CN103274992A - Method for preparing high-purity 1-deoxynojirimycin with combined membrane separation and column chromatography technology - Google Patents
Method for preparing high-purity 1-deoxynojirimycin with combined membrane separation and column chromatography technology Download PDFInfo
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Abstract
The invention relates to the field of separation and purification of active ingredients of a natural product, in particular to a method for preparing high-purity 1-deoxynojirimycin with a combined membrane separation and column chromatography technology. According to the method, raw materials of silkworm and white mulberry is subjected to water extraction in aid of ultrasonic waves; an ultrafiltration feeding liquid is obtained through centrifugation and micro-filtration membrane treatment; and a 1-deoxynojirimycin product with purity larger than 80% is obtained through steps of secondary ultrafiltration, nanofiltration, cation exchange resin chromatography and dextrane gel chromatography. The method has the advantages of simple process, high efficiency, safety and environment 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 uniting membrane sepn and column chromatography technology enrichment 1-S-GI.
Background technology
1-S-GI (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, influence processes such as the sugar metabolism of organism and biological processing, have 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.
Studies show that the natural origin of DNJ mainly contains silkworm and mulberry resources such as mulberry leaf, ramulus mori, Sang Pi and silkworm.Yet material composition kind is many in the silkworm and mulberry resource, and the content of DNJ is few, and separation and purification exists very big challenge.In the report, methods such as water extraction and alcohol precipitation method, reduced vacuum method of evaporation, absorption method, ion exchange method and gel filtration method generally are used to separation and purification mulberry leaf DNJ at present.The patent of invention of CN102101840A is to extract by water solvent, centrifuging, and ethanol sedimentation is crossed steps such as highly acidic cation exchange column, macroporous resin and alkaline adsorption column, finally obtains DNJ.Though can obtain the DNJ of higher degree, exist complex treatment process, elution time is long, is mixed with shortcomings such as organic solvent.The CN101654428A patent is used gel filtration method purifying DNJ, and each applied sample amount is limited, and cost is higher relatively.Though used micro-filtration, ultra-filtration membrane among the patent of invention CN102718697A, just as a kind of pretreatment process, mainly also still used macroporous resin regulating YIN and YANG ion exchange resin and carry out separation and purification.By contrast, membrane separation technique has advantages such as treatment condition gentleness, flexible operation, environmental friendliness, efficient and safety, can realize multi-component solute is separated, purifies with solvent and concentrated.At home, the multiple membrane technique of combined utilization micro-filtration, ultrafiltration and nanofiltration and column chromatography technology enrichment DNJ yet there are no report.
Summary of the invention
For improving deficiency and the shortcoming that exists in the prior art processes, the invention provides a kind of method of uniting membrane sepn and column chromatography technology enrichment 1-S-GI.
The objective of the invention is to realize by following technological method.
A kind of method of uniting membrane sepn and column chromatography technology enrichment 1-S-GI, described method is:
(1) ultrasonic water is carried: with silkworm, the mulberry raw material pulverizing of drying, sieve, obtain coarse meal; Take by weighing an amount of coarse meal, press 40:1-10:1 liquid ratio (mL/g) and add distilled water, ultrasonication 30min under 70 ℃ of heating in water bath, supernatant liquor is collected in centrifuging, will precipitate by above-mentioned steps to repeat to extract once, merges supernatant liquor twice, namely gets extracting solution;
(2) pre-treatment: with the extracting solution of step (1) gained, handle by centrifugal, microfiltration membrane, obtain the ultrafiltration feeding liquid;
(3) second ultrafiltration: with step (2) the gained ultrafiltration feeding liquid ultra-filtration membrane first time, in the ultra-filtration process, in the feed liquid bucket, add deionized water, collect ultrafiltration for the first time and see through liquid; To collect ultrafiltration for the second time and see through liquid through liquid with the littler ultrafiltration membrane treatment of molecular weight cut-off again;
(4) nanofiltration: see through liquid with nanofiltration membrane treatment step (3) gained ultrafiltration, collect the liquid that dams, namely get 1-S-GI nanofiltration concentrated solution;
(5) cation exchange resin layer is analysed: the 1-S-GI nanofiltration concentrated solution of gained in (4) is carried out the Zeo-karb separation, wash with water, use the ammonia soln wash-out again, collect the ammoniacal liquor elutriant, and its deammoniation is concentrated, obtain 1-S-GI crude samples;
(6) sephadex chromatography: further adopt Sephadex LH-20 resin to carry out purifying positively charged ion refined solution in (5), acquisition purity is the smart sample of 1-S-GI more than 80% after the lyophilize.
Further, raw material described in (1) is mulberry leaf, ramulus mori, Cortex Mori, mulberry tree root and silkworm.
Further, microfiltration membrane described in (2) is the hollow fiber type filter membrane of 0.5-5 μ m or the millipore filtration of 0.45-2.0 μ m.
Further, twice used ultra-filtration membrane is poly-PES or compound rolled film described in (3), and preferred molecular weight cut-off is the ultra-filtration membrane of 8.0-50kDa during ultrafiltration for the first time, and working pressure is 0.5-1.2MPa, and feed temperature is 30 ℃; Preferred molecular weight cut-off is the ultra-filtration membrane of 1.0-2.0kDa during ultrafiltration for the second time, and working pressure is 0.5-2.7MPa, preferred 1.2-1.8MPa.
Further, nanofiltration membrane described in (4) is the 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 go up the 1-S-GI nanofiltration concentrated solution that sample 2-4 doubly adorns post resin volume, the water washing volume is 1-2 times of column volume at every turn, and the ammoniacal liquor elution volume is 4-6 times of column volume.
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, selects for use the methanol solution of 10-50% to carry out wash-out with the 0.25-0.5mL/min flow velocity, and elution volume is 6-8 times of column volume.
The method of measuring DNJ content in the method for the invention is FMOC-Cl column front derivation-high performance liquid chromatography-fluorescence detection, concrete testing conditions: detector is the RF-10AXL fluorimetric detector, and chromatographic column is Inertsil ODS-SP C18 (4.6mm * 250mm, 5 μ m), moving phase is acetonitrile: 0.1% aqueous acetic acid (50:50, V/V), 25 ℃ of column temperatures, 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) membrane separation process is reasonable in the method for the present invention, and the second ultrafiltration step that adopts is macromole impurity such as protein isolate, polysaccharide effectively, and separation selectivity is good, efficient is high;
(2) the selected nanofiltration membrane of method of the present invention can be removed small molecular weight impurities such as metal ion, and can concentrate the DNJ active principle by fast enriching, can avoid steps such as vacuum concentration, the applied sample amount of sample in the time of increasing the subsequent columns chromatography again more is conducive to follow-up processing;
(3) the membrane separation process whole process is physical sepn in the method for the present invention, mild condition, and can realize serialization and automated operation;
(4) the used resin of chromatographic separation is renewable, repeated use resin in the method for the present invention, and used elute soln is ammoniacal liquor and methyl alcohol, and is volatile, recyclable when concentrating, safety and environmental protection;
(5) method of the present invention is not single ground component films isolation technique and chromatographic technique, but probe into by experiment, unite optimised process and parameter that membrane sepn and column chromatography technology can draw effectively, can realize physical sepn and concentrated DNJ efficiently, can realize single-minded again and purifying effectively, obtain highly purified DNJ.
Description of drawings
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 among the 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 such as Fig. 1, concrete steps are as follows:
(1) ultrasonic water is carried: dried feed is pulverized, sieved, obtain coarse meal.Take by weighing an amount of coarse meal, press 40:1 liquid ratio (mL/g) and add distilled water, ultrasonication 30min under 70 ℃ of heating in water bath, supernatant liquor is collected in centrifuging, will precipitate by above-mentioned steps to repeat to extract once, merges supernatant liquor twice, namely gets extracting solution.
(2) pre-treatment: with the extracting solution of step (1) gained, by centrifugal, handle through the millipore filtration of 2.0 μ m again, obtain the ultrafiltration feeding liquid.
(3) second ultrafiltration: with step (2) the gained ultrafiltration feeding liquid ultra-filtration membrane first time, in the ultra-filtration process, in the feed liquid bucket, add deionized water, collect ultrafiltration for the first time and see through liquid; To collect ultrafiltration for the second time and see through liquid through liquid with the littler ultrafiltration membrane treatment of molecular weight cut-off again.Preferred molecular weight cut-off is the ultra-filtration membrane of 50kDa during ultrafiltration for the first time, and working pressure is 1.2MPa, and feed temperature is 30 ℃; Preferred molecular weight cut-off is the ultra-filtration membrane of 2.0kDa during ultrafiltration for the second time, preferred 1.8MPa.
(4) nanofiltration: the nanofiltration membrane that preferred molecular weight cut-off is 200Da sees through liquid to step (3) gained ultrafiltration and handles, and preferred operations pressure is 2.5MPa, collects the liquid that dams, and namely gets 1-S-GI (DNJ) nanofiltration concentrated solution.
(5) cation exchange resin layer is analysed: select for use strongly acidic cation-exchange that gained DNJ nanofiltration concentrated solution in (4) is separated, the DNJ nanofiltration concentrated solution of measuring 2 times of column volumes is gone up sample, with 1 times of column volume water washing, ammonia soln with 6 times of column volumes carries out wash-out by the 0.5mol/L flow velocity again, collect the ammoniacal liquor elutriant, and its deammoniation is concentrated.
(6) sephadex chromatography: further adopt Sephadex LH-20 resin to carry out purifying positively charged ion refined solution in (5), during the dress post, the control blade diameter length ratio is 1:20, selects 10% methanol solution to carry out wash-out with the 0.25mL/min flow velocity, and elution volume is 6 times of column volumes.Acquisition purity is 81.83% the smart sample of DNJ (Fig. 2 is the HPLC collection of illustrative plates of DNJ reference substance, and Fig. 3 is the HPLC collection of illustrative plates of the smart sample of DNJ) after the lyophilize.
Embodiment 2
During the ultrasonic water of step (1) is carried, use liquid ratio to be 10:1 liquid ratio (mL/g), preferred molecular weight cut-off is the ultra-filtration membrane of 20kDa during again with the first time ultrafiltration in the step (3), and working pressure is 0.8MPa, and feed temperature is 30 ℃; Preferred molecular weight cut-off is the ultra-filtration membrane of 1.0kDa during ultrafiltration for the second time, preferred 1.2MPa.Other operations are with embodiment 1.The smart sample purity of the DNJ that finally obtains is 83.56%.
Embodiment 3
During the ultrasonic water of step (1) is carried, use liquid ratio to be 20:1 liquid ratio (mL/g), preferred molecular weight cut-off is the ultra-filtration membrane of 8kDa during again with the first time ultrafiltration in the step (3), and working pressure is 0.8MPa, and feed temperature is 30 ℃; Preferred molecular weight cut-off is the ultra-filtration membrane of 1.0kDa during ultrafiltration for the second time, preferred 1.2MPa; The nanofiltration membrane that preferred molecular weight cut-off is 150Da sees through liquid to step (3) gained ultrafiltration and handles, and preferred operations pressure is 1.2MPa.Other operations are with embodiment 1.The smart sample purity of the DNJ that finally obtains is 85.24%
Embodiment 4
During step (5) cation exchange resin layer is analysed, the DNJ nanofiltration concentrated solution of measuring 4 times of column volumes is gone up sample, with 1 times of column volume water washing, ammonia soln with 8 times of column volumes carries out wash-out by the 0.5mol/L flow velocity again, collect the ammoniacal liquor elutriant, and its deammoniation is concentrated, other are operated with embodiment 1.The smart sample purity of the DNJ that finally obtains is 84.62%.
Embodiment 5
In step (6) sephadex chromatography, the blade diameter length ratio during with the dress post controls to 1:40, selects 50% methanol solution to carry out wash-out with the 0.5mL/min flow velocity, and elution volume is 8 times of column volumes, and other are operated with embodiment 1.Acquisition purity is 87.15% the smart sample of DNJ after the lyophilize.
Claims (7)
1. unite the method that membrane sepn and column chromatography technology prepare high purity 1-S-GI for one kind, it is characterized in that may further comprise the steps:
(1) ultrasonic auxiliary water is carried: with silkworm, the mulberry raw material pulverizing of drying, sieve, obtain coarse meal; Take by weighing an amount of coarse meal, press 40:1-10:1 liquid ratio (mL/g) and add distilled water, ultrasonication 30min under 70 ℃ of heating in water bath, supernatant liquor is collected in centrifuging, will precipitate by above-mentioned steps to repeat to extract once, merges supernatant liquor twice, namely gets extracting solution;
(2) pre-treatment: with the extracting solution of step (1) gained, handle by centrifugal, microfiltration membrane, obtain the ultrafiltration feeding liquid
(3) second ultrafiltration: with step (2) the gained ultrafiltration feeding liquid ultra-filtration membrane first time, in the ultra-filtration process, in the feed liquid bucket, add deionized water, collect ultrafiltration for the first time and see through liquid; To collect ultrafiltration for the second time and see through liquid through liquid with the littler ultrafiltration membrane treatment of molecular weight cut-off again;
(4) nanofiltration: see through liquid with nanofiltration membrane treatment step (3) gained ultrafiltration, collect the liquid that dams, namely get 1-S-GI nanofiltration concentrated solution;
(5) cation exchange resin layer is analysed: the 1-S-GI nanofiltration concentrated solution of gained in (4) is carried out the Zeo-karb separation, wash with water, use the ammonia soln wash-out again, collect the ammoniacal liquor elutriant, and its deammoniation is concentrated, obtain 1-S-GI crude samples;
(6) sephadex chromatography: further adopt Sephadex LH-20 resin to carry out purifying positively charged ion refined solution in (5), acquisition purity is the 1-S-GI product more than 80% after the lyophilize.
2. require a kind of method of uniting membrane sepn and column chromatography technology enrichment 1-S-GI of 1 according to authority, it is characterized in that raw material is mulberry leaf, ramulus mori, Cortex Mori, mulberry tree root and silkworm in the described step (1).
3. require a kind of method of uniting membrane sepn and column chromatography technology enrichment 1-S-GI of 1 according to authority, it is characterized in that microfiltration membrane is the hollow fiber type filter membrane of 0.5-5 μ m or the millipore filtration of 0.45-2.0 μ m in the described step (2).
4. require a kind of method of uniting membrane sepn and column chromatography technology enrichment 1-S-GI of 1 according to authority, it is characterized in that 2 used ultra-filtration membranes are poly-PES or compound rolled film in the described step (3), preferred molecular weight cut-off is the ultra-filtration membrane of 8.0-50kDa during ultrafiltration for the first time, working pressure is 0.5-1.2MPa, feed temperature is 25-40 ℃, and the deionized water volume of adding is 1-2 times of feeding liquid volume; Preferred molecular weight cut-off is the ultra-filtration membrane of 1.0-2.0kDa during ultrafiltration for the second time, and working pressure is 0.5-2.7MPa, preferred 1.2-1.8MPa.
5. require a kind of method of uniting membrane sepn and column chromatography technology enrichment 1-S-GI of 1 according to authority, it is characterized in that nanofiltration membrane is the rolling organic membrane in the described step (4), molecular weight cut-off is 100-200Da, and working pressure is 0.5-4.0MPa, preferred 1.2-2.5MPa.
6. require a kind of method of uniting membrane sepn and column chromatography technology enrichment 1-S-GI of 1 according to authority, it is characterized in that Zeo-karb is strongly acidic cation-exchange in the described step (5), can go up the DNJ nanofiltration concentrated solution that sample 2-4 doubly adorns post resin volume at every turn, the water washing volume is 1-2 times of column volume, and the ammoniacal liquor elution volume is 4-6 times of column volume.
7. require a kind of method of uniting membrane sepn and column chromatography technology enrichment 1-S-GI of 1 according to authority, it is characterized in that the dextrane gel resin is Sephadex LH-20 dextrane gel resin in the described step (6), the blade diameter length ratio of dress post is 1:20-1:40, select for use the methanol solution of 10-50% to carry out wash-out with the 0.25-0.5mL/min flow velocity, elution volume is 6-8 times of column volume.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103965096A (en) * | 2014-05-09 | 2014-08-06 | 湖南华诚生物资源有限公司 | 1-deoxynojirimycin preparing method suitable for industrial production |
| CN104007205A (en) * | 2014-06-13 | 2014-08-27 | 漳州片仔癀药业股份有限公司 | Method for detecting pharmaceutical preparation for treating xiaoke disease |
| CN106083697A (en) * | 2016-06-29 | 2016-11-09 | 新疆医科大学 | The extracting method of 1 deoxynojirimycin in medicine Mulberry |
| CN107162955A (en) * | 2017-06-30 | 2017-09-15 | 中国科学院过程工程研究所 | A kind of extracting method of DNJ |
| CN107721909A (en) * | 2017-11-02 | 2018-02-23 | 厦门福美科技有限公司 | DNJ, flavones, the method and system of polysaccharide are continuously extracted from moraceae plants |
| 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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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103965096A (en) * | 2014-05-09 | 2014-08-06 | 湖南华诚生物资源有限公司 | 1-deoxynojirimycin preparing method suitable for industrial production |
| 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 |
| CN104007205A (en) * | 2014-06-13 | 2014-08-27 | 漳州片仔癀药业股份有限公司 | Method for detecting pharmaceutical preparation for treating xiaoke disease |
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| CN107162955A (en) * | 2017-06-30 | 2017-09-15 | 中国科学院过程工程研究所 | A kind of extracting method of DNJ |
| CN107162955B (en) * | 2017-06-30 | 2020-02-18 | 中国科学院过程工程研究所 | Method for extracting deoxynojirimycin |
| CN107721909A (en) * | 2017-11-02 | 2018-02-23 | 厦门福美科技有限公司 | DNJ, flavones, the method and system of polysaccharide are continuously extracted from moraceae plants |
| 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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