 4.0～5.0mbar) to constant weight, collects the freeze-drying sample, can obtain sulforaphen through vacuum lyophilization again after concentrating.
Embodiment 1
(1) takes by weighing 1.0g Cauliflower blade dry powder, (enzyme activity unit is 1.0units to the external source myrosin of adding 8mL, adopt 0.05mM, the preparation of the PBS damping fluid of pH=7.0) and deionized water or the ultrapure water (pH7.0～8.0) of 4mL, thorough mixing becomes homogenate on the vortex instrument, 40 ℃ of water bath processing 30min finish enzyme digestion reaction.
(2) solvent extraction, filtration
After enzyme digestion reaction finishes, actual addition 12mL according to exogenous enzyme and deionized water or ultrapure water, fast (3min) methylene dichloride of adding 120mL mixes and shakes up in homogenate, extraction 15min, and be 5.0～8.0 (W according to the weight ratio of blade dry powder and anhydrous sodium sulphate
Blade dry powder: W
Anhydrous sodium sulphate=5.0～8.0) add the dehydration of 6.0g anhydrous sodium sulfate drying, medical absorbent cotton filters, and collects filtrate.
(3) concentrated, vacuum lyophilization
Filtrate adopts BUCHI Rotary Evaporators (30 ℃ water-bath, 110mba vacuum tightness) to carry out underpressure distillation, and (35～-40 ℃, 4.0～5.0mbar) to constant weight, collects the freeze-drying sample, can obtain sulforaphen through vacuum lyophilization again after concentrating.
Embodiment 2
(1) takes by weighing 50.0g Cauliflower blade dry powder, (enzyme activity unit is 1.0units to the external source myrosin of adding 500mL, adopt 0.05mM, the preparation of the PBS damping fluid of pH=7.0) and deionized water or the ultrapure water (pH7.0～8.0) of 200mL, thorough mixing becomes homogenate on the vortex instrument, 40 ℃ of water bath processing 40min finish enzyme digestion reaction.
(2) solvent extraction, filtration
After enzyme digestion reaction finishes, actual addition 700mL according to exogenous enzyme and deionized water or ultrapure water, fast (3min) methylene dichloride of adding 5.6L mixes and shakes up in homogenate, extraction 20min, and be 5.0～8.0 (W according to the weight ratio of blade dry powder and anhydrous sodium sulphate
Blade dry powder: W
Anhydrous sodium sulphate=5.0～8.0) add the dehydration of 300.0g anhydrous sodium sulfate drying, medical absorbent cotton filters, and collects filtrate.
(3) concentrated, vacuum lyophilization
Filtrate adopts BUCHI Rotary Evaporators (40 ℃ water-bath, 120mba vacuum tightness) to carry out underpressure distillation, and (35～-40 ℃, 4.0～5.0mbar) to constant weight, collects the freeze-drying sample, can obtain sulforaphen through vacuum lyophilization again after concentrating.
Embodiment 3
(1) takes by weighing 15.0g～25.0g Cauliflower blade dry powder, (enzyme activity unit is 1.0units to the external source myrosin of adding 100～250mL, adopt 0.05mM, the preparation of the PBS damping fluid of pH=7.0) and deionized water or the ultrapure water (pH7.0～8.0) of 60～150mL, thorough mixing becomes homogenate on the vortex instrument, 40 ℃ of water bath processing 30～50min finish enzyme digestion reaction.
(2) solvent extraction, filtration
After enzyme digestion reaction finishes, actual addition according to exogenous enzyme and deionized water or ultrapure water, fast (3min) methylene dichloride of adding 1000mL～4000mL mixes and shakes up in homogenate, extraction 20～30min, and be 5.0～8.0 (W according to the weight ratio of blade dry powder and anhydrous sodium sulphate
Blade dry powder: W
Anhydrous sodium sulphate=5.0～8.0) add the anhydrous sodium sulfate drying dehydration, medical absorbent cotton filters, and collects filtrate.
(3) concentrated, vacuum lyophilization
Filtrate adopts BUCHI Rotary Evaporators (30 ℃ water-bath, 100mba vacuum tightness) to carry out underpressure distillation, and (35～-40 ℃, 4.0～5.0mbar) to constant weight, collects the freeze-drying sample, can obtain sulforaphen through vacuum lyophilization again after concentrating.
The above-mentioned description to embodiment is can understand and apply the invention for ease of those skilled in the art.The person skilled in the art obviously can easily make various modifications to these embodiment, and needn't pass through performing creative labour being applied in the General Principle of this explanation among other embodiment.Therefore, the invention is not restricted to the embodiment here, those skilled in the art are according to the above content that discloses, and improvement of being made under the situation that does not break away from category of the present invention and modification all should be within protection scope of the present invention.
Claims (5)
1. the extracting method of a sulforaphen is characterized in that: by extracting sulforaphen in the Cauliflower blade.
2. extraction process according to claim 1 is characterized in that: may further comprise the steps: to cleaning, freeze-drying, the pulverizing of Cauliflower blade; Homogenate, enzyme digestion reaction, solvent extraction, filtration, concentrated, vacuum lyophilization.
3. extraction process according to claim 1 is characterized in that: comprising:
1. the cleaning of blade, freeze-drying, pulverizing
Put into the Ultralow Temperature Freezer freezing treatment after fresh blade cleaned, through vacuum lyophilization to constant weight, take out be ground into Powdered;
2. homogenate, enzyme digestion reaction
Take out a blade dry powder, add 6～10 times external source myrosin and 4～6 times deionized water or ultrapure water by weight, be mixed into homogenate on the vortex instrument, 40～50 ℃ of water bath processing, fully enzyme digestion reaction 30～40min;
3. solvent extraction, filtration
Enzyme digestion reaction according to the actual addition of exogenous enzyme and deionized water or ultrapure water, adds 8～10 times dichloromethane extraction after finishing, and adds an amount of anhydrous sodium sulfate drying dehydration, mixes the back and filters with medical absorbent cotton, collects filtrate;
4. concentrated, vacuum lyophilization
Filtrate can obtain sulforaphen through vacuum lyophilization after concentrating by underpressure distillation again.
4. extracting method according to claim 1 is characterized in that: wherein 2. to go on foot used exogenous enzyme be special, and enzyme activity unit is 1.0units.
5. extracting method according to claim 1 is characterized in that: wherein the 4. in the step underpressure distillation condition be 30 ℃ of-40 ℃ of water-baths, vacuum tightness 100mba-120mba.
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102688219A (en) * | 2012-02-03 | 2012-09-26 | 马建华 | Sulforaphane microencapsulation method |
| CN103852361A (en) * | 2014-03-10 | 2014-06-11 | 河南中烟工业有限责任公司 | Closed device for circularly extracting crop leaf surface secreta |
| CN104059947A (en) * | 2014-06-03 | 2014-09-24 | 重庆海巨农业发展有限公司 | Method for preparing high-purity sulforaphane |
| CN104086467A (en) * | 2014-07-08 | 2014-10-08 | 北京化工大学 | Method for preparing sulforaphene by adopting solvent extraction method and molecular distillation method |
| CN104131047A (en) * | 2014-01-14 | 2014-11-05 | 南京农业大学 | High-efficiency enrichment technology for isothiocyanate in brassica bud seedlings |
| CN106588724A (en) * | 2016-12-02 | 2017-04-26 | 湖南农业大学 | Method for preparing sulforaphen by virtue of subcritical fluid extraction |
| CN106982856A (en) * | 2016-01-21 | 2017-07-28 | 黄柏园 | Natural bactericidal agent and its preparation method |
| CN109875011A (en) * | 2019-04-04 | 2019-06-14 | 西南医科大学 | A kind of natural anti-cancer food |
| CN113699192A (en) * | 2021-08-23 | 2021-11-26 | 南开大学 | Method for extracting sulforaphane from broccoli |
| CN113908294A (en) * | 2021-11-02 | 2022-01-11 | 成都格纯生物医药有限公司 | Preparation method and application of double inclusion compound containing refined cauliflower extract |
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Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102688219A (en) * | 2012-02-03 | 2012-09-26 | 马建华 | Sulforaphane microencapsulation method |
| CN104131047A (en) * | 2014-01-14 | 2014-11-05 | 南京农业大学 | High-efficiency enrichment technology for isothiocyanate in brassica bud seedlings |
| CN103852361B (en) * | 2014-03-10 | 2016-08-24 | 河南中烟工业有限责任公司 | A kind of closed crop leaf surface secretions circulation extracting apparatus |
| CN103852361A (en) * | 2014-03-10 | 2014-06-11 | 河南中烟工业有限责任公司 | Closed device for circularly extracting crop leaf surface secreta |
| CN104059947A (en) * | 2014-06-03 | 2014-09-24 | 重庆海巨农业发展有限公司 | Method for preparing high-purity sulforaphane |
| CN104086467A (en) * | 2014-07-08 | 2014-10-08 | 北京化工大学 | Method for preparing sulforaphene by adopting solvent extraction method and molecular distillation method |
| 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 |
| CN106982856A (en) * | 2016-01-21 | 2017-07-28 | 黄柏园 | Natural bactericidal agent and its preparation method |
| CN106588724A (en) * | 2016-12-02 | 2017-04-26 | 湖南农业大学 | Method for preparing sulforaphen by virtue of subcritical fluid extraction |
| CN109875011A (en) * | 2019-04-04 | 2019-06-14 | 西南医科大学 | A kind of natural anti-cancer food |
| CN113699192A (en) * | 2021-08-23 | 2021-11-26 | 南开大学 | Method for extracting sulforaphane from broccoli |
| CN113699192B (en) * | 2021-08-23 | 2023-08-11 | 南开大学 | Method for extracting sulforaphane from broccoli |
| CN113908294A (en) * | 2021-11-02 | 2022-01-11 | 成都格纯生物医药有限公司 | Preparation method and application of double inclusion compound containing refined cauliflower extract |
| CN113908294B (en) * | 2021-11-02 | 2023-09-12 | 成都格纯生物医药有限公司 | Preparation method and application of double inclusion compound containing refined cauliflower extract |
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Application publication date: 20110622 |