CN106632520A - Method for preparing high-purity reduced glucoraphanin from radish - Google Patents
Method for preparing high-purity reduced glucoraphanin from radish Download PDFInfo
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- CN106632520A CN106632520A CN201610865375.3A CN201610865375A CN106632520A CN 106632520 A CN106632520 A CN 106632520A CN 201610865375 A CN201610865375 A CN 201610865375A CN 106632520 A CN106632520 A CN 106632520A
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- C07H1/00—Processes for the preparation of sugar derivatives
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- C07H1/08—Separation; Purification from natural products
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Abstract
The invention discloses a method for preparing high-purity reduced glucoraphanin from radish; the method comprises: drying radish, and crushing; extracting radish powder by reflowing through an extracting agent; dissolving in water to extract radish extract, and repeating the dissolution extraction; preliminarily purifying through acidic alumina low-pressure column chromatography; further purifying through hydrophilic C-18 low-pressure column chromatography to obtain dried high-purity reduced glucoraphanin. The raw material, radish, is low in price and easy to obtain and is widely applicable; the reduced glucoraphanin extract is extracted by using the extracting through water dissolution, mass lipophilic impurities are removed by precipitating, and total process yield and the content of reduced glucoraphanin in extract are significantly increased; by combining acidic alumina low-pressure column chromatography and hydrophilic C-18 low-pressure column chromatography, the purity and yield of reduced glucoraphanin are significantly increased, production cost is lowered, the extractive purification process is simplified, and industrial large-scale production is facilitated.
Description
Technical field
The invention belongs to the extraction purification technical field of plant actives, and in particular to one kind is with radish as raw material system
The method of standby high-purity reduced form glucorphanin.
Background technology
In recent years, epidemiological studies show that brassicaceous vegetable has the effect of significant cancer chemo-preventive,
The Jing probability that often in a large number the crowd of edible brassicaceous vegetable suffers from kinds cancer does not substantially less than eat or eats on a small quantity cruciate flower
The intake of the crowd of section vegetables, i.e. brassicaceous vegetable is presented significant negative correlation, explanation with the incidence of disease of kinds cancer
The active skull cap components contained in brassicaceous vegetable have functions that pre- anti-cancer.Further study showed that, Cruciferae vegetable
Containing substantial amounts of glucosinolate and myrosin in dish, glucosinolate is distributed in cell liquid, and myrosin point
In being distributed in specific proteosome.In the case where brassicaceous vegetable histocyte is intact, glucosinolate and myrosin
Can not contact with each other, glucosinolate can stable existence.When histocyte is damaged, myrosin can be with thio grape
Glucosides is contacted, and glucosinolate forms unstable aglycone by fast hydrolyzing, and the aglycone is by non-enzymatic catalysis
Intramolecular rearrangement reacts, and is converted into the isosulfocyanate compound with efficient active anticancer, therefore, in people edible ten
During Zi Hua sections vegetables, brassicaceous vegetable can generate and discharge the isosulfocyanate with efficient active anticancer
Compound, so as to be provided with effect of anti-cancer and cancer-preventing.
Radish is a kind of daily a large amount of edible brassicaceous vegetables of people, in radish sprout, radish rhizome and radish leaf all
Containing abundant reduced form glucorphanin (4- methyl mercapto -3- cyclobutenyl glucosinolates, chemical constitution is shown in Fig. 2) and black mustard
Enzyme, myrosin can hydrolyze reduced form glucorphanin, and be converted into reduced form by the intramolecular rearrangement reaction of non-enzymatic catalysis
Sulforaphen (4- methyl mercapto -3- cyclobutenyl isothiocyanates).Reduced form sulforaphen is with II phase detoxification enzyme activity of induction
A kind of isosulfocyanate compound, it can not only improve human body to carcinogenic Scavenging activity, may also suppress and to kill cancer thin
Born of the same parents.Research shows that reduced form sulforaphen can hinder tumour by blocking Cancer Cell cycle circulation and promoting cancer cell-apoptosis
The growth of tissue；Reduced form sulforaphen can specifically be adjusted as cell carcinogenic metabolic pathway repressor by mechanism and multiple
The carcinogenic metabolic process of section histocyte；Reduced form sulforaphen can efficiently induce the expression of quinone oxidoreductase, in accelerating cell
The metabolism discharge process of carcinogen, so as to have functions that cancer chemo-preventive；Reduced form sulforaphen has suppression pylorus spiral shell
The activity of bacillus growing multiplication and mouse gastritis, and pylorus spiral shell can be prevented by suppressing and eliminating helicobacter pylori infections
The cancer of the stomach that bacillus infection causes.Reduced form sulforaphen is the most strong phytochemical components of the active anticancer found in radish, to breast
The kinds cancers such as gland cancer, lung cancer, prostate cancer and intestinal cancer all have very strong suppression effect.But reduced form sulforaphen is stablized
Property extreme difference, it is degradable, and easily with the reaction of the compound containing sulfydryl and lose active anticancer, and its precursor substance reduced form radish sulphur
Glycosides stable chemical nature, it is not degradable, and after human body intake, can be converted in the presence of enteric microorganism with efficient
The reduced form sulforaphen of active anticancer, therefore, reduced form glucorphanin can as anticancer and anti-cancer active skull cap components, add to
In food, functional food, health products and medicine.At present, in the world can be for sale the high sterling of glucosinolate (purity >=
98%) mainly there are 4- methylsulfinyl -3- butyl glucosinolates, 4- methyl mercapto -3- butyl glucosinolates, 4- first
Base sulfinyl -3- cyclobutenyl glucosinolates and pi-allyl glucosinolate, are provided by the U.S. and German company, and
It is expensive, and there is efficient anticancer and the high sterling of reduced form glucorphanin of anti-cancer effect there is no Company.
The content of the invention
The present invention for current reduced form glucorphanin rely on external import, the complicated shortcoming of expensive, technology,
There is provided a kind of method for preparing high-purity reduced form glucorphanin as raw material with radish, the method efficient can prepare high-purity
The reduced form glucorphanin of degree, greatly reduces production cost, realizes the industrialized production of the high sterling of reduced form glucorphanin.
The purpose of the present invention is achieved by the following technical solution：
A kind of method for preparing high-purity reduced form glucorphanin as raw material with radish, is prepared by following steps
's：
(1) by radish raw material drying, crush, obtain radish raw material powder；
(2) with extractant refluxing extraction radish raw material powder, filter, collect filtrate, repeat refluxing extraction, be filtered to remove
Solid impurity, collects and merging filtrate, and extractant is reclaimed in vacuum distillation, obtains dry Turnip extractive；
(3) Turnip extractive is extracted with water dissolves, is filtered, collect filtrate, repeated dissolving and extract, solids removed by filtration impurity,
Collect and merging filtrate, vacuum distillation obtains dry reduced form glucorphanin extract；
(4) acidic alumina low-pressure column chromatography preliminary purification reduced form glucorphanin extract：
A. the acidic alumina by particle diameter for 25-250 μm is soaked in deionized water, soaks 10-120min, repeated washing
1-5 time, after deionized water washing, in filling in the stainless steel column of low pressure that ratio of height to diameter is 2-30；
B. the dissolving of reduced form glucorphanin extract deionized water is configured to into the solution that concentration is 25-750mg/mL,
5000rpm is centrifuged 20min, removes solid impurity, obtains reduced form glucorphanin extract solution；
C. by reduced form glucorphanin extract solution loading, sample size is every milliliter of acidic alumina loading absorption 0.25-
20 milligrams of reduced form glucorphanin extracts, deionized water wash-out removes the impurity that can not be adsorbed by acidic alumina；
D. with the potassium sulfate-aqueous solution wash-out of 5-20g/L, in flow velocity be 1-15BV/h and Detection wavelength is 210-245nm
Under, reduced form glucorphanin chromatographic peak is collected, vacuum distillation obtains dry reduced form glucorphanin and potassium sulfate mixture；
E. with absolute methanol stirring leaching reduced form glucorphanin and potassium sulfate mixture, it is filtered to remove potassium sulfate solid miscellaneous
Matter, collects and merging filtrate, and methyl alcohol is reclaimed in vacuum distillation, obtains dry reduced form glucorphanin semi-finished product；
(5) hydrophilic C-18 low-pressure column chromatographies are further purified：
A. the hydrophilic C-18 by particle diameter for 10-140 μm is soaked in absolute methanol, soaks 10-120min, repeated washing 1-
5 times, after absolute methanol washing, in filling in the stainless steel column of low pressure that ratio of height to diameter is 2-30；
B., reduced form glucorphanin semi-finished product are configured to the solution of 10-750mg/mL with ultrapure water dissolves, 5000rpm from
Heart 20min, removes solid impurity, obtains reduced form glucorphanin semi-finished product solution；
C. by reduced form glucorphanin semi-finished product solution loading, sample size is that per milliliter of hydrophilic C-18 loading adsorbs 0.1-15
Milligram reduced form glucorphanin semi-finished product；
D. ultrapure water elution, under being 210-245nm for 1-15BV/h and Detection wavelength in flow velocity, collects reduced form radish sulphur
Glycosides chromatographic peak, it is freeze-dried to dry high-purity reduced form glucorphanin.
Described, in step (1), radish raw material are the one kind in radish sprout, radish rhizome and radish leaf；The drying
It is 1.5-7.5% for oven drying under the conditions of 40-65 DEG C to water content；The powder size of crushing is 30-300 mesh.
Described, in step (2), extractant is 70-100% methanol-water solutions, and the addition of extractant is radish former material
5-30 times of material powder weight, refluxing extraction 0.5-3h repeats refluxing extraction 1-5 time.
Preferably, in step (2), the addition of extractant is 10-20 times of radish raw material powder weight, refluxing extraction
1-2h, repeats refluxing extraction 2-3 time.
Described, in step (3), the addition of water is 5-30 times of Turnip extractive weight, dissolves and extracts 10-180min,
Repeat dissolving to extract 1-5 time.
Preferably, in step (3), the addition of water is 10-20 times of Turnip extractive weight, dissolves and extracts 30-60min,
Repeat dissolving to extract 2-3 time.
Preferably, in step (4), the particle diameter of acidic alumina is 50-200 μm, and soak time is 30-60min, and repetition is washed
Wash 2-3 time, the ratio of height to diameter of the stainless steel column of low pressure is 5-20, and reduced form glucorphanin extract solution concentration is 50-500mg/mL,
Sample size is every milliliter of acidic alumina loading absorption 0.5-15 milligram reduced form glucorphanin extract, and potassium sulfate-aqueous solution is dense
Spend for 5-15g/L, flow velocity is 3-10BV/h, and Detection wavelength is 220-240nm.
Described, in step (4), the addition of absolute methanol is reduced form glucorphanin and potassium sulfate mixture gross mass
10-30 times, stirring leaching 10-60min, repeat leaching 1-3 time.
Preferably, in step (5), the particle diameter of hydrophilic C-18 is 25-100 μm；Soak time is 30-60min；Repeated washing
2-3 time；Low pressure stainless steel post ratio of height to diameter is 5-20；Reduced form glucorphanin semi-finished product solution concentration is 25-500mg/mL；Sample introduction
Amount is that per milliliter of hydrophilic C-18 loading adsorbs 0.25-10 milligram reduced form glucorphanin semi-finished product；Flow velocity is 3-10BV/h；Detection
Wavelength is 220-240nm.
Beneficial effects of the present invention：The raw material of the present invention are cheap and easy to get, applied widely, using extractant and water dissolves
Reduced form glucorphanin extract is extracted, while precipitate removing a large amount of lipophilic contaminant compositions, overall craft yield is significantly improved
With the content of reduced form glucorphanin in extract；Phase is adsorbed using the absorption of acidic alumina lower pressure column and hydrophilic C-18 lower pressure columns
With reference to, the purity and yield of reduced form glucorphanin product are significantly improved, production cost is reduced, simplify extraction and purification process
Process, it is easy to industrial amplification production.
Description of the drawings
Fig. 1 is the process chart of the present invention；
Fig. 2 is the chemical constitution of reduced form glucorphanin prepared by the present invention；
Fig. 3 is the high-efficient liquid phase chromatogram of reduced form glucorphanin extract prepared by the present invention；
Fig. 4 is the chromatogram of acidic alumina lower pressure column preliminary purification reduced form glucorphanin extract of the present invention；
Fig. 5 is the chromatogram that hydrophilic C-18 lower pressure columns of the invention purify reduced form glucorphanin semi-finished product；
Fig. 6 is the high-efficient liquid phase chromatogram of high-purity reduced form glucorphanin prepared by the present invention.
Specific embodiment
Below in conjunction with specific embodiment, the process technology scheme of the present invention is further described, but, these embodiments
The restriction to the claimed scope of the invention is not constituted.
Embodiment 1
A kind of method for preparing high-purity reduced form glucorphanin as raw material with radish, is prepared by following steps
's：
(1) fresh radish sprout is harvested, 50 DEG C of oven dryings obtain dry radish sprout, and water content is 3.5%；It is high
Fast pulverizer is crushed and sieved, and obtains radish sprout powder of the granularity in the range of 50-300 mesh；
(2) using 100% methanol-water solution refluxing extraction radish sprout powder, the addition of 100% methanol-water solution is
5 times of radish sprout powder weight, refluxing extraction 1h repeats to extract 5 times, and solids removed by filtration impurity is collected and merging filtrate,
Under the conditions of 50 DEG C, methyl alcohol is reclaimed in vacuum distillation, obtains dry Turnip extractive, and recovery rate is 25.3%；
(3) Turnip extractive is dissolved in the water, the addition of water is 20 times of Turnip extractive weight, dissolves and extracts
60min, repeats to extract 3 times, and solids removed by filtration impurity is collected and merging filtrate, and the vacuum distillation under the conditions of 55 DEG C obtains drying
Reduced form glucorphanin extract, total recovery rate be 16.4%；
(4) using acidic alumina low-pressure column chromatography preliminary purification reduced form glucorphanin extract：
A. the acidic alumina by particle diameter for 75-150 μm is soaked in deionized water, immersion 10min, repeated washing 5 times,
In filling in the stainless steel column of low pressure that ratio of height to diameter is 30；
B. the dissolving of reduced form glucorphanin extract deionized water is configured to into the solution that concentration is 25mg/mL,
5000rpm is centrifuged 20min, removes solid impurity, obtains reduced form glucorphanin extract solution；
C. by reduced form glucorphanin extract solution loading, sample size is every milliliter of acidic alumina loading absorption 0.25
Milligram reduced form glucorphanin extract, deionized water wash-out fully removes the impurity that can not be adsorbed by acidic alumina；
D. with the potassium sulfate-aqueous solution wash-out of 20g/L, under being 235nm for 1BV/h and Detection wavelength in flow velocity, collect also
Prototype glucorphanin chromatographic peak, the vacuum distillation under the conditions of 55 DEG C obtains dry reduced form glucorphanin and potassium sulfate mixture；
E. leached with absolute methanol stirring, the addition of absolute methanol is that reduced form glucorphanin and potassium sulfate mixture are total
10 times of quality, stirring leaching 60min, repeat leaching 2 times, are filtered to remove potassium sulfate solid impurity, collect and merging filtrate,
Methyl alcohol is reclaimed in vacuum distillation under the conditions of 50 DEG C, and obtains dry reduced form glucorphanin semi-finished product, reduced form glucorphanin content
For 44.2%, yield is 96.5%；
(5) hydrophilic C-18 low-pressure column chromatographies are further purified：
A. the hydrophilic C-18 that particle diameter is 10 μm is soaked in absolute methanol, soaks 120min, absolute methanol repeated washing 3
It is secondary, in filling in the stainless steel column of low pressure that ratio of height to diameter is 2；
B., reduced form glucorphanin semi-finished product are configured to the solution of 500mg/mL, 5000rpm centrifugations with ultrapure water dissolves
20min, removes solid impurity, obtains reduced form glucorphanin semi-finished product solution；
C. by reduced form glucorphanin semi-finished product solution loading, sample size is that per milliliter of hydrophilic C-18 loading adsorbs 10 milligrams
Reduced form glucorphanin semi-finished product；
D. ultrapure water elution is used, under being 235nm for 3BV/h and Detection wavelength in flow velocity, reduced form glucorphanin chromatogram is collected
Peak, freeze-dried to obtain high-purity reduced form glucorphanin, reduced form glucorphanin content is 99.6%, and yield is 91.3%.
Embodiment 2
A kind of method for preparing high-purity reduced form glucorphanin as raw material with radish, is prepared by following steps
's：
(1) fresh radish sprout is harvested, 40 DEG C of oven dryings obtain dry radish sprout, and water content is 7.5%；It is high
Fast pulverizer is crushed and sieved, and obtains radish sprout powder of the granularity in the range of 30-300 mesh；
(2) using 80% methanol-water solution refluxing extraction radish sprout powder, the addition of 80% methanol-water solution is trailing plants
Foretell sprout powder weight 30 times, refluxing extraction 0.5h repeats to extract 3 times, and solids removed by filtration impurity is collected and merging filtrate,
Under the conditions of 50 DEG C, methyl alcohol is reclaimed in vacuum distillation, obtains dry Turnip extractive, and recovery rate is 27.5%；
(3) Turnip extractive is dissolved in the water, the addition of water is 5 times of Turnip extractive weight, dissolves and extracts
10min, repeats to extract 5 times, and solids removed by filtration impurity is collected and merging filtrate, and the vacuum distillation under the conditions of 55 DEG C obtains drying
Reduced form glucorphanin extract, total recovery rate be 19.2%；
(4) using acidic alumina low-pressure column chromatography preliminary purification reduced form glucorphanin extract：
A. the acidic alumina by particle diameter for 25-50 μm is soaked in deionized water, immersion 60min, repeated washing 3 times,
In filling in the stainless steel column of low pressure that ratio of height to diameter is 20；
B. the dissolving of reduced form glucorphanin extract deionized water is configured to into the solution that concentration is 50mg/mL,
5000rpm is centrifuged 20min, removes solid impurity, obtains reduced form glucorphanin extract solution；
C. by reduced form glucorphanin extract solution loading, sample size is every milliliter of milli of acidic alumina loading absorption 0.5
Gram reduced form glucorphanin extract, deionized water wash-out fully removes the impurity that can not be adsorbed by acidic alumina；
D. with the potassium sulfate-aqueous solution wash-out of 10g/L, under being 210nm for 6BV/h and Detection wavelength in flow velocity, collect also
Prototype glucorphanin chromatographic peak, the vacuum distillation under the conditions of 55 DEG C obtains dry reduced form glucorphanin and potassium sulfate mixture；
E. leached with absolute methanol stirring, the addition of absolute methanol is that reduced form glucorphanin and potassium sulfate mixture are total
30 times of quality, stirring leaching 30min, repeat leaching 1 time, are filtered to remove potassium sulfate solid impurity, collect and merging filtrate,
Methyl alcohol is reclaimed in vacuum distillation under the conditions of 50 DEG C, and obtains dry reduced form glucorphanin semi-finished product, reduced form glucorphanin content
For 50.3%, yield is 93.6%；
(5) hydrophilic C-18 low-pressure column chromatographies are further purified：
A. the hydrophilic C-18 that particle diameter is 50 μm is soaked in absolute methanol, soaks 60min, absolute methanol repeated washing 5
It is secondary, in filling in the stainless steel column of low pressure that ratio of height to diameter is 5；
B., reduced form glucorphanin semi-finished product are configured to the solution of 10mg/mL, 5000rpm centrifugations with ultrapure water dissolves
20min, removes solid impurity, obtains reduced form glucorphanin semi-finished product solution；
C. by reduced form glucorphanin semi-finished product solution loading, sample size is that per milliliter of hydrophilic C-18 loading adsorbs 0.1 milligram
Reduced form glucorphanin semi-finished product；
D. ultrapure water elution is used, under being 210nm for 1BV/h and Detection wavelength in flow velocity, reduced form glucorphanin chromatogram is collected
Peak, freeze-dried to obtain high-purity reduced form glucorphanin, reduced form glucorphanin content is 99.2%, and yield is 92.5%.
Embodiment 3
A kind of method for preparing high-purity reduced form glucorphanin as raw material with radish, is prepared by following steps
's：
(1) fresh radish sprout is harvested, 65 DEG C of oven dryings obtain dry radish sprout, and water content is 1.5%；It is high
Fast pulverizer is crushed and sieved, and obtains radish sprout powder of the granularity in the range of 60-300 mesh；
(2) using 70% methanol-water solution refluxing extraction radish sprout powder, the addition of 70% methanol-water solution is trailing plants
Foretell sprout powder weight 20 times, refluxing extraction 3h repeats to extract 1 time, and solids removed by filtration impurity is collected and merging filtrate,
Under the conditions of 50 DEG C, methyl alcohol is reclaimed in vacuum distillation, obtains dry Turnip extractive, and recovery rate is 27.6%；
(3) Turnip extractive is dissolved in the water, the addition of water is 30 times of Turnip extractive weight, dissolves and extracts
180min, repeats to extract 1 time, and solids removed by filtration impurity is collected and merging filtrate, and the vacuum distillation under the conditions of 55 DEG C obtains dry
Dry reduced form glucorphanin extract, total recovery rate is 17.1%；
(4) using acidic alumina low-pressure column chromatography preliminary purification reduced form glucorphanin extract：
A. the acidic alumina by particle diameter for 150-250 μm is soaked in deionized water, soaks 120min, repeated washing 5
It is secondary, in filling in the stainless steel column of low pressure that ratio of height to diameter is 15；
B. the dissolving of reduced form glucorphanin extract deionized water is configured to into the solution that concentration is 350mg/mL,
5000rpm is centrifuged 20min, removes solid impurity, obtains reduced form glucorphanin extract solution；
C. by reduced form glucorphanin extract solution loading, sample size is that every milliliter of acidic alumina loading adsorbs 7 milligrams
Reduced form glucorphanin extract, deionized water wash-out fully removes the impurity that can not be adsorbed by acidic alumina；
D. with the potassium sulfate-aqueous solution wash-out of 15g/L, under being 245nm for 10BV/h and Detection wavelength in flow velocity, collect also
Prototype glucorphanin chromatographic peak, the vacuum distillation under the conditions of 55 DEG C obtains dry reduced form glucorphanin and potassium sulfate mixture；
E. leached with absolute methanol stirring, the addition of absolute methanol is that reduced form glucorphanin and potassium sulfate mixture are total
20 times of quality, stirring leaching 10min, repeat leaching 3 times, are filtered to remove potassium sulfate solid impurity, collect and merging filtrate,
Methyl alcohol is reclaimed in vacuum distillation under the conditions of 50 DEG C, and obtains dry reduced form glucorphanin semi-finished product, reduced form glucorphanin content
For 46.7%, yield is 94.8%；
(5) hydrophilic C-18 low-pressure column chromatographies are further purified：
A. the hydrophilic C-18 that particle diameter is 100 μm is soaked in absolute methanol, soaks 30min, absolute methanol repeated washing 2
It is secondary, in filling in the stainless steel column of low pressure that ratio of height to diameter is 30；
B., reduced form glucorphanin semi-finished product are configured to the solution of 25mg/mL, 5000rpm centrifugations with ultrapure water dissolves
20min, removes solid impurity, obtains reduced form glucorphanin semi-finished product solution；
C. by reduced form glucorphanin semi-finished product solution loading, sample size is per milliliter of hydrophilic milli of C-18 loadings absorption 0.25
Gram reduced form glucorphanin semi-finished product；
D. ultrapure water elution is used, under being 245nm for 10BV/h and Detection wavelength in flow velocity, reduced form glucorphanin color is collected
Spectral peak, freeze-dried to obtain high-purity reduced form glucorphanin, reduced form glucorphanin content is 99.5%, and yield is
90.8%.
Embodiment 4
A kind of method for preparing high-purity reduced form glucorphanin as raw material with radish, is prepared by following steps
's：
(1) fresh radish rhizome is harvested, is cut into the square cylinder fritter of 10 × 10 × 20mm, 50 DEG C of oven dryings are obtained
Dry radish rhizome, water content is 4.0%；High speed disintegrator is crushed and sieved, and obtains trailing plants of the granularity in the range of 40-300 mesh
Foretell rhizome powder；
(2) using 90% methanol-water solution refluxing extraction radish rhizome powder, the addition of 90% methanol-water solution is trailing plants
Foretell rhizome powder weight 10 times, refluxing extraction 2h repeats to extract 3 times, and solids removed by filtration impurity is collected and merging filtrate,
Under the conditions of 50 DEG C, methyl alcohol is reclaimed in vacuum distillation, obtains dry Turnip extractive, and recovery rate is 22.1%；
(3) Turnip extractive is dissolved in the water, the addition of water is 20 times of Turnip extractive weight, dissolves and extracts
120min, repeats to extract 2 times, and solids removed by filtration impurity is collected and merging filtrate, and the vacuum distillation under the conditions of 55 DEG C obtains dry
Dry reduced form glucorphanin extract, total recovery rate is 15.2%；
(4) using acidic alumina low-pressure column chromatography preliminary purification reduced form glucorphanin extract：
A. the acidic alumina by particle diameter for 50-200 μm is soaked in deionized water, soaks 120min, repeated washing 1
It is secondary, in filling in the stainless steel column of low pressure that ratio of height to diameter is 2；
B. the dissolving of reduced form glucorphanin extract deionized water is configured to into the solution that concentration is 750mg/mL,
5000rpm is centrifuged 20min, removes solid impurity, obtains reduced form glucorphanin extract solution；
C. by reduced form glucorphanin extract solution loading, sample size is every milliliter of milli of acidic alumina loading absorption 15
Gram reduced form glucorphanin extract, deionized water wash-out fully removes the impurity that can not be adsorbed by acidic alumina；
D. with the potassium sulfate-aqueous solution wash-out of 5g/L, under being 220nm for 15BV/h and Detection wavelength in flow velocity, collect also
Prototype glucorphanin chromatographic peak, the vacuum distillation under the conditions of 55 DEG C obtains dry reduced form glucorphanin and potassium sulfate mixture；
E. leached with absolute methanol stirring, the addition of absolute methanol is that reduced form glucorphanin and potassium sulfate mixture are total
10 times of quality, stirring leaching 20min, repeat leaching 2 times, are filtered to remove potassium sulfate solid impurity, collect and merging filtrate,
Methyl alcohol is reclaimed in vacuum distillation under the conditions of 50 DEG C, and obtains dry reduced form glucorphanin semi-finished product, reduced form glucorphanin content
For 43.5%, yield is 93.2%；
(5) hydrophilic C-18 low-pressure column chromatographies are further purified：
A. the hydrophilic C-18 that particle diameter is 25 μm is soaked in absolute methanol, soaks 10min, absolute methanol repeated washing 3
It is secondary, in filling in the stainless steel column of low pressure that ratio of height to diameter is 10；
B., reduced form glucorphanin semi-finished product are configured to the solution of 750mg/mL, 5000rpm centrifugations with ultrapure water dissolves
20min, removes solid impurity, obtains reduced form glucorphanin semi-finished product solution；
C. by reduced form glucorphanin semi-finished product solution loading, sample size is that per milliliter of hydrophilic C-18 loading adsorbs 15 milligrams
Reduced form glucorphanin semi-finished product；
D. ultrapure water elution is used, under being 220nm for 15BV/h and Detection wavelength in flow velocity, reduced form glucorphanin color is collected
Spectral peak, freeze-dried to obtain high-purity reduced form glucorphanin, reduced form glucorphanin content is 98.5%, and yield is
86.7%.
Embodiment 5
A kind of method for preparing high-purity reduced form glucorphanin as raw material with radish, is prepared by following steps
's：
(1) fresh radish rhizome is harvested, is cut into the square cylinder fritter of 10 × 10 × 20mm, 55 DEG C of oven dryings are obtained
Dry radish rhizome, water content is 2.5%；High speed disintegrator is crushed and sieved, and obtains trailing plants of the granularity in the range of 60-300 mesh
Foretell rhizome powder；
(2) using 100% methanol-water solution refluxing extraction radish rhizome powder, the addition of 100% methanol-water solution is
20 times of radish rhizome powder weight, refluxing extraction 0.5h repeats to extract 5 times, and filter is collected and merged to solids removed by filtration impurity
Liquid, under the conditions of 50 DEG C, methyl alcohol is reclaimed in vacuum distillation, obtains dry Turnip extractive, and recovery rate is 21.5%；
(3) Turnip extractive is dissolved in the water, the addition of water is 10 times of Turnip extractive weight, dissolves and extracts
60min, repeats to extract 3 times, and solids removed by filtration impurity is collected and merging filtrate, and the vacuum distillation under the conditions of 55 DEG C obtains drying
Reduced form glucorphanin extract, total recovery rate be 13.8%；
(4) using acidic alumina low-pressure column chromatography preliminary purification reduced form glucorphanin extract：
A. the acidic alumina by particle diameter for 75-150 μm is soaked in deionized water, immersion 90min, repeated washing 4 times,
In filling in the stainless steel column of low pressure that ratio of height to diameter is 5；
B. the dissolving of reduced form glucorphanin extract deionized water is configured to into the solution that concentration is 250mg/mL,
5000rpm is centrifuged 20min, removes solid impurity, obtains reduced form glucorphanin extract solution；
C. by reduced form glucorphanin extract solution loading, sample size is every milliliter of milli of acidic alumina loading absorption 10
Gram reduced form glucorphanin extract, deionized water wash-out fully removes the impurity that can not be adsorbed by acidic alumina；
D. with the potassium sulfate-aqueous solution wash-out of 15g/L, under being 230nm for 3BV/h and Detection wavelength in flow velocity, collect also
Prototype glucorphanin chromatographic peak, the vacuum distillation under the conditions of 55 DEG C obtains dry reduced form glucorphanin and potassium sulfate mixture；
E. leached with absolute methanol stirring, the addition of absolute methanol is that reduced form glucorphanin and potassium sulfate mixture are total
30 times of quality, stirring leaching 40min, repeat leaching 1 time, are filtered to remove potassium sulfate solid impurity, collect and merging filtrate,
Methyl alcohol is reclaimed in vacuum distillation under the conditions of 50 DEG C, and obtains dry reduced form glucorphanin semi-finished product, reduced form glucorphanin content
For 40.9%, yield is 95.6%；
(5) hydrophilic C-18 low-pressure column chromatographies are further purified：
A. the hydrophilic C-18 that particle diameter is 140 μm is soaked in absolute methanol, soaks 90min, absolute methanol repeated washing 1
It is secondary, in filling in the stainless steel column of low pressure that ratio of height to diameter is 20；
B., reduced form glucorphanin semi-finished product are configured to the solution of 250mg/mL, 5000rpm centrifugations with ultrapure water dissolves
20min, removes solid impurity, obtains reduced form glucorphanin semi-finished product solution；
C. by reduced form glucorphanin semi-finished product solution loading, sample size is that per milliliter of hydrophilic C-18 loading adsorbs 10 milligrams
Reduced form glucorphanin semi-finished product；
D. ultrapure water elution is used, under being 230nm for 10BV/h and Detection wavelength in flow velocity, reduced form glucorphanin color is collected
Spectral peak, freeze-dried to obtain high-purity reduced form glucorphanin, reduced form glucorphanin content is 98.8%, and yield is
90.5%.
Embodiment 6
A kind of method for preparing high-purity reduced form glucorphanin as raw material with radish, is prepared by following steps
's：
(1) fresh radish leaf is harvested, is cut into the square turnip leaves of 10 × 10mm, 45 DEG C of oven dryings obtain drying
Radish leaf, water content is 5.5%；High speed disintegrator is crushed and sieved, and obtains radish leaves of the granularity in the range of 30-300 mesh
Sub- powder；
(2) using 80% methanol-water solution refluxing extraction radish leaf powder, the addition of 80% methanol-water solution is trailing plants
30 times of Bu Yezi powder weights, refluxing extraction 2h repeats to extract 2 times, and solids removed by filtration impurity is collected and merging filtrate,
Under the conditions of 50 DEG C, methyl alcohol is reclaimed in vacuum distillation, obtains dry Turnip extractive, and recovery rate is 18.5%；
(3) Turnip extractive is dissolved in the water, the addition of water is 30 times of Turnip extractive weight, dissolves and extracts
30min, repeats to extract 2 times, and solids removed by filtration impurity is collected and merging filtrate, and the vacuum distillation under the conditions of 55 DEG C obtains drying
Reduced form glucorphanin extract, total recovery rate be 9.51%；
(4) using acidic alumina low-pressure column chromatography preliminary purification reduced form glucorphanin extract：
A. the acidic alumina by particle diameter for 50-75 μm is soaked in deionized water, immersion 30min, repeated washing 2 times,
In filling in the stainless steel column of low pressure that ratio of height to diameter is 20；
B. the dissolving of reduced form glucorphanin extract deionized water is configured to into the solution that concentration is 500mg/mL,
5000rpm is centrifuged 20min, removes solid impurity, obtains reduced form glucorphanin extract solution；
C. by reduced form glucorphanin extract solution loading, sample size is every milliliter of milli of acidic alumina loading absorption 20
Gram reduced form glucorphanin extract, deionized water wash-out fully removes the impurity that can not be adsorbed by acidic alumina；
D. with the potassium sulfate-aqueous solution wash-out of 10g/L, under being 240nm for 10BV/h and Detection wavelength in flow velocity, collect also
Prototype glucorphanin chromatographic peak, the vacuum distillation under the conditions of 55 DEG C obtains dry reduced form glucorphanin and potassium sulfate mixture；
E. leached with absolute methanol stirring, the addition of absolute methanol is that reduced form glucorphanin and potassium sulfate mixture are total
20 times of quality, stirring leaching 50min, repeat leaching 2 times, are filtered to remove potassium sulfate solid impurity, collect and merging filtrate,
Methyl alcohol is reclaimed in vacuum distillation under the conditions of 50 DEG C, and obtains dry reduced form glucorphanin semi-finished product, reduced form glucorphanin content
For 38.5%, yield is 96.8%；
(5) hydrophilic C-18 low-pressure column chromatographies are further purified：
A. the hydrophilic C-18 that particle diameter is 50 μm is soaked in absolute methanol, soaks 60min, absolute methanol repeated washing 3
It is secondary, in filling in the stainless steel column of low pressure that ratio of height to diameter is 15；
B., reduced form glucorphanin semi-finished product are configured to the solution of 100mg/mL, 5000rpm centrifugations with ultrapure water dissolves
20min, removes solid impurity, obtains reduced form glucorphanin semi-finished product solution；
C. by reduced form glucorphanin semi-finished product solution loading, sample size is that per milliliter of hydrophilic C-18 loading adsorbs 5 milligrams also
Prototype glucorphanin semi-finished product；
D. ultrapure water elution is used, under being 240nm for 6BV/h and Detection wavelength in flow velocity, reduced form glucorphanin chromatogram is collected
Peak, freeze-dried to obtain high-purity reduced form glucorphanin, reduced form glucorphanin content is 98.2%, and yield is 85.6%.
Claims (9)
1. it is a kind of with radish as raw material prepare high-purity reduced form glucorphanin method, it is characterised in that be by following steps
Prepare：
(1) by radish raw material drying, crush, obtain radish raw material powder；
(2) with extractant refluxing extraction radish raw material powder, filter, collect filtrate, repeat refluxing extraction, solids removed by filtration
Impurity, collects and merging filtrate, and extractant is reclaimed in vacuum distillation, obtains dry Turnip extractive；
(3) Turnip extractive is extracted with water dissolves, is filtered, collect filtrate, repeated dissolving and extract, solids removed by filtration impurity is collected
And merging filtrate, vacuum distillation, obtain dry reduced form glucorphanin extract；
(4) acidic alumina low-pressure column chromatography preliminary purification reduced form glucorphanin extract：
A. the acidic alumina by particle diameter for 25-250 μm is soaked in deionized water, soaks 10-120min, repeated washing 1-5
It is secondary, after deionized water washing, in filling in the stainless steel column of low pressure that ratio of height to diameter is 2-30；
B. the dissolving of reduced form glucorphanin extract deionized water is configured to into the solution that concentration is 25-750mg/mL,
5000rpm is centrifuged 20min, removes solid impurity, obtains reduced form glucorphanin extract solution；
C. by reduced form glucorphanin extract solution loading, sample size is every milliliter of acidic alumina loading absorption 0.25-20 milli
Gram reduced form glucorphanin extract, deionized water wash-out removes the impurity that can not be adsorbed by acidic alumina；
D. with the potassium sulfate-aqueous solution wash-out of 5-20g/L, under being 210-245nm for 1-15BV/h and Detection wavelength in flow velocity, receive
Collection reduced form glucorphanin chromatographic peak, vacuum distillation obtains dry reduced form glucorphanin and potassium sulfate mixture；
E. with absolute methanol stirring leaching reduced form glucorphanin and potassium sulfate mixture, potassium sulfate solid impurity is filtered to remove, is received
Methyl alcohol is reclaimed in collection and merging filtrate, vacuum distillation, obtains dry reduced form glucorphanin semi-finished product；
(5) hydrophilic C-18 low-pressure column chromatographies are further purified：
A. the hydrophilic C-18 by particle diameter for 10-140 μm is soaked in absolute methanol, immersion 10-120min, repeated washing 1-5 time,
After absolute methanol washing, in filling in the stainless steel column of low pressure that ratio of height to diameter is 2-30；
B., reduced form glucorphanin semi-finished product are configured to the solution of 10-750mg/mL, 5000rpm centrifugations with ultrapure water dissolves
20min, removes solid impurity, obtains reduced form glucorphanin semi-finished product solution；
C. by reduced form glucorphanin semi-finished product solution loading, sample size is that per milliliter of hydrophilic C-18 loading adsorbs 0.1-15 milligrams
Reduced form glucorphanin semi-finished product；
D. ultrapure water elution, under being 210-245nm for 1-15BV/h and Detection wavelength in flow velocity, collects reduced form glucorphanin color
Spectral peak, it is freeze-dried to dry high-purity reduced form glucorphanin.
2. method according to claim 1, it is characterised in that in the step (1), radish raw material are radish sprout, radish
One kind in rhizome and radish leaf；The drying is 1.5-7.5% for oven drying under the conditions of 40-65 DEG C to water content；Crush
Powder size be 30-300 mesh.
3. method according to claim 1, it is characterised in that in the step (2), extractant be 70-100% methyl alcohol-
The aqueous solution, the addition of extractant is 5-30 times of radish raw material powder weight, and refluxing extraction 0.5-3h repeats refluxing extraction
1-5 time.
4. method according to claim 3, it is characterised in that in the step (2), the addition of extractant is that radish is former
10-20 times of material powder weight, refluxing extraction 1-2h repeats refluxing extraction 2-3 time.
5. method according to claim 1, it is characterised in that in the step (3), the addition of water is Turnip extractive
10-180min is extracted in 5-30 times of weight, dissolving, is repeated dissolving and is extracted 1-5 time.
6. method according to claim 5, it is characterised in that in the step (3), the addition of water is Turnip extractive
30-60min is extracted in 10-20 times of weight, dissolving, is repeated dissolving and is extracted 2-3 time.
7. method according to claim 1, it is characterised in that in the step (4), the particle diameter of acidic alumina is 50-
200 μm, soak time is 30-60min, repeated washing 2-3 time, and the ratio of height to diameter of the stainless steel column of low pressure is 5-20, reduced form radish sulphur
Glucoside extract solution concentration is 50-500mg/mL, and sample size is every milliliter of acidic alumina loading absorption 0.5-15 milligram reduction
Type glucorphanin extract, potassium sulfate-concentration of aqueous solution is 5-15g/L, and flow velocity is 3-10BV/h, and Detection wavelength is 220-
240nm。
8. method according to claim 1, it is characterised in that in the step (4), the addition of absolute methanol is reduction
10-30 times of type glucorphanin and potassium sulfate mixture gross mass, stirring leaching 10-60min, repeats leaching 1-3 time.
9. method according to claim 1, it is characterised in that in the step (5), the particle diameter of hydrophilic C-18 is 25-100
μm；Soak time is 30-60min；Repeated washing 2-3 time；Low pressure stainless steel post ratio of height to diameter is 5-20；Reduced form glucorphanin half
Final mean annual increment solution concentration is 25-500mg/mL；Sample size is that per milliliter of hydrophilic C-18 loading adsorbs 0.25-10 milligram reduced form radish
Sulphur glycosides semi-finished product；Flow velocity is 3-10BV/h；Detection wavelength is 220-240nm.
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| CN111227262A (en) * | 2020-02-11 | 2020-06-05 | 临沂大学 | Preparation method of green radish dietary fiber rich in oxidized and reduced glucoraphanin |
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