CN103936664A - Method for extracting deoxynojirimycin from white mulberry and purifying deoxynojirimycin - Google Patents
Method for extracting deoxynojirimycin from white mulberry and purifying deoxynojirimycin Download PDFInfo
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- C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
- C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
- C07D211/36—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
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Abstract
The invention relates to a method for extracting ingredients of white mulberry and in particular relates to a method for extracting deoxynojirimycin from the white mulberry and purifying deoxynojirimycin. The method for extracting deoxynojirimycin from the white mulberry and purifying deoxynojirimycin comprises the following steps: carrying out water extraction, namely airing leaves of the white mulberry, then adding deionized water, boiling at constant temperature and extracting, filtering and collecting filtrate, adding the deionized water into filter residue and then extracting again, putting filtrate together, adding trichloroacetic acid and centrifuging, so that first supernate is obtained; loading onto a column, namely purifying the first supernate by adopting a reversed phase chromatography column, so that liquid containing deoxynojirimycin is obtained; carrying out ethanol precipitation and taking supernate, namely firstly adding ethanol with alcohol content of 55-65% into the liquid containing deoxynojirimycin obtained after the step of loading onto the column is finished, stirring for 3-8 minutes, then adding ethanol with alcohol content of 70-80%, and stirring for 3-5 minutes, so that second supernate is obtained; concentrating, namely concentrating the second supernate, so that concentrated liquor is obtained; and carrying out microwave vacuum drying, namely carrying out microwave vacuum drying on the concentrated liquor, so that a powder product containing deoxynojirimycin is obtained. The method for extracting deoxynojirimycin from the white mulberry and purifying deoxynojirimycin has the advantages of high extraction and purification efficiency and good separating effect.
Description
Technical field
The present invention relates to a kind of extracting method of mulberry tree composition, relate in particular to the extracting and purifying method of S-GI in mulberry tree.
Background technology
Mulberry leaf contain plurality of active ingredients as medicine food dual purpose plant, have the effects such as good hypoglycemic, reducing blood-fat, fat-reducing.In recent years, Chinese scholars conducts in-depth research mulberry leaf, proves that polysaccharide, flavones and the alkaloids in mulberry leaf all has good hypoglycemic activity.Chen Fujun etc. reported the mulberry leaf total polysaccharides (TPM) extracting in mulberry leaf to four oxygen uh steep diabetic mice have remarkable hypoglycemic activity; Alkaloid in mulberry leaf can significantly reduce glucose level, and wherein DNJ hypoglycemic activity is especially obvious.The wild long-standing mycin of 1-deoxidation (DNJ), chemical name is 3,4,5-trihydroxy--2 methylol tetrahydropyridines, is distinctive active skull cap components in mulberry leaf, has efficient pharmacologically active, can be used for treating the diseases such as diabetes, obesity, virus infection.
CN102276515B (2013-5-8) discloses a kind of extracting method of S-GI, and mulberry raw material is soaked and extracted 1-3 time with aqueous acid; Extracting solution Shangyang, from exchange resin absorption, after washing, is used ammoniacal liquor wash-out; After reverse osmosis pre-concentration elutriant, reclaim ammonia, in ask that body is dissolved in hydrophilic solvent, catalyzed conversion, gel chromatography column on solution, hydrophilic solvent wash-out; Reclaim after solvent, be dissolved in mixed solvent, upper chromatography column, mixed solvent wash-out; Reclaim mixed solvent, obtain the wild mycin sterling that belongs to of deoxidation.But the extraction yield of the method and separating effect still have much room for improvement.
Summary of the invention
The object of this invention is to provide that extraction yield is high, the extracting and purifying method of S-GI in the mulberry tree of good separating effect.
Above-mentioned technical purpose of the present invention is achieved by the following technical programs:
An extracting and purifying method for S-GI in mulberry tree, it comprises the following steps:
(1) water extraction: get mulberry leaf and dry, then add deionized water, constant temperature boiling is extracted, and filters and collects filtrate, and filter residue adds deionized water and extracts, and merging filtrate adds trichoroacetic acid(TCA) centrifugal, obtains the first supernatant liquor;
(2) upper prop: described the first supernatant liquor is carried out to purifying by reverse column chromatography, obtain the liquid that contains S-GI;
(3) alcohol precipitation is got supernatant: after upper prop, in the liquid that contains S-GI of gained, first add containing the ethanol of alcohol quality 55-65% and stir 3-8min, and then add containing alcohol quality 70-80% ethanol stirring 3-5min, obtain the second supernatant liquor;
(4) concentrated: will after described the second supernatant concentration, to obtain concentrated solution;
(5) microwave vacuum drying: described concentrated solution microwave vacuum drying is obtained to the powder-product that contains S-GI.
As preferably, described step (1) water extraction is specifically got mulberry leaf and is exposed to the sun and dries in the sun, then add the deionized water that 8-12 doubly dries rear mulberry leaf weight, be placed in pot group type counterflow extraction apparatus and comprise three times of at least the first circulation, the second circulation and the 3rd circulation extractions that circulate, described filling group is that every group of three extractors series connection share, countercurrent extraction flow velocity is 25-35min/ tank, and extraction temperature is 80-110 DEG C;
Described the first circulation is since the first extractor countercurrent extraction: specifically the deionized water that dries rear mulberry leaf and 8-12 and doubly dry rear mulberry leaf weight is added in the first extractor, the second extractor and the 3rd extractor by equal portions respectively; After the medical filtration that the first extractor extracts for the first time, put into container for storing liquid for subsequent use, the extraction for the second time of the first extractor and the extraction for the third time of the first extractor add respectively 4-5 times of distilled water; Then the solvent extracting for the first time using the second extracting solution of the first extractor as the second extractor, the first extracting liquid filtering of the second extractor is for subsequent use; The solvent extracting for the second time using the 3rd extracting solution of the first extractor as the second extractor, adds 4-5 times of distilled water in the extraction for the second time of the second extractor; The second extracting solution of the second extractor is as the solvent extracting for the first time of the 3rd extractor;
Described the second circulation is since the second extractor countercurrent extraction: specifically merge the first extracting solution in the first extractor, the second extractor and the 3rd extractor in the first circulation to the second extractor of the mulberry leaves slag after including the first circulation and extracting for the second time, then add 4-5 times of distilled water to extract for the first time; In the 3rd extractor of the mulberry leaves slag using the first extracting solution of the second extractor after including the first circulation and extracting for the first time, then add 4-5 times of distilled water to extract for the first time; Then the first extracting solution of the 3rd extractor is extracted in the first extractor of the mulberry leaves slag after including the first circulation and extracting for the third time for the first time;
Described the 3rd circulation is since the 3rd extractor countercurrent extraction: specifically merge the first extracting solution in the first extractor, the second extractor and the 3rd extractor in the second circulation to the 3rd extractor of the mulberry leaves slag after including the second circulation and extracting for the first time, then add 4-5 times of water to extract for the first time; In the first extractor of the mulberry leaves slag using the first extracting solution of the 3rd extractor after including the second circulation and extracting for the first time, then add 4-5 times of distilled water to extract for the first time; Then the first extracting solution of the first extractor is extracted in the second extractor of the mulberry tree slag after including the second circulation and extracting for the first time for the first time;
Merge the first extracting solution in the first extractor, the second extractor and the 3rd extractor in the 3rd circulation and filter and collect filtrate, adding trichoroacetic acid(TCA) centrifugal, obtaining the first supernatant liquor.
More preferably, described three circulations disconnect first extractor discharging in every 8 hours in extracting, and after again feeding intake, connect successively.
As preferably, described step (2) is specifically crossed described the first supernatant liquor C18 post or MCI gel CHP 20P column chromatography, then carries out gradient elution by the moving phase of different concns, collects the elutriant containing S-GI.
More preferably, in the time that the stationary phase of selecting is C18 post, mobile phase A is the phosphate buffered saline buffer of pH value 3.5-4.5, and Mobile phase B is acetonitrile, presses the flow rate gradient 55-65min of 0.4-0.5ml/min under column temperature 28-32 DEG C, the condition of pH value 5-6; Described condition of gradient elution is: at 0-35min, the volume ratio of described mobile phase A and Mobile phase B is (90-95)/(5-10); At 36-45min, the volume ratio of described mobile phase A and Mobile phase B is (80-85)/(15-20); At 46-55min, the volume ratio of described mobile phase A and Mobile phase B is (68-72)/(28-32); At 56-65min, the volume ratio of described mobile phase A and Mobile phase B is (90-95)/(5-10).
More preferably, in the time that the stationary phase of selecting is MCI gel CHP 20P column chromatography, selecting methanol-water is moving phase.
As preferably, described step (3) adopts the slow mode of accelerating to stir to add ethanol: drip the ethanol containing alcohol quality 55-65%, then rapid stirring 3-8min under 200-300r/min according to 3-7ml/s.
The slow mode of accelerating to stir can make impurity rapid precipitation, and effective constituent is unlikely to again to be wrapped up by ethanol, improves the active constituent content in supernatant liquor.
As preferably, described extracting and purifying method is also included in step (3) alcohol precipitation and gets before supernatant, by following be evacuated to-the 0.09MPa of the liquid that contains S-GI of gained after upper prop and keep 15-25min, then add deionized water 800kg, cooling standing 8-15 hour pretreatment liquid.
By vacuumizing adjusting vacuum tightness, follow-up concentrated and purifying is more easily carried out.
As preferably, concentrated specifically described the second supernatant liquor reduced vacuum of described step (4) is concentrated into the 10-40% of original volume, obtains concentrated solution.
More preferably, described step (4) is concentrated is specifically the 20-30% that under-0.075～-0.06MPa, reduced vacuum is concentrated into described the second supernatant liquor volume by described the second supernatant liquor in vacuum tightness, obtains concentrated solution.
As preferably, described extracting and purifying method is also included in described in the concentrated forward direction of step (4) and adds and account for the sherwood oil of the second supernatant liquor 1.5-3 times quality and the mixed solution of methyl alcohol in the second supernatant liquor liquid, mix and soak 0.5-0.8h, supersound extraction 2-4min under 40-50 DEG C, 50-70KHz.
Adopt this technique can make the S-GI in mulberry leaves obtain more effective extraction, thereby further improve extraction efficiency; And be conducive to follow-up concentrated.
As preferably, it is-0.04～-0.01MPa that described microwave vacuum drying is specifically evacuated to vacuum tightness, is then that 100-260MHz microwave treatment 80-90s obtains the powder-product that contains S-GI in microwave frequency.
Microwave vacuum drying can make the finished product moderate purity improve, and this may be owing to making some the impurity volatilizations in extract under this condition or decomposing.
Embodiment
embodiment mono-
Water extraction: get 100kg mulberry leaf and be exposed to the sun and dry in the sun, then add 800kg deionized water, be placed in pot group type counterflow extraction apparatus and comprise three times of the first circulation, the second circulation and the 3rd circulation extractions that circulate, filling group is that every group of three extractors series connection share, countercurrent extraction flow velocity is 25min/ tank, and extracting temperature is 80 DEG C;
The first circulation is since the first extractor countercurrent extraction: specifically will dry rear mulberry leaf and 800kg deionized water and add in the first extractor, the second extractor and the 3rd extractor by equal portions respectively; After the medical filtration that the first extractor extracts for the first time, put into container for storing liquid for subsequent use, the extraction for the second time of the first extractor and the extraction for the third time of the first extractor add respectively 400kg distilled water; Then the solvent extracting for the first time using the second extracting solution of the first extractor as the second extractor, the first extracting liquid filtering of the second extractor is for subsequent use; The solvent extracting for the second time using the 3rd extracting solution of the first extractor as the second extractor, adds 400kg distilled water in the extraction for the second time of the second extractor; The second extracting solution of the second extractor is as the solvent extracting for the first time of the 3rd extractor;
The second circulation is since the second extractor countercurrent extraction: specifically merge the first extracting solution in the first extractor, the second extractor and the 3rd extractor in the first circulation to the second extractor of the mulberry leaves slag after including the first circulation and extracting for the second time, then add 4-5 times of distilled water to extract for the first time; In the 3rd extractor of the mulberry leaves slag using the first extracting solution of the second extractor after including the first circulation and extracting for the first time, then add 400kg distilled water to extract for the first time; Then the first extracting solution of the 3rd extractor is extracted in the first extractor of the mulberry leaves slag after including the first circulation and extracting for the third time for the first time;
The 3rd circulation is since the 3rd extractor countercurrent extraction: specifically merge the first extracting solution in the first extractor, the second extractor and the 3rd extractor in the second circulation to the 3rd extractor of the mulberry leaves slag after including the second circulation and extracting for the first time, then add 400kg water to extract for the first time; In the first extractor of the mulberry leaves slag using the first extracting solution of the 3rd extractor after including the second circulation and extracting for the first time, then add 4-5 times of distilled water to extract for the first time; Then the first extracting solution of the first extractor is extracted in the second extractor of the mulberry tree slag after including the second circulation and extracting for the first time for the first time;
Merge the first extracting solution in the first extractor, the second extractor and the 3rd extractor in the 3rd circulation and filter and collect filtrate, add trichoroacetic acid(TCA) centrifugal, the first supernatant liquor is got mulberry leaf and is dried, then add deionized water, constant temperature boiling is extracted, and filters and collects filtrate, filter residue adds deionized water and extracts, merging filtrate, adds trichoroacetic acid(TCA) centrifugal, obtains the first supernatant liquor;
Upper prop: the first supernatant liquor is crossed to C18 post, then carry out gradient elution by the moving phase of different concns, collect the elutriant containing S-GI; Mobile phase A is wherein the phosphate buffered saline buffer of pH value 3.5, and Mobile phase B is acetonitrile, presses the flow rate gradient 55min of 0.4ml/min under the condition of 28 DEG C of column temperatures, pH value 5; Condition of gradient elution is: at 0-35min, the volume ratio of mobile phase A and Mobile phase B is 90/10; At 36-45min, the volume ratio of mobile phase A and Mobile phase B is 80/15; At 46-55min, the volume ratio of mobile phase A and Mobile phase B is 68/32.
Alcohol precipitation is got supernatant: after upper prop, in the liquid that contains S-GI of gained, first add containing the ethanol of alcohol quality 5% and stir 3min, and then add containing alcohol quality 70% ethanol stirring 3min, obtain the second supernatant liquor;
Concentrated: the second supernatant liquor reduced vacuum to be concentrated into 10% of original volume, to obtain concentrated solution;
Microwave vacuum drying: concentrated solution is evacuated to vacuum tightness is-0.04MPa, is then that 100MHz microwave treatment 80s obtains the powder-product that contains S-GI in microwave frequency, and detected result is in table 1.
Embodiment bis-
Water extraction: get 100kg mulberry leaf and be exposed to the sun and dry in the sun, then add the deionized water that 1200kg dries rear mulberry leaf weight, be placed in pot group type counterflow extraction apparatus and comprise three times of at least the first circulation, the second circulation and the 3rd circulation extractions that circulate, filling group is that every group of three extractors series connection share, countercurrent extraction flow velocity is 35min/ tank, and extracting temperature is 110 DEG C;
The first circulation is since the first extractor countercurrent extraction: specifically will dry rear mulberry leaf and 1200kg deionized water and add in the first extractor, the second extractor and the 3rd extractor by equal portions respectively; After the medical filtration that the first extractor extracts for the first time, put into container for storing liquid for subsequent use, the extraction for the second time of the first extractor and the extraction for the third time of the first extractor add respectively 500kg distilled water; Then the solvent extracting for the first time using the second extracting solution of the first extractor as the second extractor, the first extracting liquid filtering of the second extractor is for subsequent use; The solvent extracting for the second time using the 3rd extracting solution of the first extractor as the second extractor, adds 500kg distilled water in the extraction for the second time of the second extractor; The second extracting solution of the second extractor is as the solvent extracting for the first time of the 3rd extractor;
The second circulation is since the second extractor countercurrent extraction: specifically merge the first extracting solution in the first extractor, the second extractor and the 3rd extractor in the first circulation to the second extractor of the mulberry leaves slag after including the first circulation and extracting for the second time, then add 4-5 times of distilled water to extract for the first time; In the 3rd extractor of the mulberry leaves slag using the first extracting solution of the second extractor after including the first circulation and extracting for the first time, then add 500kg distilled water to extract for the first time; Then the first extracting solution of the 3rd extractor is extracted in the first extractor of the mulberry leaves slag after including the first circulation and extracting for the third time for the first time;
The 3rd circulation is since the 3rd extractor countercurrent extraction: specifically merge the first extracting solution in the first extractor, the second extractor and the 3rd extractor in the second circulation to the 3rd extractor of the mulberry leaves slag after including the second circulation and extracting for the first time, then add 500kg water to extract for the first time; In the first extractor of the mulberry leaves slag using the first extracting solution of the 3rd extractor after including the second circulation and extracting for the first time, then add 500kg distilled water to extract for the first time; Then the first extracting solution of the first extractor is extracted in the second extractor of the mulberry tree slag after including the second circulation and extracting for the first time for the first time;
Merge the first extracting solution in the first extractor, the second extractor and the 3rd extractor in the 3rd circulation and filter and collect filtrate, add trichoroacetic acid(TCA) centrifugal, the first supernatant liquor is got mulberry leaf and is dried, then add deionized water, constant temperature boiling is extracted, and filters and collects filtrate, filter residue adds deionized water and extracts, merging filtrate, adds trichoroacetic acid(TCA) centrifugal, obtains the first supernatant liquor;
Upper prop: the first supernatant liquor is crossed to C18 post, then carry out gradient elution by the moving phase of different concns, collect the elutriant containing S-GI; Mobile phase A is wherein the phosphate buffered saline buffer of pH value 4.5, and Mobile phase B is acetonitrile, presses the flow rate gradient 65min of 0.5ml/min under column temperature 28-32 DEG C, the condition of pH value 5-6; Condition of gradient elution is: at 35min, the volume ratio of mobile phase A and Mobile phase B is 95/5; At 45min, the volume ratio of mobile phase A and Mobile phase B is 85/15; At 46-55min, the volume ratio of mobile phase A and Mobile phase B is 72/28; At 56-65min, the volume ratio of mobile phase A and Mobile phase B is 95/5.
Alcohol precipitation is got supernatant: after upper prop, in the liquid that contains S-GI of gained, first add containing the ethanol of alcohol quality 65% and stir 8min, and then add containing alcohol quality 80% ethanol stirring 5min, obtain the second supernatant liquor;
Concentrated: the second supernatant liquor reduced vacuum to be concentrated into 40% of original volume, to obtain concentrated solution;
Microwave vacuum drying: it is-0.01MPa to be then that 260MHz microwave treatment 90s obtains the powder-product that contains S-GI in microwave frequency that concentrated solution is evacuated to vacuum tightness.
Embodiment tri-
Water extraction: get 100kg mulberry leaf and be exposed to the sun and dry in the sun, then add the deionized water that 1000kg dries rear mulberry leaf weight, be placed in pot group type counterflow extraction apparatus and comprise three times of at least the first circulation, the second circulation and the 3rd circulation extractions that circulate, filling group is that every group of three extractors series connection share, countercurrent extraction flow velocity is 30min/ tank, and extracting temperature is 100 DEG C;
The first circulation is since the first extractor countercurrent extraction: specifically will dry rear mulberry leaf and 1000kg deionized water and add in the first extractor, the second extractor and the 3rd extractor by equal portions respectively; After the medical filtration that the first extractor extracts for the first time, put into container for storing liquid for subsequent use, the extraction for the second time of the first extractor and the extraction for the third time of the first extractor add respectively 4-5 times of distilled water; Then the solvent extracting for the first time using the second extracting solution of the first extractor as the second extractor, the first extracting liquid filtering of the second extractor is for subsequent use; The solvent extracting for the second time using the 3rd extracting solution of the first extractor as the second extractor, adds 450kg distilled water in the extraction for the second time of the second extractor; The second extracting solution of the second extractor is as the solvent extracting for the first time of the 3rd extractor;
The second circulation is since the second extractor countercurrent extraction: specifically merge the first extracting solution in the first extractor, the second extractor and the 3rd extractor in the first circulation to the second extractor of the mulberry leaves slag after including the first circulation and extracting for the second time, then add 450kg distilled water to extract for the first time; In the 3rd extractor of the mulberry leaves slag using the first extracting solution of the second extractor after including the first circulation and extracting for the first time, then add 450kg distilled water to extract for the first time; Then the first extracting solution of the 3rd extractor is extracted in the first extractor of the mulberry leaves slag after including the first circulation and extracting for the third time for the first time;
The 3rd circulation is since the 3rd extractor countercurrent extraction: specifically merge the first extracting solution in the first extractor, the second extractor and the 3rd extractor in the second circulation to the 3rd extractor of the mulberry leaves slag after including the second circulation and extracting for the first time, then add 450kg water to extract for the first time; In the first extractor of the mulberry leaves slag using the first extracting solution of the 3rd extractor after including the second circulation and extracting for the first time, then add 450kg distilled water to extract for the first time; Then the first extracting solution of the first extractor is extracted in the second extractor of the mulberry tree slag after including the second circulation and extracting for the first time for the first time;
Merge the first extracting solution in the first extractor, the second extractor and the 3rd extractor in the 3rd circulation and filter and collect filtrate, add trichoroacetic acid(TCA) centrifugal, the first supernatant liquor is got mulberry leaf and is dried, then add deionized water, constant temperature boiling is extracted, and filters and collects filtrate, filter residue adds deionized water and extracts, merging filtrate, adds trichoroacetic acid(TCA) centrifugal, obtains the first supernatant liquor;
Upper prop: the first supernatant liquor is crossed to C18 post, then carry out gradient elution by the moving phase of different concns, collect the elutriant containing S-GI; Mobile phase A is wherein the phosphate buffered saline buffer of pH value 4, and Mobile phase B is acetonitrile, presses the flow rate gradient 60min of 0.45ml/min under the condition of 30 DEG C of column temperatures, pH value 5.5; Condition of gradient elution is: at 0-35min, the volume ratio of mobile phase A and Mobile phase B is 93/7; At 36-45min, the volume ratio of mobile phase A and Mobile phase B is 83/17; At 46-55min, the volume ratio of mobile phase A and Mobile phase B is 70/30; At 56-60min, the volume ratio of mobile phase A and Mobile phase B is 93/7.
Alcohol precipitation is got supernatant: after upper prop, in the liquid that contains S-GI of gained, first add containing the ethanol of alcohol quality 60% and stir 5min, and then add containing alcohol quality 78% ethanol stirring 4min, obtain the second supernatant liquor;
Concentrated: the second supernatant liquor reduced vacuum to be concentrated into 30% of original volume, to obtain concentrated solution;
Microwave vacuum drying: it is-0.03MPa to be then that 160MHz microwave treatment 88s obtains the powder-product that contains S-GI in microwave frequency that concentrated solution is evacuated to vacuum tightness.
Embodiment tetra-
With embodiment mono-, different is in three circulations of water extraction are extracted, and within every 8 hours, disconnects first extractor discharging, after again feeding intake, connects successively.
When the stationary phase that upper prop is selected is MCI gel CHP 20P column chromatography, selecting methanol-water is moving phase.Adopt the slow mode of accelerating to stir to add ethanol, drip the ethanol containing alcohol quality 60%, then rapid stirring 5min under 250r/min according to 5ml/s.。
After upper prop, the be evacuated to-0.09MPa of the liquid that contains S-GI of gained is following and keep 20min, then adds deionized water 800kg, the cooling pretreatment liquid that must contain S-GI for 10 hours that leaves standstill.And then alcohol precipitation is got cleer and peaceful concentrated.
Concentrated by the second supernatant liquor in vacuum tightness be specifically-0.07MPa under reduced vacuum be concentrated into 25% of the second supernatant liquor volume, obtain concentrated solution.
Embodiment five
With embodiment bis-, different is in three circulations of water extraction are extracted, and within every 8 hours, disconnects first extractor discharging, after again feeding intake, connects successively.
When the stationary phase that upper prop is selected is MCI gel CHP 20P column chromatography, selecting methanol-water is moving phase.Adopt the slow mode of accelerating to stir to add ethanol, drip the ethanol containing alcohol quality 55%, then rapid stirring 3min under 200r/min according to 3ml/s.。
After upper prop, the be evacuated to-0.09MPa of the liquid that contains S-GI of gained is following and keep 15min, then adds deionized water 800kg, the cooling pretreatment liquid that must contain S-GI for 8 hours that leaves standstill.And then alcohol precipitation is got cleer and peaceful concentrated.
Concentrated by the second supernatant liquor in vacuum tightness be specifically-0.075MPa under reduced vacuum be concentrated into 20% of the second supernatant liquor volume, obtain concentrated solution.
Embodiment six
With embodiment tri-, different is in three circulations of water extraction are extracted, and within every 8 hours, disconnects first extractor discharging, after again feeding intake, connects successively.
When the stationary phase that upper prop is selected is MCI gel CHP 20P column chromatography, selecting methanol-water is moving phase.Adopt the slow mode of accelerating to stir to add ethanol, drip the ethanol containing alcohol quality 65%, then rapid stirring 8min under 300r/min according to 7ml/s.。
After upper prop, the be evacuated to-0.09MPa of the liquid that contains S-GI of gained is following and keep 25min, then adds deionized water 800kg, the cooling pretreatment liquid that must contain S-GI for 15 hours that leaves standstill.And then alcohol precipitation is got cleer and peaceful concentrated.
Concentrated by the second supernatant liquor in vacuum tightness be specifically-0.06MPa under reduced vacuum be concentrated into 30% of the second supernatant liquor volume, obtain concentrated solution.
Embodiment seven
Alcohol precipitation is got in backward the second supernatant liquor liquid of supernatant and is added and account for the sherwood oil of 1.5 times of quality of the second supernatant liquor and the mixed solution of methyl alcohol, mixes and soaks 0.5h, supersound extraction 2min under 40 DEG C, 50KHz; And then concentrate.
Embodiment eight
Alcohol precipitation is got in backward the second supernatant liquor liquid of supernatant and is added and account for the sherwood oil of 3 times of quality of the second supernatant liquor and the mixed solution of methyl alcohol, mixes and soaks 0.8h, supersound extraction 4min under 50 DEG C, 70KHz; And then concentrate.
Embodiment nine
Alcohol precipitation is got in backward the second supernatant liquor liquid of supernatant and is added and account for the sherwood oil of 2 times of quality of the second supernatant liquor and the mixed solution of methyl alcohol, mixes and soaks 0.7h, supersound extraction 3min under 45 DEG C, 60KHz; And then concentrate.
Comparative example one
With embodiment mono-, different is to get mulberry leaf to dry, and then adds water, and constant temperature boiling is extracted, and filters and collects filtrate, and filter residue adds deionized water and extracts, and merging filtrate adds methyl alcohol centrifugal, obtains the first supernatant liquor; Then by the first supernatant liquor spent ion exchange resin purifying, obtain the liquid that contains S-GI; After upper prop, the liquid that contains S-GI of gained adds and stirs 9min containing the ethanol of alcohol quality 70% and obtain the second supernatant liquor; Concentrated solution will be obtained after the second supernatant concentration; Concentrated solution oven dry is obtained to the powder-product that contains S-GI.
Table 1 embodiment and the comparison of comparative example's S-GI product
This specific embodiment is only explanation of the invention; it is not limitation of the present invention; those skilled in the art are reading after this specification sheets and can make to the present embodiment the amendment that there is no creative contribution as required, but as long as within the scope of claim of the present invention, are all subject to the protection of patent law.
Claims (10)
1. an extracting and purifying method for S-GI in mulberry tree, is characterized in that: it comprises the following steps:
(1) water extraction: get mulberry leaf and dry, then add deionized water, constant temperature boiling is extracted, and filters and collects filtrate, and filter residue adds deionized water and extracts, and merging filtrate adds trichoroacetic acid(TCA) centrifugal, obtains the first supernatant liquor;
(2) upper prop: described the first supernatant liquor is carried out to purifying by reverse column chromatography, obtain the liquid that contains S-GI;
(3) alcohol precipitation is got supernatant: after upper prop, in the liquid that contains S-GI of gained, first add containing the ethanol of alcohol quality 55-65% and stir 3-8min, and then add containing alcohol quality 70-80% ethanol stirring 3-5min, obtain the second supernatant liquor;
(4) concentrated: will after described the second supernatant concentration, to obtain concentrated solution;
(5) microwave vacuum drying: described concentrated solution microwave vacuum drying is obtained to the powder-product that contains S-GI.
2. the extracting and purifying method of S-GI in a kind of mulberry tree according to claim 1, it is characterized in that: described step (1) water extraction is specifically got mulberry leaf and is exposed to the sun and dries in the sun, then add the deionized water that 8-12 doubly dries rear mulberry leaf weight, be placed in pot group type counterflow extraction apparatus and comprise three times of at least the first circulation, the second circulation and the 3rd circulation extractions that circulate, described filling group is that every group of three extractors series connection share, countercurrent extraction flow velocity is 25-35min/ tank, and extraction temperature is 80-110 DEG C;
Described the first circulation is since the first extractor countercurrent extraction: specifically the deionized water that dries rear mulberry leaf and 8-12 and doubly dry rear mulberry leaf weight is added in the first extractor, the second extractor and the 3rd extractor by equal portions respectively; After the medical filtration that the first extractor extracts for the first time, put into container for storing liquid for subsequent use, the extraction for the second time of the first extractor and the extraction for the third time of the first extractor add respectively 4-5 times of distilled water; Then the solvent extracting for the first time using the second extracting solution of the first extractor as the second extractor, the first extracting liquid filtering of the second extractor is for subsequent use; The solvent extracting for the second time using the 3rd extracting solution of the first extractor as the second extractor, adds 4-5 times of distilled water in the extraction for the second time of the second extractor; The second extracting solution of the second extractor is as the solvent extracting for the first time of the 3rd extractor;
Described the second circulation is since the second extractor countercurrent extraction: specifically merge the first extracting solution in the first extractor, the second extractor and the 3rd extractor in the first circulation to the second extractor of the mulberry leaves slag after including the first circulation and extracting for the second time, then add 4-5 times of distilled water to extract for the first time; In the 3rd extractor of the mulberry leaves slag using the first extracting solution of the second extractor after including the first circulation and extracting for the first time, then add 4-5 times of distilled water to extract for the first time; Then the first extracting solution of the 3rd extractor is extracted in the first extractor of the mulberry leaves slag after including the first circulation and extracting for the third time for the first time;
Described the 3rd circulation is since the 3rd extractor countercurrent extraction: specifically merge the first extracting solution in the first extractor, the second extractor and the 3rd extractor in the second circulation to the 3rd extractor of the mulberry leaves slag after including the second circulation and extracting for the first time, then add 4-5 times of water to extract for the first time; In the first extractor of the mulberry leaves slag using the first extracting solution of the 3rd extractor after including the second circulation and extracting for the first time, then add 4-5 times of distilled water to extract for the first time; Then the first extracting solution of the first extractor is extracted in the second extractor of the mulberry tree slag after including the second circulation and extracting for the first time for the first time;
Merge the first extracting solution in the first extractor, the second extractor and the 3rd extractor in the 3rd circulation and filter and collect filtrate, adding trichoroacetic acid(TCA) centrifugal, obtaining the first supernatant liquor.
3. the extracting and purifying method of S-GI in a kind of mulberry tree according to claim 2, is characterized in that: described three circulations disconnect first extractor discharging in every 8 hours in extracting, and after again feeding intake, connect successively.
4. the extracting and purifying method of S-GI in a kind of mulberry tree according to claim 1, it is characterized in that: described step (2) is specifically crossed described the first supernatant liquor C18 post or MCI gel CHP 20P column chromatography, then carry out gradient elution by the moving phase of different concns, collect the elutriant containing S-GI.
5. the extracting and purifying method of S-GI in a kind of mulberry tree according to claim 4, it is characterized in that: in the time that the stationary phase of selecting is C18 post, mobile phase A is the phosphate buffered saline buffer of pH value 3.5-4.5, Mobile phase B is acetonitrile, presses the flow rate gradient 55-65min of 0.4-0.5ml/min under column temperature 28-32 DEG C, the condition of pH value 5-6; Described condition of gradient elution is: at 0-35min, the volume ratio of described mobile phase A and Mobile phase B is (90-95)/(5-10); At 36-45min, the volume ratio of described mobile phase A and Mobile phase B is (80-85)/(15-20); At 46-55min, the volume ratio of described mobile phase A and Mobile phase B is (68-72)/(28-32); At 56-65min, the volume ratio of described mobile phase A and Mobile phase B is (90-95)/(5-10).
6. according to the extracting and purifying method of S-GI in a kind of mulberry tree described in claim 1-5 any one, it is characterized in that: described step (3) adopts the slow mode of accelerating to stir to add ethanol: drip the ethanol containing alcohol quality 55-65%, then rapid stirring 3-8min under 200-300r/min according to 3-7ml/s.
7. the extracting and purifying method of S-GI in a kind of mulberry tree according to claim 6, it is characterized in that: described extracting and purifying method is also included in step (3) alcohol precipitation and gets before supernatant, by following be evacuated to-the 0.09MPa of the liquid that contains S-GI of gained after upper prop and keep 15-25min, then add deionized water 800kg, within cooling standing 8-15 hour, obtain pretreatment liquid.
8. the extracting and purifying method of S-GI in a kind of mulberry tree according to claim 7, is characterized in that: concentrated specifically described the second supernatant liquor reduced vacuum of described step (4) is concentrated into the 10-40% of original volume, obtains concentrated solution.
9. the extracting and purifying method of S-GI in a kind of mulberry tree according to claim 8, it is characterized in that: described extracting and purifying method is also included in described in the concentrated forward direction of step (4) and adds and account for the sherwood oil of the second supernatant liquor 1.5-3 times quality and the mixed solution of methyl alcohol in the second supernatant liquor liquid, mix and soak 0.5-0.8h, supersound extraction 2-4min under 40-50 DEG C, 50-70KHz.
10. the extracting and purifying method of S-GI in a kind of mulberry tree according to claim 9, it is characterized in that: it is-0.04～-0.01MPa that described microwave vacuum drying is specifically evacuated to vacuum tightness, is then that 100-260MHz microwave treatment 80-90s obtains the powder-product that contains S-GI in microwave frequency.
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Denomination of invention: A method for extracting and purifying deoxynojirimycin from Mulberry Effective date of registration: 20210727 Granted publication date: 20150916 Pledgee: Zhejiang Nanxun Rural Commercial Bank Co.,Ltd. Changrui sub branch Pledgor: HUIZHOU LIUYIN BIOLOGICAL TECHNOLOGY Co.,Ltd. Registration number: Y2021330000993 |