CN106632520B - A method of high-purity reduced form glucorphanin is prepared by raw material of radish - Google Patents
A method of high-purity reduced form glucorphanin is prepared by raw material of radish Download PDFInfo
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Abstract
The invention discloses a kind of methods for preparing high-purity reduced form glucorphanin as raw material using radish: radish raw material drying crushes；Extractant refluxing extraction radish raw material powder；Turnip extractive is extracted in water dissolution, repeats dissolution and extracts；Acidic alumina low-pressure column chromatography preliminary purification；Hydrophilic C-18 low-pressure column chromatography is further purified, and obtains dry high-purity reduced form glucorphanin.Raw material of the invention are cheap and easy to get, it is applied widely, reduced form glucorphanin extract is extracted using extractant and water dissolution, while precipitating and removing a large amount of lipophilic contaminant ingredients, significantly improves the content of reduced form glucorphanin in overall craft yield and extract；It is combined using the absorption of acidic alumina lower pressure column and the absorption of hydrophilic C-18 lower pressure column, significantly improves the purity and yield of reduced form glucorphanin product, reduce production cost, simplify extraction and purification process process, be easy to industrial amplification production.
Description
Technical field
The invention belongs to the extraction purification technical fields of plant actives, and in particular to one kind is using radish as raw material system
The method of standby high-purity reduced form glucorphanin.
Background technique
In recent years, epidemiological studies show that brassicaceous vegetable has the function of significant cancer chemo-preventive,
The probability that the crowd of frequent large amounts of food brassicaceous vegetable suffers from kinds cancer is not substantially less than edible or eats cruciate flower on a small quantity
Significant negative correlation, explanation is presented in the crowd of section vegetables, the i.e. disease incidence of the intake of brassicaceous vegetable and kinds cancer
The active skull cap components contained in brassicaceous vegetable have the effect of pre- anti-cancer.Further study showed that Cruciferae vegetable
Contain a large amount of glucosinolate and myrosin in dish, glucosinolate is distributed in cell liquid, and myrosin point
It is distributed in specific proteosome.In the case where brassicaceous vegetable histocyte is intact, glucosinolate and myrosin
It cannot contact with each other, glucosinolate can be stabilized.When histocyte damages, myrosin can be with thio grape
Glucosides contact, glucosinolate form unstable aglycone by fast hydrolyzing, which passes through non-enzymatic
Intramolecular rearrangement reaction, is converted into the isosulfocyanate compound with efficient anticancer activity, therefore, eats ten in people
During Zi Hua section vegetables, brassicaceous vegetable can generate and discharge the isosulfocyanate with efficient anticancer activity
Object is closed, thus the effect of being provided with anti-cancer and cancer-preventing.
Radish is a kind of brassicaceous vegetable of daily large amounts of food of people, radish sprout, radish rhizome and radish leaf
In reduced form glucorphanin (4- methyl mercapto -3- cyclobutenyl glucosinolates, chemical structure are shown in Fig. 2) all rich in and black
Myrosase, myrosin can hydrolyze reduced form glucorphanin, and are converted by the reaction of non-enzymatic intramolecular rearrangement
Reduced form sulforaphen (4- methyl mercapto -3- cyclobutenyl isothiocyanates).Reduced form sulforaphen is that have induction II phase removing toxic substances
A kind of isosulfocyanate compound of enzymatic activity not only can be improved human body to carcinogenic Scavenging activity, may also suppress simultaneously
Kill cancer cell.Studies have shown that reduced form sulforaphen can be by blocking Cancer Cell cycle circulation and promoting cancer cell-apoptosis
And hinder the growth of tumor tissues；Reduced form sulforaphen can be used as cell carcinogenic metabolic pathway repressor, pass through multiple machine
System specifically adjusts the carcinogenic metabolic process of histocyte；Reduced form sulforaphen can efficiently induce the table of quinone oxidoreductase
It reaches, accelerates the metabolism discharge process of carcinogen in cell, to have effects that cancer chemo-preventive；Reduced form sulforaphen
With the activity for inhibiting growth of H. pylori proliferation and mouse gastritis, and can be by inhibiting and eliminating Helicobacter pylori
Dye, to prevent gastric cancer caused by helicobacter pylori infections.Reduced form sulforaphen is that the anticancer activity that finds in radish is strongest
Phytochemical components all have very strong inhibition effect to kinds cancers such as breast cancer, lung cancer, prostate cancer and intestinal cancer.But
Reduced form sulforaphen stability is very poor, degradable, and the compound of Yi Yuhan sulfydryl reacts and loses anticancer activity, and before it
Body substance reduced form glucorphanin chemical property is stablized, not degradable, and after human body intake, can be in the effect of enteric microorganism
Under be converted into the reduced form sulforaphen with efficient anticancer activity, therefore, reduced form glucorphanin can be used as anticancer and anti-
Cancer active skull cap components are added in food, functional food, health care product and drug.Currently, in the world can be for sale it is thio
Glucoside high sterling (purity >=98%) mainly has 4- methylsulfinyl -3- butyl glucosinolate, 4- methyl mercapto -3-
Butyl glucosinolate, 4-methylsulfinyl-3-butenyl glucosinolate and allyl glucosinolate, by
The U.S. and German company provide, and expensive, and the reduced form glucorphanin high sterling with efficient anticancer and the effect that gives protection against cancer
Company there is no to sell.
Summary of the invention
The present invention relies on external import for current reduced form glucorphanin, the disadvantage expensive, technology is complicated,
A kind of method that high-purity reduced form glucorphanin is prepared as raw material using radish is provided, this method can efficient preparation height
The reduced form glucorphanin of purity, greatly reduces production cost, realizes the industrial metaplasia of reduced form glucorphanin high sterling
It produces.
The purpose of the present invention is what is be achieved through the following technical solutions:
A method of high-purity reduced form glucorphanin being prepared by raw material of radish, is to be prepared by the following steps to obtain
:
(1) it by radish raw material drying, crushes, obtains radish raw material powder；
(2) with extractant refluxing extraction radish raw material powder, filtrate is collected in filtering, is repeated refluxing extraction, is filtered to remove
Solid impurity, is collected and merging filtrate, vacuum distillation recycling extractant obtain dry Turnip extractive；
(3) it is dissolved with water and extracts Turnip extractive, filtrate is collected in filtering, is repeated dissolution and is extracted, it is miscellaneous to be filtered to remove solid
Matter, 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 that partial size is 25-250 μm is soaked in deionized water, impregnates 10-120min, repeated washing
It 1-5 times, after deionized water washing, is loaded in the low pressure stainless steel column that ratio of height to diameter is 2-30；
B. reduced form glucorphanin extract is configured to the solution that concentration is 25-750mg/mL with deionized water dissolving,
5000rpm is centrifuged 20min, removes solid impurity, obtains reduced form glucorphanin extract solution；
C. by reduced form glucorphanin extract solution loading, sample volume is every milliliter of acidic alumina loading absorption
0.25-20 milligrams of reduced form glucorphanin extracts, deionized water elution remove the impurity that cannot be adsorbed by acidic alumina；
D. with the potassium sulfate of 5-20g/L-aqueous solution elution, 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. leaching reduced form glucorphanin and potassium sulfate mixture are stirred with anhydrous methanol, it is miscellaneous is filtered to remove potassium sulfate solid
Matter, is collected and merging filtrate, vacuum distillation recycling methanol obtain dry reduced form glucorphanin semi-finished product；
(5) hydrophilic C-18 low-pressure column chromatography is further purified:
A. the hydrophilic C-18 that partial size is 10-140 μm is soaked in anhydrous methanol, impregnates 10-120min, wash repeatedly 1-
It 5 times, after anhydrous methanol washing, is loaded in the low pressure stainless steel column that ratio of height to diameter is 2-30；
B. the dissolution of reduced form glucorphanin semi-finished product ultrapure water is configured to the solution of 10-750mg/mL, 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 volume is that every milliliter of hydrophilic C-18 loading adsorbs 0.1-15
Milligram reduced form glucorphanin semi-finished product；
D. ultrapure water elution collects reduced form radish sulphur in the case where flow velocity is 1-15BV/h and Detection wavelength is 210-245nm
Glycosides chromatographic peak, it is freeze-dried to dry high-purity reduced form glucorphanin.
Described, in step (1), radish raw material are one of radish sprout, radish rhizome and radish leaf；It is described
Oven drying to water content is 1.5-7.5% under the conditions of drying is 40-65 DEG C；The powder size of crushing is 30-300 mesh.
Described, in step (2), extractant is 70-100% methanol-water solution, and the additional amount of extractant is radish former material
5-30 times, refluxing extraction 0.5-3h for expecting powder weight repeats refluxing extraction 1-5 times.
Preferably, in step (2), the additional amount of extractant is 10-20 times of radish raw material powder weight, refluxing extraction
1-2h is repeated refluxing extraction 2-3 times.
Described, in step (3), the additional amount of water is 5-30 times of Turnip extractive weight, and 10- is extracted in dissolution
180min repeats dissolution and extracts 1-5 times.
Preferably, in step (3), the additional amount of water is 10-20 times of Turnip extractive weight, and 30- is extracted in dissolution
60min repeats dissolution and extracts 2-3 times.
Preferably, in step (4), the partial size of acidic alumina is 50-200 μm, soaking time 30-60min, and repetition is washed
It washs 2-3 times, the ratio of height to diameter of low pressure stainless steel column is 5-20, and reduced form glucorphanin extract solution concentration is 50-500mg/mL,
Sample volume is that every milliliter of acidic alumina loading adsorbs 0.5-15 milligrams of reduced form glucorphanin extracts, potassium sulfate-aqueous solution
Concentration is 5-15g/L, flow velocity 3-10BV/h, Detection wavelength 220-240nm.
Described, in step (4), the additional amount of anhydrous methanol is reduced form glucorphanin and potassium sulfate mixture gross mass
10-30 times, stirring leaching 10-60min, repeat leaching 1-3 times.
Preferably, in step (5), the partial size of hydrophilic C-18 is 25-100 μm；Soaking time is 30-60min；Repeated washing
2-3 times；Low pressure stainless steel column ratio of height to diameter is 5-20；Reduced form glucorphanin semi-finished product solution concentration is 25-500mg/mL；Sample introduction
Amount adsorbs 0.25-10 milligrams of reduced form glucorphanin semi-finished product for every milliliter of hydrophilic C-18 loading；Flow velocity is 3-10BV/h；Detection
Wavelength is 220-240nm.
Beneficial effects of the present invention: raw material of the invention are cheap and easy to get, applied widely, dissolved using extractant and water
Reduced form glucorphanin extract is extracted, while precipitating and removing a large amount of lipophilic contaminant ingredients, significantly improves overall craft production
The content of reduced form glucorphanin in rate and extract；Using the absorption of acidic alumina lower pressure column and the absorption of hydrophilic C-18 lower pressure column
It combines, significantly improves the purity and yield of reduced form glucorphanin product, reduce production cost, simplify extraction purification work
Skill process, is easy to industrial amplification production.
Detailed description of the invention
Fig. 1 is process flow chart of the invention；
Fig. 2 is the chemical structure 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 of the hydrophilic C-18 low pressure column purification reduced form glucorphanin semi-finished product of the present invention；
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, process technology scheme of the invention is further described, but, these embodiments
The limitation to the claimed scope of the invention is not constituted.
Embodiment 1
A method of high-purity reduced form glucorphanin being prepared by raw material of radish, is to be prepared by the following steps to obtain
:
(1) fresh radish sprout is harvested, 50 DEG C of oven dryings obtain dry radish sprout, water content 3.5%；
High speed disintegrator is crushed and is sieved, and obtains radish sprout powder of the granularity within the scope of 50-300 mesh；
(2) 100% methanol-water solution refluxing extraction radish sprout powder, the additional amount of 100% methanol-water solution are used
It is 5 times of radish sprout powder weight, refluxing extraction 1h, repeats to extract 5 times, be filtered to remove solid impurity, collect and merge filter
Liquid, under the conditions of 50 DEG C, vacuum distillation recycling methanol obtains dry Turnip extractive, recovery rate 25.3%；
(3) Turnip extractive is dissolved in the water, the additional amount of water is 20 times of Turnip extractive weight, and dissolution is extracted
60min repeats to extract 3 times, is filtered to remove solid impurity, collects simultaneously merging filtrate, is evaporated under reduced pressure under the conditions of 55 DEG C, obtains drying
Reduced form glucorphanin extract, total recovery rate be 16.4%；
(4) acidic alumina low-pressure column chromatography preliminary purification reduced form glucorphanin extract is used:
A. the acidic alumina that partial size is 75-150 μm is soaked in deionized water, impregnates 10min, washed repeatedly 5 times,
It is loaded in the low pressure stainless steel column that ratio of height to diameter is 30；
B. reduced form glucorphanin extract is configured to the solution that concentration is 25mg/mL with deionized water dissolving,
5000rpm is centrifuged 20min, removes solid impurity, obtains reduced form glucorphanin extract solution；
C. by reduced form glucorphanin extract solution loading, sample volume is every milliliter of acidic alumina loading absorption 0.25
Milligram reduced form glucorphanin extract, deionized water elution sufficiently remove the impurity that cannot be adsorbed by acidic alumina；
D. it is collected also with the potassium sulfate of 20g/L-aqueous solution elution in the case where flow velocity is 1BV/h and Detection wavelength is 235nm
Prototype glucorphanin chromatographic peak is evaporated under reduced pressure under the conditions of 55 DEG C, obtains dry reduced form glucorphanin and potassium sulfate mixing
Object；
E. it is stirred and is leached with anhydrous methanol, the additional amount of anhydrous 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, collection and merging filtrate,
Vacuum distillation recycling methanol under the conditions of 50 DEG C, and dry reduced form glucorphanin semi-finished product are obtained, reduced form glucorphanin contains
Amount is 44.2%, yield 96.5%；
(5) hydrophilic C-18 low-pressure column chromatography is further purified:
A. the hydrophilic C-18 that partial size is 10 μm is soaked in anhydrous methanol, impregnates 120min, anhydrous methanol repeated washing 3
It is secondary, it is loaded in the low pressure stainless steel column that ratio of height to diameter is 2；
B., the dissolution of reduced form glucorphanin semi-finished product ultrapure water is configured to the solution of 500mg/mL, 5000rpm centrifugation
20min removes solid impurity, obtains reduced form glucorphanin semi-finished product solution；
C. by reduced form glucorphanin semi-finished product solution loading, sample volume is that every milliliter of hydrophilic C-18 loading adsorbs 10 milligrams
Reduced form glucorphanin semi-finished product；
D. reduced form glucorphanin color is collected in the case where flow velocity is 3BV/h and Detection wavelength is 235nm with ultrapure water elution
Spectral 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 method of high-purity reduced form glucorphanin being prepared by raw material of radish, is to be prepared by the following steps to obtain
:
(1) fresh radish sprout is harvested, 40 DEG C of oven dryings obtain dry radish sprout, water content 7.5%；
High speed disintegrator is crushed and is sieved, and obtains radish sprout powder of the granularity within the scope of 30-300 mesh；
(2) 80% methanol-water solution refluxing extraction radish sprout powder is used, the additional amount of 80% methanol-water solution is
30 times of radish sprout powder weight, refluxing extraction 0.5h are repeated to extract 3 times, are filtered to remove solid impurity, collect and merge filter
Liquid, under the conditions of 50 DEG C, vacuum distillation recycling methanol obtains dry Turnip extractive, recovery rate 27.5%；
(3) Turnip extractive is dissolved in the water, the additional amount of water is 5 times of Turnip extractive weight, and dissolution is extracted
10min repeats to extract 5 times, is filtered to remove solid impurity, collects simultaneously merging filtrate, is evaporated under reduced pressure under the conditions of 55 DEG C, obtains drying
Reduced form glucorphanin extract, total recovery rate be 19.2%；
(4) acidic alumina low-pressure column chromatography preliminary purification reduced form glucorphanin extract is used:
A. the acidic alumina that partial size is 25-50 μm is soaked in deionized water, impregnates 60min, washed repeatedly 3 times,
It is loaded in the low pressure stainless steel column that ratio of height to diameter is 20；
B. reduced form glucorphanin extract is configured to the solution that concentration is 50mg/mL with deionized water dissolving,
5000rpm is centrifuged 20min, removes solid impurity, obtains reduced form glucorphanin extract solution；
C. by reduced form glucorphanin extract solution loading, sample volume is every milliliter of acidic alumina loading absorption 0.5
Milligram reduced form glucorphanin extract, deionized water elution sufficiently remove the impurity that cannot be adsorbed by acidic alumina；
D. it is collected also with the potassium sulfate of 10g/L-aqueous solution elution in the case where flow velocity is 6BV/h and Detection wavelength is 210nm
Prototype glucorphanin chromatographic peak is evaporated under reduced pressure under the conditions of 55 DEG C, obtains dry reduced form glucorphanin and potassium sulfate mixing
Object；
E. it is stirred and is leached with anhydrous methanol, the additional amount of anhydrous 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, collection and merging filtrate,
Vacuum distillation recycling methanol under the conditions of 50 DEG C, and dry reduced form glucorphanin semi-finished product are obtained, reduced form glucorphanin contains
Amount is 50.3%, yield 93.6%；
(5) hydrophilic C-18 low-pressure column chromatography is further purified:
A. the hydrophilic C-18 that partial size is 50 μm is soaked in anhydrous methanol, impregnates 60min, anhydrous methanol repeated washing 5
It is secondary, it is loaded in the low pressure stainless steel column that ratio of height to diameter is 5；
B., the dissolution of reduced form glucorphanin semi-finished product ultrapure water is configured to the solution of 10mg/mL, 5000rpm centrifugation
20min removes solid impurity, obtains reduced form glucorphanin semi-finished product solution；
C. by reduced form glucorphanin semi-finished product solution loading, sample volume is every milliliter of hydrophilic 0.1 milli of C-18 loading absorption
Gram reduced form glucorphanin semi-finished product；
D. reduced form glucorphanin color is collected in the case where flow velocity is 1BV/h and Detection wavelength is 210nm with ultrapure water elution
Spectral 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 method of high-purity reduced form glucorphanin being prepared by raw material of radish, is to be prepared by the following steps to obtain
:
(1) fresh radish sprout is harvested, 65 DEG C of oven dryings obtain dry radish sprout, water content 1.5%；
High speed disintegrator is crushed and is sieved, and obtains radish sprout powder of the granularity within the scope of 60-300 mesh；
(2) 70% methanol-water solution refluxing extraction radish sprout powder is used, the additional amount of 70% methanol-water solution is
20 times of radish sprout powder weight, refluxing extraction 3h are repeated to extract 1 time, are filtered to remove solid impurity, collect and merge filter
Liquid, under the conditions of 50 DEG C, vacuum distillation recycling methanol obtains dry Turnip extractive, recovery rate 27.6%；
(3) Turnip extractive is dissolved in the water, the additional amount of water is 30 times of Turnip extractive weight, and dissolution is extracted
180min repeats to extract 1 time, is filtered to remove solid impurity, collects simultaneously merging filtrate, is evaporated under reduced pressure, obtains dry under the conditions of 55 DEG C
Dry reduced form glucorphanin extract, total recovery rate are 17.1%；
(4) acidic alumina low-pressure column chromatography preliminary purification reduced form glucorphanin extract is used:
A. the acidic alumina that partial size is 150-250 μm is soaked in deionized water, impregnates 120min, repeated washing 5
It is secondary, it is loaded in the low pressure stainless steel column that ratio of height to diameter is 15；
B. reduced form glucorphanin extract is configured to the solution that concentration is 350mg/mL with deionized water dissolving,
5000rpm is centrifuged 20min, removes solid impurity, obtains reduced form glucorphanin extract solution；
C. by reduced form glucorphanin extract solution loading, sample volume is every milliliter of 7 milli of acidic alumina loading absorption
Gram reduced form glucorphanin extract, deionized water elution sufficiently remove the impurity that cannot be adsorbed by acidic alumina；
D. it is collected also with the potassium sulfate of 15g/L-aqueous solution elution in the case where flow velocity is 10BV/h and Detection wavelength is 245nm
Prototype glucorphanin chromatographic peak is evaporated under reduced pressure under the conditions of 55 DEG C, obtains dry reduced form glucorphanin and potassium sulfate mixing
Object；
E. it is stirred and is leached with anhydrous methanol, the additional amount of anhydrous 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, collection and merging filtrate,
Vacuum distillation recycling methanol under the conditions of 50 DEG C, and dry reduced form glucorphanin semi-finished product are obtained, reduced form glucorphanin contains
Amount is 46.7%, yield 94.8%；
(5) hydrophilic C-18 low-pressure column chromatography is further purified:
A. the hydrophilic C-18 that partial size is 100 μm is soaked in anhydrous methanol, impregnates 30min, anhydrous methanol repeated washing 2
It is secondary, it is loaded in the low pressure stainless steel column that ratio of height to diameter is 30；
B., the dissolution of reduced form glucorphanin semi-finished product ultrapure water is configured to the solution of 25mg/mL, 5000rpm centrifugation
20min removes solid impurity, obtains reduced form glucorphanin semi-finished product solution；
C. by reduced form glucorphanin semi-finished product solution loading, sample volume is every milliliter of hydrophilic 0.25 milli of C-18 loading absorption
Gram reduced form glucorphanin semi-finished product；
D. reduced form glucorphanin color is collected in the case where flow velocity is 10BV/h and Detection wavelength is 245nm with ultrapure water elution
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 method of high-purity reduced form glucorphanin being prepared by raw material of radish, is to be prepared by the following steps to obtain
:
(1) fresh radish rhizome is harvested, is cut into the square cylinder fritter of 10 × 10 × 20mm, 50 DEG C of oven dryings obtain
Obtain dry radish rhizome, water content 4.0%；High speed disintegrator is crushed and is sieved, and obtains granularity within the scope of 40-300 mesh
Radish rhizome powder；
(2) 90% methanol-water solution refluxing extraction radish rhizome powder is used, the additional amount of 90% methanol-water solution is
10 times of radish rhizome powder weight, refluxing extraction 2h are repeated to extract 3 times, are filtered to remove solid impurity, collect and merge filter
Liquid, under the conditions of 50 DEG C, vacuum distillation recycling methanol obtains dry Turnip extractive, recovery rate 22.1%；
(3) Turnip extractive is dissolved in the water, the additional amount of water is 20 times of Turnip extractive weight, and dissolution is extracted
120min repeats to extract 2 times, is filtered to remove solid impurity, collects simultaneously merging filtrate, is evaporated under reduced pressure, obtains dry under the conditions of 55 DEG C
Dry reduced form glucorphanin extract, total recovery rate are 15.2%；
(4) acidic alumina low-pressure column chromatography preliminary purification reduced form glucorphanin extract is used:
A. the acidic alumina that partial size is 50-200 μm is soaked in deionized water, impregnates 120min, repeated washing 1
It is secondary, it is loaded in the low pressure stainless steel column that ratio of height to diameter is 2；
B. reduced form glucorphanin extract is configured to the solution that concentration is 750mg/mL with deionized water dissolving,
5000rpm is centrifuged 20min, removes solid impurity, obtains reduced form glucorphanin extract solution；
C. by reduced form glucorphanin extract solution loading, sample volume is every milliliter of 15 milli of acidic alumina loading absorption
Gram reduced form glucorphanin extract, deionized water elution sufficiently remove the impurity that cannot be adsorbed by acidic alumina；
D. it is collected also with the potassium sulfate of 5g/L-aqueous solution elution in the case where flow velocity is 15BV/h and Detection wavelength is 220nm
Prototype glucorphanin chromatographic peak is evaporated under reduced pressure under the conditions of 55 DEG C, obtains dry reduced form glucorphanin and potassium sulfate mixing
Object；
E. it is stirred and is leached with anhydrous methanol, the additional amount of anhydrous 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, collection and merging filtrate,
Vacuum distillation recycling methanol under the conditions of 50 DEG C, and dry reduced form glucorphanin semi-finished product are obtained, reduced form glucorphanin contains
Amount is 43.5%, yield 93.2%；
(5) hydrophilic C-18 low-pressure column chromatography is further purified:
A. the hydrophilic C-18 that partial size is 25 μm is soaked in anhydrous methanol, impregnates 10min, anhydrous methanol repeated washing 3
It is secondary, it is loaded in the low pressure stainless steel column that ratio of height to diameter is 10；
B., the dissolution of reduced form glucorphanin semi-finished product ultrapure water is configured to the solution of 750mg/mL, 5000rpm centrifugation
20min removes solid impurity, obtains reduced form glucorphanin semi-finished product solution；
C. by reduced form glucorphanin semi-finished product solution loading, sample volume is that every milliliter of hydrophilic C-18 loading adsorbs 15 milligrams
Reduced form glucorphanin semi-finished product；
D. reduced form glucorphanin color is collected in the case where flow velocity is 15BV/h and Detection wavelength is 220nm with ultrapure water elution
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 method of high-purity reduced form glucorphanin being prepared by raw material of radish, is to be prepared by the following steps to obtain
:
(1) fresh radish rhizome is harvested, is cut into the square cylinder fritter of 10 × 10 × 20mm, 55 DEG C of oven dryings obtain
Obtain dry radish rhizome, water content 2.5%；High speed disintegrator is crushed and is sieved, and obtains granularity within the scope of 60-300 mesh
Radish rhizome powder；
(2) 100% methanol-water solution refluxing extraction radish rhizome powder, the additional amount of 100% methanol-water solution are used
It is 20 times of radish rhizome powder weight, refluxing extraction 0.5h, repeats to extract 5 times, be filtered to remove solid impurity, collect and merge
Filtrate, under the conditions of 50 DEG C, vacuum distillation recycling methanol obtains dry Turnip extractive, recovery rate 21.5%；
(3) Turnip extractive is dissolved in the water, the additional amount of water is 10 times of Turnip extractive weight, and dissolution is extracted
60min repeats to extract 3 times, is filtered to remove solid impurity, collects simultaneously merging filtrate, is evaporated under reduced pressure under the conditions of 55 DEG C, obtains drying
Reduced form glucorphanin extract, total recovery rate be 13.8%；
(4) acidic alumina low-pressure column chromatography preliminary purification reduced form glucorphanin extract is used:
A. the acidic alumina that partial size is 75-150 μm is soaked in deionized water, impregnates 90min, washed repeatedly 4 times,
It is loaded in the low pressure stainless steel column that ratio of height to diameter is 5；
B. reduced form glucorphanin extract is configured to the solution that concentration is 250mg/mL with deionized water dissolving,
5000rpm is centrifuged 20min, removes solid impurity, obtains reduced form glucorphanin extract solution；
C. by reduced form glucorphanin extract solution loading, sample volume is every milliliter of 10 milli of acidic alumina loading absorption
Gram reduced form glucorphanin extract, deionized water elution sufficiently remove the impurity that cannot be adsorbed by acidic alumina；
D. it is collected also with the potassium sulfate of 15g/L-aqueous solution elution in the case where flow velocity is 3BV/h and Detection wavelength is 230nm
Prototype glucorphanin chromatographic peak is evaporated under reduced pressure under the conditions of 55 DEG C, obtains dry reduced form glucorphanin and potassium sulfate mixing
Object；
E. it is stirred and is leached with anhydrous methanol, the additional amount of anhydrous 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, collection and merging filtrate,
Vacuum distillation recycling methanol under the conditions of 50 DEG C, and dry reduced form glucorphanin semi-finished product are obtained, reduced form glucorphanin contains
Amount is 40.9%, yield 95.6%；
(5) hydrophilic C-18 low-pressure column chromatography is further purified:
A. the hydrophilic C-18 that partial size is 140 μm is soaked in anhydrous methanol, impregnates 90min, anhydrous methanol repeated washing 1
It is secondary, it is loaded in the low pressure stainless steel column that ratio of height to diameter is 20；
B., the dissolution of reduced form glucorphanin semi-finished product ultrapure water is configured to the solution of 250mg/mL, 5000rpm centrifugation
20min removes solid impurity, obtains reduced form glucorphanin semi-finished product solution；
C. by reduced form glucorphanin semi-finished product solution loading, sample volume is that every milliliter of hydrophilic C-18 loading adsorbs 10 milligrams
Reduced form glucorphanin semi-finished product；
D. reduced form glucorphanin color is collected in the case where flow velocity is 10BV/h and Detection wavelength is 230nm with ultrapure water elution
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 method of high-purity reduced form glucorphanin being prepared by raw material of radish, is to be prepared by the following steps to obtain
:
(1) fresh radish leaf is harvested, is cut into the rectangular turnip leaves of 10 × 10mm, 45 DEG C of oven dryings are done
Dry radish leaf, water content 5.5%；High speed disintegrator is crushed and is sieved, and obtains trailing plants of the granularity within the scope of 30-300 mesh
Bu Yezi powder；
(2) 80% methanol-water solution refluxing extraction radish leaf powder is used, the additional amount of 80% methanol-water solution is
30 times of radish leaf powder weight, refluxing extraction 2h are repeated to extract 2 times, are filtered to remove solid impurity, collect and merge filter
Liquid, under the conditions of 50 DEG C, vacuum distillation recycling methanol obtains dry Turnip extractive, recovery rate 18.5%；
(3) Turnip extractive is dissolved in the water, the additional amount of water is 30 times of Turnip extractive weight, and dissolution is extracted
30min repeats to extract 2 times, is filtered to remove solid impurity, collects simultaneously merging filtrate, is evaporated under reduced pressure under the conditions of 55 DEG C, obtains drying
Reduced form glucorphanin extract, total recovery rate be 9.51%；
(4) acidic alumina low-pressure column chromatography preliminary purification reduced form glucorphanin extract is used:
A. the acidic alumina that partial size is 50-75 μm is soaked in deionized water, impregnates 30min, washed repeatedly 2 times,
It is loaded in the low pressure stainless steel column that ratio of height to diameter is 20；
B. reduced form glucorphanin extract is configured to the solution that concentration is 500mg/mL with deionized water dissolving,
5000rpm is centrifuged 20min, removes solid impurity, obtains reduced form glucorphanin extract solution；
C. by reduced form glucorphanin extract solution loading, sample volume is every milliliter of 20 milli of acidic alumina loading absorption
Gram reduced form glucorphanin extract, deionized water elution sufficiently remove the impurity that cannot be adsorbed by acidic alumina；
D. it is collected also with the potassium sulfate of 10g/L-aqueous solution elution in the case where flow velocity is 10BV/h and Detection wavelength is 240nm
Prototype glucorphanin chromatographic peak is evaporated under reduced pressure under the conditions of 55 DEG C, obtains dry reduced form glucorphanin and potassium sulfate mixing
Object；
E. it is stirred and is leached with anhydrous methanol, the additional amount of anhydrous 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, collection and merging filtrate,
Vacuum distillation recycling methanol under the conditions of 50 DEG C, and dry reduced form glucorphanin semi-finished product are obtained, reduced form glucorphanin contains
Amount is 38.5%, yield 96.8%；
(5) hydrophilic C-18 low-pressure column chromatography is further purified:
A. the hydrophilic C-18 that partial size is 50 μm is soaked in anhydrous methanol, impregnates 60min, anhydrous methanol repeated washing 3
It is secondary, it is loaded in the low pressure stainless steel column that ratio of height to diameter is 15；
B., the dissolution of reduced form glucorphanin semi-finished product ultrapure water is configured to the solution of 100mg/mL, 5000rpm centrifugation
20min removes solid impurity, obtains reduced form glucorphanin semi-finished product solution；
C. by reduced form glucorphanin semi-finished product solution loading, sample volume is that every milliliter of hydrophilic C-18 loading adsorbs 5 milligrams
Reduced form glucorphanin semi-finished product；
D. reduced form glucorphanin color is collected in the case where flow velocity is 6BV/h and Detection wavelength is 240nm with ultrapure water elution
Spectral 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. a kind of method for preparing high-purity reduced form glucorphanin as raw material using radish, which is characterized in that be by following step
Suddenly it is prepared:
(1) it by radish raw material drying, crushes, obtains radish raw material powder；
(2) with extractant refluxing extraction radish raw material powder, filtrate is collected in filtering, is repeated refluxing extraction, is filtered to remove solid
Impurity, is collected and merging filtrate, vacuum distillation recycling extractant obtain dry Turnip extractive；
(3) it is dissolved with water and extracts Turnip extractive, filtrate is collected in filtering, is repeated dissolution and is extracted, be filtered to remove solid impurity, receives
Collect 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 that partial size is 25-250 μm is soaked in deionized water, impregnates 10-120min, wash repeatedly 1-5
It is secondary, after deionized water washing, it is loaded in the low pressure stainless steel column that ratio of height to diameter is 2-30；
B. reduced form glucorphanin extract is configured to the solution that concentration is 25-750mg/mL with deionized water dissolving,
5000rpm is centrifuged 20min, removes solid impurity, obtains reduced form glucorphanin extract solution；
C. by reduced form glucorphanin extract solution loading, sample volume is that every milliliter of acidic alumina loading adsorbs 0.25-20
Milligram reduced form glucorphanin extract, deionized water elution remove the impurity that cannot be adsorbed by acidic alumina；
D. it is received with the potassium sulfate of 5-20g/L-aqueous solution elution in the case where flow velocity is 1-15BV/h and Detection wavelength is 210-245nm
Collect reduced form glucorphanin chromatographic peak, vacuum distillation obtains dry reduced form glucorphanin and potassium sulfate mixture；
E. leaching reduced form glucorphanin and potassium sulfate mixture are stirred with anhydrous methanol, are filtered to remove potassium sulfate solid impurity,
It collects and merging filtrate, vacuum distillation recycling methanol obtains dry reduced form glucorphanin semi-finished product；
(5) hydrophilic C-18 low-pressure column chromatography is further purified:
A. the hydrophilic C-18 that partial size is 10-140 μm is soaked in anhydrous methanol, impregnates 10-120min, washed repeatedly 1-5 times,
After anhydrous methanol washing, it is loaded in the low pressure stainless steel column that ratio of height to diameter is 2-30；
B., the dissolution of reduced form glucorphanin semi-finished product ultrapure water is configured to the solution of 10-750mg/mL, 5000rpm centrifugation
20min removes solid impurity, obtains reduced form glucorphanin semi-finished product solution；
C. by reduced form glucorphanin semi-finished product solution loading, sample volume is that every milliliter of hydrophilic C-18 loading adsorbs 0.1-15 milligrams
Reduced form glucorphanin semi-finished product；
D. ultrapure water elution collects reduced form glucorphanin color in the case where flow velocity is 1-15BV/h and Detection wavelength is 210-245nm
Spectral peak, it is freeze-dried to obtain dry high-purity 4- methyl mercapto -3- cyclobutenyl glucosinolates.
2. the method according to claim 1, wherein in the step (1), radish raw material be radish sprout,
One of radish rhizome and radish leaf；Oven drying to water content is 1.5-7.5% under the conditions of the drying is 40-65 DEG C；
The powder size of crushing is 30-300 mesh.
3. the method according to claim 1, wherein extractant is 70-100% methanol-water in the step (2)
Solution, the additional amount of extractant are 5-30 times, refluxing extraction 0.5-3h of radish raw material powder weight, repeat refluxing extraction 1-
5 times.
4. according to the method described in claim 3, it is characterized in that, the additional amount of extractant is that radish is former in the step (2)
10-20 times of material powder weight, refluxing extraction 1-2h are repeated refluxing extraction 2-3 times.
5. the method according to claim 1, wherein the additional amount of water is Turnip extractive in the step (3)
10-180min is extracted in 5-30 times of weight, dissolution, is repeated dissolution and is extracted 1-5 times.
6. according to the method described in claim 5, it is characterized in that, the additional amount of water is Turnip extractive in the step (3)
30-60min is extracted in 10-20 times of weight, dissolution, is repeated dissolution and is extracted 2-3 times.
7. the method according to claim 1, wherein the partial size of acidic alumina is 50- in the step (4)
It 200 μm, soaking time 30-60min, washes repeatedly 2-3 times, the ratio of height to diameter of low pressure stainless steel column is 5-20, reduced form radish
Sulphur glucoside extract solution concentration is 50-500mg/mL, and sample volume is that every milliliter of acidic alumina loading adsorbs 0.5-15 milligrams also
Prototype glucorphanin extract, potassium sulfate-concentration of aqueous solution are 5-15g/L, flow velocity 3-10BV/h, Detection wavelength 220-
240nm。
8. the method according to claim 1, wherein the additional amount of anhydrous methanol is reduction in the step (4)
10-30 times of type glucorphanin and potassium sulfate mixture gross mass, stirring leaching 10-60min, repeats leaching 1-3 times.
9. the method according to claim 1, wherein the partial size of hydrophilic C-18 is 25-100 in the step (5)
μm；Soaking time is 30-60min；Repeated washing 2-3 times；Low pressure stainless steel column ratio of height to diameter is 5-20；Reduced form glucorphanin half
Final mean annual increment solution concentration is 25-500mg/mL；Sample volume is that every milliliter of hydrophilic C-18 loading adsorbs 0.25-10 milligrams of reduced form radish
Sulphur glycosides semi-finished product；Flow velocity is 3-10BV/h；Detection wavelength is 220-240nm.
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