CN103274992B - A kind of method of combining membrane sepn and column chromatography technology and preparing high purity 1-DNJ - Google Patents
A kind of method of combining membrane sepn and column chromatography technology and preparing high purity 1-DNJ Download PDFInfo
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Abstract
The present invention relates to the separation and purification field of active ingredient of natural product, be specifically related to a kind of method of combining membrane sepn and column chromatography technology enrichment 1-DNJ.The method is by silkworm, mulberry raw material ultrasonic wave added water extraction; Then by centrifugal, microfiltration membrane process, ultrafiltration feeding liquid is obtained; Analyse the step with sephadex chromatography through second ultrafiltration, nanofiltration, cation exchange resin layer again, obtain the 1-DNJ product that purity is more than 80%.Method of the present invention has that technique is simple, efficiency is high, the advantage of safety and environmental protection.
Description
Technical field
The present invention relates to the separation and purification field of active ingredient of natural product, be specifically related to a kind of method of combining membrane sepn and column chromatography technology enrichment 1-DNJ.
Background technology
1-DNJ (1-deoxynojirimycin is called for short DNJ) is a kind of piperidines polyhydroxylated alkaloid.The structure of DNJ is similar to glucose, can competitive inhibition alpha-glucosidase, affects the processes such as the sugar metabolism of organism and biological processing, has the multiple pharmacological activities such as hypoglycemic, reducing blood-fat, virus, antitumor and anti-inflammatory.Therefore, the development research of its related products has vast potential for future development.
Research shows, the natural origin of DNJ mainly contains the silkworm and mulberry resources such as mulberry leaf, ramulus mori, Sang Pi and silkworm.But material composition kind is many in silkworm and mulberry resource, the content of DNJ is few, and separation and purification then also exists very large challenge.In current report, the methods such as water extraction and alcohol precipitation method, reduced vacuum method of evaporation, absorption method, ion exchange method and gel filtration method are generally used to separation and purification mulberry leaf DNJ.The patent of invention of CN102101840A is extracted by water solvent, centrifuging, alcohol settling, crosses the steps such as highly acidic cation exchange column, macroporous resin and alkaline adsorption post, finally obtain DNJ.Though can obtain the DNJ of higher degree, there is complex treatment process, elution time is long, is mixed with the shortcomings such as organic solvent.CN101654428A patent uses gel filtration method purifying DNJ, and each applied sample amount is limited, and cost is relatively high.Although used micro-filtration, ultra-filtration membrane in patent of invention CN102718697A, just as a kind of pretreatment process, mainly also still use macroporous resin regulating YIN and YANG ion exchange resin and carried out separation and purification.By contrast, membrane separation technique has the advantages such as treatment condition gentleness, flexible operation, environmental friendliness, efficient and safety, can realize being separated with solvent multi-component solute, purifying and concentrating.At home, combined utilization micro-filtration, ultrafiltration and nanofiltration multiple film technology and column chromatography technology enrichment DNJ have not been reported.
Summary of the invention
For improving the deficiency and shortcoming that exist in prior art processes, the invention provides a kind of method of combining membrane sepn and column chromatography technology enrichment 1-DNJ.
The object of the invention is to be realized by following technological method.
Combine a method for membrane sepn and column chromatography technology enrichment 1-DNJ, described method is:
(1) ultrasonic water extraction: by the silkworm of drying, mulberry raw material pulverizing, sieve, obtains coarse meal; Take appropriate coarse meal, add distilled water by 40:1-10:1 liquid ratio (mL/g), ultrasonication 30min under 70 DEG C of heating in water bath, centrifuging, collect supernatant liquor, precipitation is repeated to extract once by above-mentioned steps, merges twice supernatant liquor, obtain extracting solution;
(2) pre-treatment: by the extracting solution of step (1) gained, by centrifugal, microfiltration membrane process, obtains ultrafiltration feeding liquid;
(3) second ultrafiltration: by step (2) gained ultrafiltration feeding liquid first time ultra-filtration membrane, in ultra-filtration process, add deionized water in feed liquid bucket, collect first time ultrafiltration permeate; Again by ultrafiltration membrane treatment less for permeate molecular weight cut-off, collect second time ultrafiltration permeate;
(4) nanofiltration: with nanofiltration membrane treatment step (3) gained ultrafiltration permeate, collect the liquid that dams, obtain 1-DNJ nanofiltration concentrated solution;
(5) cation exchange resin layer is analysed: the 1-DNJ nanofiltration concentrated solution of gained in (4) is carried out Zeo-karb separation, wash with water, then use ammonia soln wash-out, collect ammoniacal liquor elutriant, and its deammoniation is concentrated, obtain 1-DNJ crude samples;
(6) sephadex chromatography: adopt Sephadex LH-20 resin to carry out purifying further (5) cationic refined solution, obtains the 1-DNJ essence sample that purity is more than 80% after lyophilize.
Further, raw material described in (1) is mulberry leaf, ramulus mori, Cortex Mori, Mulberry Roots and silkworm.
Further, microfiltration membrane described in (2) is the hollow fiber type filter membrane of 0.5-5 μm or the millipore filtration of 0.45-2.0 μm.
Further, described in (3), twice ultra-filtration membrane used is poly-PES or compound rolled film, and during first time ultrafiltration, preferred molecular weight cut-off is the ultra-filtration membrane of 8.0-50kDa, and working pressure is 0.5-1.2MPa, and feed temperature is 30 DEG C; During second time ultrafiltration, preferred molecular weight cut-off is the ultra-filtration membrane of 1.0-2.0kDa, and working pressure is 0.5-2.7MPa, preferred 1.2-1.8MPa.
Further, nanofiltration membrane described in (4) is rolling organic membrane, and molecular weight cut-off is 100-200Da, and working pressure is 0.5-4.0MPa, preferred 1.2-2.5MPa.
Further, (5) Zeo-karb described in is strongly acidic cation-exchange, can doubly fill the 1-DNJ nanofiltration concentrated solution of post resin volume by loading 2-4, water washing volume is 1-2 times of column volume, and ammoniacal liquor elution volume is 4-6 times of column volume at every turn.
Further, dextrane gel resin described in (6) is Sephadex LH-20 dextrane gel resin, and the blade diameter length ratio of dress post is 1:20-1:40, and select the methanol solution of 10-50% to carry out wash-out with 0.25-0.5mL/min flow velocity, elution volume is 6-8 times of column volume.
The method measuring DNJ content in the method for the invention is FMOC-Cl column front derivation-High Performance Liquid Chromatography with Fluorescence Detection, concrete testing conditions: detector is RF-10AXL fluorimetric detector, chromatographic column is Inertsil ODS-SP C18 (4.6mm × 250mm, 5 μm), moving phase is acetonitrile: 0.1% aqueous acetic acid (50:50, V/V), column temperature 25 DEG C, flow velocity 1.0mL/min, analysis time 25min, excitation wavelength is 254nm, and emission wavelength is 322nm, and sample size is 10 μ L.
Compared with prior art, beneficial effect of the present invention be mainly reflected in following some:
(1) in method of the present invention, membrane separation process is reasonable, and the second ultrafiltration step adopted can the effective macromole such as protein isolate, polysaccharide impurity, and separation selectivity is good, efficiency is high;
(2) nanofiltration membrane selected by method of the present invention can remove the small molecular weight impurities such as metal ion, and DNJ active principle can be concentrated by fast enriching, can the steps such as vacuum concentration be avoided, the applied sample amount of sample during subsequent columns chromatography can be increased again, be more conducive to follow-up processing;
(3) in method of the present invention, membrane separation process whole process is physical sepn, mild condition, and can realize serialization and automated operation;
(4) resin that in method of the present invention, chromatographic separation is used be renewable, reuse resin, elute soln used is ammoniacal liquor and methyl alcohol, volatile, recyclable time concentrated, safety and environmental protection;
(5) method of the present invention is not solely component films isolation technique and chromatographic technique, but probe into by experiment, effectively combine optimised process and parameter that membrane sepn and column chromatography technology can draw, physical sepn and concentrated DNJ efficiently can be realized, single-minded and purifying effectively can be realized again, obtain highly purified DNJ.
Accompanying drawing explanation
Fig. 1 is the process flow sheet of the inventive method;
Fig. 2 is the HPLC collection of illustrative plates of DNJ reference substance standard specimen;
Fig. 3 is the HPLC collection of illustrative plates of DNJ purifying product in embodiment 1.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in further details, but protection scope of the present invention is not limited in this:
Embodiment 1
Technical process is as Fig. 1, and concrete steps are as follows:
(1) ultrasonic water extraction: dried feed is pulverized, sieved, obtains coarse meal.Take appropriate coarse meal, add distilled water by 40:1 liquid ratio (mL/g), ultrasonication 30min under 70 DEG C of heating in water bath, centrifuging, collect supernatant liquor, precipitation is repeated to extract once by above-mentioned steps, merges twice supernatant liquor, obtain extracting solution.
(2) pre-treatment: by the extracting solution of step (1) gained, by centrifugal, then process through the millipore filtration of 2.0 μm, obtains ultrafiltration feeding liquid.
(3) second ultrafiltration: by step (2) gained ultrafiltration feeding liquid first time ultra-filtration membrane, in ultra-filtration process, add deionized water in feed liquid bucket, collect first time ultrafiltration permeate; Again by ultrafiltration membrane treatment less for permeate molecular weight cut-off, collect second time ultrafiltration permeate.During first time ultrafiltration, preferred molecular weight cut-off is the ultra-filtration membrane of 50kDa, and working pressure is 1.2MPa, and feed temperature is 30 DEG C; During second time ultrafiltration, preferred molecular weight cut-off is the ultra-filtration membrane of 2.0kDa, preferred 1.8MPa.
(4) nanofiltration: preferred molecular weight cut-off is that the nanofiltration membrane of 200Da processes step (3) gained ultrafiltration permeate, and preferred operations pressure is 2.5MPa, collects the liquid that dams, obtains 1-DNJ (DNJ) nanofiltration concentrated solution.
(5) cation exchange resin layer is analysed: select strongly acidic cation-exchange to be separated gained DNJ nanofiltration concentrated solution in (4), the DNJ nanofiltration concentrated solution measuring 2 times of column volumes carries out loading, with 1 times of column volume water washing, wash-out is carried out by 0.5mol/L flow velocity again with the ammonia soln of 6 times of column volumes, collect ammoniacal liquor elutriant, and its deammoniation is concentrated.
(6) sephadex chromatography: adopt Sephadex LH-20 resin to carry out purifying further (5) cationic refined solution, during dress post, control blade diameter length ratio is 1:20, selects 10% methanol solution to carry out wash-out with 0.25mL/min flow velocity, and elution volume is 6 times of column volumes.The DNJ essence sample (Fig. 2 is the HPLC collection of illustrative plates of DNJ reference substance, and Fig. 3 is the HPLC collection of illustrative plates of DNJ essence sample) that purity is 81.83% is obtained after lyophilize.
Embodiment 2
In step (1) ultrasonic water extraction, use liquid ratio to be 10:1 liquid ratio (mL/g), then be the ultra-filtration membrane of 20kDa by preferred molecular weight cut-off during first time ultrafiltration in step (3), working pressure is 0.8MPa, and feed temperature is 30 DEG C; During second time ultrafiltration, preferred molecular weight cut-off is the ultra-filtration membrane of 1.0kDa, preferred 1.2MPa.Other operations are with embodiment 1.The DNJ essence sample purity finally obtained is 83.56%.
Embodiment 3
In step (1) ultrasonic water extraction, use liquid ratio to be 20:1 liquid ratio (mL/g), then be the ultra-filtration membrane of 8kDa by preferred molecular weight cut-off during first time ultrafiltration in step (3), working pressure is 0.8MPa, and feed temperature is 30 DEG C; During second time ultrafiltration, preferred molecular weight cut-off is the ultra-filtration membrane of 1.0kDa, preferred 1.2MPa; Preferred molecular weight cut-off is that the nanofiltration membrane of 150Da processes step (3) gained ultrafiltration permeate, and preferred operations pressure is 1.2MPa.Other operations are with embodiment 1.The DNJ essence sample purity finally obtained is 85.24%
Embodiment 4
During step (5) cation exchange resin layer is analysed, the DNJ nanofiltration concentrated solution measuring 4 times of column volumes carries out loading, with 1 times of column volume water washing, wash-out is carried out by 0.5mol/L flow velocity again with the ammonia soln of 8 times of column volumes, collect ammoniacal liquor elutriant, and its deammoniation is concentrated, other operations are with embodiment 1.The DNJ essence sample purity finally obtained is 84.62%.
Embodiment 5
In step (6) sephadex chromatography, blade diameter length ratio during dress post is controlled to 1:40, and select 50% methanol solution to carry out wash-out with 0.5mL/min flow velocity, elution volume is 8 times of column volumes, and other operations are with embodiment 1.The DNJ essence sample that purity is 87.15% is obtained after lyophilize.
Claims (7)
1. combine membrane sepn and column chromatography technology prepares the method that purity is the 1-DNJ of more than 80%, it is characterized in that comprising the following steps:
(1) ultrasonic wave added water extraction: by the silkworm of drying, mulberry raw material pulverizing, sieve, obtains coarse meal; Take appropriate coarse meal, add distilled water by 40:1-10:1 liquid ratio mL/g, ultrasonication 30min under 70 DEG C of heating in water bath, centrifuging, collect supernatant liquor, precipitation is repeated to extract once by above-mentioned steps, merges twice supernatant liquor, obtain extracting solution;
(2) pre-treatment: by the extracting solution of step (1) gained, by centrifugal, microfiltration membrane process, obtains ultrafiltration feeding liquid
(3) second ultrafiltration: by step (2) gained ultrafiltration feeding liquid first time ultra-filtration membrane, in ultra-filtration process, add deionized water in feed liquid bucket, collect first time ultrafiltration permeate; Again by ultrafiltration membrane treatment less for permeate molecular weight cut-off, collect second time ultrafiltration permeate;
(4) nanofiltration: with nanofiltration membrane treatment step (3) gained ultrafiltration permeate, collect the liquid that dams, obtain 1-DNJ nanofiltration concentrated solution;
(5) cation exchange resin layer is analysed: the 1-DNJ nanofiltration concentrated solution of gained in (4) is carried out Zeo-karb separation, wash with water, use ammonia soln wash-out again, collect ammoniacal liquor elutriant, and its deammoniation is concentrated, obtain 1-DNJ crude samples;
(6) sephadex chromatography: adopt Sephadex LH-20 resin to carry out purifying further (5) cationic refined solution, obtains the 1-DNJ product that purity is more than 80% after lyophilize.
2. a kind of method of combining membrane sepn and column chromatography technology enrichment 1-DNJ according to claim 1, is characterized in that described step (1) Raw is mulberry leaf, ramulus mori, Cortex Mori, Mulberry Roots and silkworm.
3. a kind of method of combining membrane sepn and column chromatography technology enrichment 1-DNJ according to claim 1, is characterized in that in described step (2), microfiltration membrane is the hollow fiber type filter membrane of 0.5-5 μm or the millipore filtration of 0.45-2.0 μm.
4. a kind of method of combining membrane sepn and column chromatography technology enrichment 1-DNJ according to claim 1, it is characterized in that in described step (3), 2 ultra-filtration membranes used are poly-PES or compound rolled film, during first time ultrafiltration, selective retention molecular weight is the ultra-filtration membrane of 8.0-50kDa, working pressure is 0.5-1.2MPa, feed temperature is 25-40 DEG C, and the deionized water volume added is 1-2 times of feeding liquid volume; During second time ultrafiltration, selective retention molecular weight is the ultra-filtration membrane of 1.0-2.0kDa, and working pressure is 0.5-2.7MPa.
5. a kind of method of combining membrane sepn and column chromatography technology enrichment 1-DNJ according to claim 1, it is characterized in that in described step (4), nanofiltration membrane is rolling organic membrane, molecular weight cut-off is 100-200Da, and working pressure is 0.5-4.0MPa.
6. a kind of method of combining membrane sepn and column chromatography technology enrichment 1-DNJ according to claim 1, it is characterized in that described step (5) cationic exchange resin is strongly acidic cation-exchange, can doubly fill the DNJ nanofiltration concentrated solution of post resin volume by loading 2-4 at every turn, water washing volume is 1-2 times of column volume, and ammoniacal liquor elution volume is 4-6 times of column volume.
7. a kind of method of combining membrane sepn and column chromatography technology enrichment 1-DNJ according to claim 1, it is characterized in that in described step (6), dextrane gel resin is Sephadex LH-20 dextrane gel resin, the blade diameter length ratio of dress post is 1:20-1:40, select the methanol solution of 10-50% to carry out wash-out with 0.25-0.5mL/min flow velocity, elution volume is 6-8 times of column volume.
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| CN103965096B (en) * | 2014-05-09 | 2016-08-24 | 江西海富生物工程有限公司 | A kind of preparation method being applicable to industrial 1-DNJ |
| CN104007205B (en) * | 2014-06-13 | 2015-09-30 | 漳州片仔癀药业股份有限公司 | A kind of detection method for the treatment of the pharmaceutical preparation of diabete |
| CN106083697A (en) * | 2016-06-29 | 2016-11-09 | 新疆医科大学 | The extracting method of 1 deoxynojirimycin in medicine Mulberry |
| CN107162955B (en) * | 2017-06-30 | 2020-02-18 | 中国科学院过程工程研究所 | Method for extracting deoxynojirimycin |
| CN107721909B (en) * | 2017-11-02 | 2023-12-29 | 广西五和博澳药业有限公司 | Method and system for continuously extracting DNJ, flavone and polysaccharide from Moraceae plant |
| CN109180563A (en) * | 2018-08-28 | 2019-01-11 | 广东碧桑园科技有限公司 | A method of extracting DNJ from fresh mulberry leaf |
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| CN102311379A (en) * | 2011-07-05 | 2012-01-11 | 南京泽朗农业发展有限公司 | Method for preparing 1-deoxynojirimycin by membrane separation technology |
| CN102491938A (en) * | 2011-12-16 | 2012-06-13 | 北京贯虹科技集团有限公司 | Purification method of deoxynojirimycin |
| CN102718697A (en) * | 2012-06-06 | 2012-10-10 | 浙江工业大学 | Method for preparing mulberry leaf 1-deoxynojirimycin extract with filter membrane and resin |
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| KR100603892B1 (en) * | 2004-12-28 | 2006-07-24 | 사회복지법인 삼성생명공익재단 | Precess for isolating 1-deoxynojirimycin derived from powdery silkworm |
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| CN102311379A (en) * | 2011-07-05 | 2012-01-11 | 南京泽朗农业发展有限公司 | Method for preparing 1-deoxynojirimycin by membrane separation technology |
| CN102491938A (en) * | 2011-12-16 | 2012-06-13 | 北京贯虹科技集团有限公司 | Purification method of deoxynojirimycin |
| CN102718697A (en) * | 2012-06-06 | 2012-10-10 | 浙江工业大学 | Method for preparing mulberry leaf 1-deoxynojirimycin extract with filter membrane and resin |
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