CN103408627A - Method for extracting and purifying euonymin A - Google Patents
Method for extracting and purifying euonymin A Download PDFInfo
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- CN103408627A CN103408627A CN201310374054XA CN201310374054A CN103408627A CN 103408627 A CN103408627 A CN 103408627A CN 201310374054X A CN201310374054X A CN 201310374054XA CN 201310374054 A CN201310374054 A CN 201310374054A CN 103408627 A CN103408627 A CN 103408627A
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- evonymin
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Abstract
The invention relates to a method for extracting and purifying euonymin A, and belongs to the technical field of natural plant extraction. The method comprises the following steps of: (1) crushing the dried bark of Euonymus sieboldianus, and adding a biological enzyme to carry out enzymolysis for 1-3 days; (2) carrying out microwave extraction on enzymolysis raw materials by using an alcohol solution, and recovering ethanol from an extracting solution; (3) dissolving extractum by adding hot water, absorbing by adopting macroporous resin columns, eluting by adopting water-ethanol, carrying out ultrafiltration on eluent by using an ultrafiltration membrane, concentrating by adopting a nanofiltration membrane, and drying a concentrated solution to obtain a concentrate; (4) carrying out chromatographic separation on the concentrate to obtain the high-content euonymin A. The method disclosed by the invention has the advantages of less sample loss, good separation effect and low cost and can be used for obtaining the euonymin A with high content and good quality.
Description
Technical field
The invention belongs to the extracted form natural plant technical field, relate to a kind of method of extracting purifying evonymin A.
Background technology
Evonymin A(Euonymoside A), be to separate a kind of cardiac glycoside compounds obtained, molecular formula: C from the bark of western Bo Shi winged euonymus Euonymus sieboldianus Bl., extracting
35H
54O
14, molecular weight: 698.8, molecular structural formula is:
Evonymin A has cytotoxic activity.IC50 to people's lung cancer (A-549) and human ovarian cancer (SK-OV-3) is respectively 0.06 μ g/mL and 0.4 μ g/mL.
In domestic prior art, there is not yet the Patents report for preparing evonymin A production technique.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of from the bark of western Bo Shi winged euonymus, extracting the method for purifying evonymin A.
The objective of the invention is to be achieved through the following technical solutions:
A kind of method of extracting purifying evonymin A is characterized in that comprising the following steps:
1) the dry bark of getting western Bo Shi winged euonymus is pulverized, and adds biological enzyme enzymolysis 1-3 days, obtains the enzymolysis raw material;
2) with alcohol solution, the enzymolysis raw material is carried out to microwave extraction, extracting solution reclaims ethanol;
3) the medicinal extract heating water dissolves, upper macroporous resin column absorption, and the ultra-filtration membrane ultrafiltration of water-ethanol wash-out, elutriant, nanofiltration membrane is concentrated, the dry enriched material that obtains of concentrated solution;
4) enriched material is carried out to chromatographic separation, obtain high-content evonymin A.
Bark to western Bo Shi winged euonymus in described step 1) carries out pulverization process, and controlling particle diameter is the 40-60 order.
Any in described step 1) in the optional polygalacturonase of biological enzyme, cellulase and amylase, consumption are the 0.1-0.8% of raw material weight.
Described step 2) in, alcohol solution is the 70-90% aqueous ethanolic solution, and the solid-liquid volume ratio is 1:8-1:13.
Described step 2) in, microwave power is 800-1000W, extracts 2-3 time, each 20-40min.
A kind of in the optional AB-8 of macroporous resin, D101, HPD100 and HZ830 in described step 3).
In described step 3), eluent is 3-5BV water → 3-5BV20-40% ethanol → 5-7BV75-85% ethanol.
In described step 3), ultra-filtration membrane is the hollow composite membrane of molecular weight cut-off 3000-10000, and nanofiltration membrane is the hollow composite membrane of molecular weight cut-off 300-500, and intake pressure is controlled 0.5-0.9Mpa.
In described step 4), chromatographic separation adopts high speed adverse current chromatogram to carry out, its two-phase solvent system is chloroform-methanol-water, volume ratio is (11-15): (5-8): (4-9), described high-speed counter-current chromatograph rotating speed is 850-1050rpm, and flow rate of mobile phase is 3-5ml/min.
To the west of advantage of the present invention was, the bark of Bo Shi winged euonymus was raw material, and cost is low; Adopt microwave extraction, extraction time is short, extraction efficiency is high, and the solvent of employing is the organic solvent of easy recovery, nontoxic or low toxicity; Adopt the macroporous resin column chromatography enriching and purifying, can remove plurality of impurities, alleviated the workload of back operation; Adopt the high speed adverse current chromatogram good separating effect, product purity is high.
Embodiment:
Embodiment 1:
The tree bark powder of getting western Bo Shi winged euonymus is broken to 60 orders, taking 500g adds the sour water of 2 times of amount pH5 to mix wet, add the 0.5g cellulase, normal temperature enzymolysis 2 days, the enzymolysis raw material drops into the microwave extracting tank, add 4kg70% ethanolic soln microwave extraction 40min, power 800W, emit extracting solution, add again 70% extraction using alcohol 2 times, merge No. three times extracting solution, with Rotary Evaporators, reclaim ethanol to a small amount of, add AB-8 type macroporous resin column chromatography (post blade diameter length ratio 1:12), use successively 5BV water → 3BV40% ethanol → 5BV75% ethanol elution, the high performance liquid chromatography monitoring, collect flow point, elutriant adds the hollow composite membrane ultra-filtration membrane ultrafiltration of molecular weight cut-off 5000, see through liquid and add again molecular weight cut-off 300 hollow composite nanometer filter membrane concentration, intake pressure is controlled 0.7Mpa, obtain concentrated solution, the concentrated solution cryodrying is obtained to enriched material, getting chloroform-methanol-water (11:5:7) is placed in separating funnel and mixes, get and be mutually stationary phase, lower is moving phase mutually, gets above-mentioned enriched material to be dissolved in moving phase standby, open high-speed counter-current chromatograph, the setting flow rate of mobile phase is 3.0ml/min, opens simultaneously main motor, when moving phase flows out, starts sample introduction, rotating speed is controlled to be 950rpm, by detector, collect evonymin A flow point, the concentrated and dry evonymin A that obtains, the high performance liquid phase detection level is 90.6%.
Embodiment 2:
The tree bark powder of getting western Bo Shi winged euonymus is broken to 80 orders, taking 500g adds the sour water of 2.5 times of amount pH4 to mix wet, add the 4g cellulase, normal temperature enzymolysis 3 days, the enzymolysis raw material drops into the microwave extracting tank, add 6.5kg90% ethanolic soln microwave extraction 30min, power 950W, emit extracting solution, add again 90% extraction using alcohol 1 time, merge extracted twice liquid, with Rotary Evaporators, reclaim ethanol to a small amount of, add HZ830 type macroporous resin column chromatography (post blade diameter length ratio 1:12), use successively 4BV water → 5BV30% ethanol → 7BV85% ethanol elution, the high performance liquid chromatography monitoring, collect flow point, elutriant adds the hollow composite membrane ultra-filtration membrane ultrafiltration of molecular weight cut-off 5000, see through liquid and add again molecular weight cut-off 500 hollow composite nanometer filter membrane concentration, intake pressure is controlled 0.8Mpa, obtain concentrated solution, the concentrated solution cryodrying is obtained to enriched material, getting chloroform-methanol-water (12:6:7) is placed in separating funnel and mixes, get and be mutually stationary phase, lower is moving phase mutually, gets above-mentioned enriched material to be dissolved in moving phase standby, open high-speed counter-current chromatograph, the setting flow rate of mobile phase is 3.5ml/min, opens simultaneously main motor, when moving phase flows out, starts sample introduction, rotating speed is controlled to be 950rpm, by detector, collect evonymin A flow point, the concentrated and dry evonymin A that obtains, the high performance liquid phase detection level is 92.1%.
Embodiment 3:
The tree bark powder of getting western Bo Shi winged euonymus is broken to 60 orders, taking 1kg adds the sour water of 1.5 times of amount pH3.5 to mix wet, add the 5.4g polygalacturonase, normal temperature enzymolysis 2.5 days, the enzymolysis raw material drops into the microwave extracting tank, add 12kg75% ethanolic soln microwave extraction 20min, power 1000W, emit extracting solution, add again 75% extraction using alcohol 2 times, merge No. three times extracting solution, with Rotary Evaporators, reclaim ethanol to a small amount of, add D101 type macroporous resin column chromatography (post blade diameter length ratio 1:12), use successively 5BV water → 4BV20% ethanol → 7BV75% ethanol elution, the high performance liquid chromatography monitoring, collect flow point, elutriant adds the hollow composite membrane ultra-filtration membrane ultrafiltration of molecular weight cut-off 6000, see through liquid and add again molecular weight cut-off 500 hollow composite nanometer filter membrane concentration, intake pressure is controlled 0.9Mpa, obtain concentrated solution, the concentrated solution cryodrying is obtained to enriched material, getting chloroform-methanol-water (15:5:9) is placed in separating funnel and mixes, get and be mutually stationary phase, lower is moving phase mutually, gets above-mentioned enriched material to be dissolved in moving phase standby, open high-speed counter-current chromatograph, the setting flow rate of mobile phase is 3.0ml/min, opens simultaneously main motor, when moving phase flows out, starts sample introduction, rotating speed is controlled to be 1000rpm, by detector, collect evonymin A flow point, the concentrated and dry evonymin A that obtains, the high performance liquid phase detection level is 91.5%.
Embodiment 4:
The tree bark powder of getting western Bo Shi winged euonymus is broken to 40 orders, taking 1.5kg adds the sour water of 3 times of amount pH3 to mix wet, add the 10.5g polygalacturonase, normal temperature enzymolysis 2 days, the enzymolysis raw material drops into the microwave extracting tank, add 17kg80% ethanolic soln microwave extraction 35min, power 800W, emit extracting solution, add again 80% extraction using alcohol 2 times, merge No. three times extracting solution, with Rotary Evaporators, reclaim ethanol to a small amount of, add HPD100 type macroporous resin column chromatography (post blade diameter length ratio 1:12), use successively 3BV water → 4BV30% ethanol → 6BV80% ethanol elution, the high performance liquid chromatography monitoring, collect flow point, elutriant adds the hollow composite membrane ultra-filtration membrane ultrafiltration of holding back molecule 7000, see through liquid and add again molecular weight cut-off 500 hollow composite nanometer filter membrane concentration, intake pressure is controlled 0.9Mpa, obtain concentrated solution, the concentrated solution cryodrying is obtained to enriched material, getting chloroform-methanol-water (11:8:4) is placed in separating funnel and mixes, get and be mutually stationary phase, lower is moving phase mutually, gets above-mentioned enriched material to be dissolved in moving phase standby, open high-speed counter-current chromatograph, the setting flow rate of mobile phase is 5ml/min, opens simultaneously main motor, when moving phase flows out, starts sample introduction, rotating speed is controlled to be 850rpm, by detector, collect evonymin A flow point, the concentrated and dry evonymin A that obtains, the high performance liquid phase detection level is 92.5%.
Embodiment 5:
The tree bark powder of getting western Bo Shi winged euonymus is broken to 80 orders, taking 2kg adds the sour water of 3 times of amount pH3.5 to mix wet, add 14g amylase, normal temperature enzymolysis 3 days, the enzymolysis raw material drops into the microwave extracting tank, add 24kg75% ethanolic soln microwave extraction 30min, power 900W, emit extracting solution, add again 75% extraction using alcohol 1 time, merge extracted twice liquid, with Rotary Evaporators, reclaim ethanol to a small amount of, add HPD100 type macroporous resin column chromatography (post blade diameter length ratio 1:12), use successively 4BV water → 5BV35% ethanol → 7BV85% ethanol elution, the high performance liquid chromatography monitoring, collect flow point, elutriant adds the hollow composite membrane ultra-filtration membrane ultrafiltration of molecular weight cut-off 8000, see through liquid and add again molecular weight cut-off 300 hollow composite nanometer filter membrane concentration, intake pressure is controlled 0.5Mpa, obtain concentrated solution, the concentrated solution cryodrying is obtained to enriched material, getting chloroform-methanol-water (12:5:6) is placed in separating funnel and mixes, get and be mutually stationary phase, lower is moving phase mutually, gets above-mentioned enriched material to be dissolved in moving phase standby, open high-speed counter-current chromatograph, the setting flow rate of mobile phase is 4.5ml/min, opens simultaneously main motor, when moving phase flows out, starts sample introduction, rotating speed is controlled to be 1050rpm, by detector, collect evonymin A flow point, the concentrated and dry evonymin A that obtains, the high performance liquid phase detection level is 91.7%.
Claims (10)
1. method of extracting purifying evonymin A is characterized in that comprising the following steps:
1) the dry bark of getting western Bo Shi winged euonymus is pulverized, and adds biological enzyme enzymolysis 1-3 days, obtains the enzymolysis raw material;
2) with alcohol solution, the enzymolysis raw material is carried out to microwave extraction, extracting solution reclaims ethanol;
3) the medicinal extract heating water dissolves, upper macroporous resin column absorption, and the ultra-filtration membrane ultrafiltration of water-ethanol wash-out, elutriant, nanofiltration membrane is concentrated, the dry enriched material that obtains of concentrated solution;
4) enriched material is carried out to chromatographic separation, obtain high-content evonymin A.
2. the method for extraction purifying evonymin A as claimed in claim 1, it is characterized in that: the bark to western Bo Shi winged euonymus in described step 1) carries out pulverization process, and controlling particle diameter is the 40-60 order.
3. the method for extraction purifying evonymin A as claimed in claim 1, it is characterized in that: any in described step 1) in the optional polygalacturonase of biological enzyme, cellulase and amylase, consumption are the 0.1-0.8% of raw material weight.
4. the method for extraction purifying evonymin A as claimed in claim 1 is characterized in that: described step 2), alcohol solution is the 70-90% aqueous ethanolic solution, and the solid-liquid volume ratio is 1:8-1:13.
5. the method for extraction purifying evonymin A as claimed in claim 1 is characterized in that: described step 2), microwave power is 800-1000W, extracts 2-3 time, at every turn 20-40min.
6. the method for extraction purifying evonymin A as claimed in claim 1, is characterized in that: a kind of in the optional AB-8 of macroporous resin, D101, HPD100 and HZ830 in described step 3).
7. the method for extraction purifying evonymin A as claimed in claim 1, it is characterized in that: in described step 3), eluent is 3-5BV water → 3-5BV20-40% ethanol → 5-7BV75-85% ethanol.
8. the method for extraction purifying evonymin A as claimed in claim 1, it is characterized in that: in described step 3), ultra-filtration membrane is the hollow composite membrane of molecular weight cut-off 3000-10000, nanofiltration membrane is the hollow composite membrane of molecular weight cut-off 300-500, and intake pressure is controlled 0.5-0.9Mpa.
9. the method for extraction purifying evonymin A as claimed in claim 1, it is characterized in that: in described step 4), chromatographic separation adopts high speed adverse current chromatogram to carry out, its two-phase solvent system is chloroform-methanol-water, and volume ratio is (11-15): (5-8): (4-9).
10. the method for extraction purifying evonymin A as claimed in claim 9, it is characterized in that: described high-speed counter-current chromatograph rotating speed is 850-1050rpm, flow rate of mobile phase is 3-5ml/min.
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| CN201310374054XA CN103408627A (en) | 2013-08-26 | 2013-08-26 | Method for extracting and purifying euonymin A |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103965284A (en) * | 2014-05-07 | 2014-08-06 | 吉林农业大学 | Spindle tree fruit saponin and preparing method and application thereof |
| CN105901021A (en) * | 2016-06-02 | 2016-08-31 | 黄山力神日用品有限公司 | Preparation method of microcapsule liquid mosquito-repellent incense added with Euonymus alatus |
| CN107519232A (en) * | 2017-10-16 | 2017-12-29 | 铜仁职业技术学院 | One kind extraction Gueldenstaedtia verna extractive of general flavone and preparation method thereof |
-
2013
- 2013-08-26 CN CN201310374054XA patent/CN103408627A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103965284A (en) * | 2014-05-07 | 2014-08-06 | 吉林农业大学 | Spindle tree fruit saponin and preparing method and application thereof |
| CN105901021A (en) * | 2016-06-02 | 2016-08-31 | 黄山力神日用品有限公司 | Preparation method of microcapsule liquid mosquito-repellent incense added with Euonymus alatus |
| CN107519232A (en) * | 2017-10-16 | 2017-12-29 | 铜仁职业技术学院 | One kind extraction Gueldenstaedtia verna extractive of general flavone and preparation method thereof |
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