CN102827137A - Purification method of Machilin A - Google Patents
Purification method of Machilin A Download PDFInfo
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- CN102827137A CN102827137A CN 201210371887 CN201210371887A CN102827137A CN 102827137 A CN102827137 A CN 102827137A CN 201210371887 CN201210371887 CN 201210371887 CN 201210371887 A CN201210371887 A CN 201210371887A CN 102827137 A CN102827137 A CN 102827137A
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- nanmu
- ethanol
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- reduced pressure
- lignanoid
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Abstract
The invention discloses a purification method of Machilin A, which comprises the following steps: (1) taking the raw material Machilus thungbergii dry bark, pulverizing, soaking with an enzyme solution, and carrying out enzymolysis in a water bath; (2) filtering the enzymolysis solution, taking the filter residue, extracting 5-10 times of ethanol water solution 2-3 times, merging the extracting solutions, and concentrating under reduced pressure to obtain an extractum; (3) passing the extractum through a macroporous adsorbent resin column, eluting with an alcohol-water mixed solution, and collecting the eluate; (4) recyling ethanol from the eluate under reduced pressure, passing through a silica gel column, carrying out ethyl acetate-ethanol gradient elution, carrying out tracking and monitoring by TLC (thin layer chromatography), collecting the Machilin A fraction, concentrating under reduced pressure, and carrying out freeze-drying to obtain a crude product; and (5) separating the crude product by high-speed counter-current chromatography, collecting the target component, concentrating and drying to obtain the high-purity Machilin A. The invention is simple to operate, has the advantages of high product yield and low cost, and is suitable for large-scale production.
Description
Technical field
The invention belongs to technical field of plant extraction, relate to a kind of method of from the dry bark of red nanmu, extracting the purifying nanmu A of lignanoid.
Background technology
Red nanmu
Machilus thunbergiiSieb.et Zucc. is a canella, mainly is distributed in provinces and regions such as China Shandong, Jiangsu, Zhejiang, Anhui, Jiangxi, Fujian, Taiwan, Sichuan, Guangdong, Guangxi.Modern pharmacological research shows that the A of nanmu lignanoid has immunosuppressive activity, and mitogen (con A) inductive human peripheral liquid lymphocytosis is had extremely strong restraining effect, its IC
50Be 0.02 μ g/ml, suitable with Ultracortene H; Also have cytotoxic activity in addition, human-like leukemia cell line HL-60 and Molt-4 cell are had cytotoxic activity, its IC
50(ng/ml) be respectively 26 and 54.
The A of nanmu lignanoid (Machilin A), molecular formula is C
20H
22O
4, molecular weight is 326.39, is colourless needle, is from the isolated a kind of Lignanoids compounds of the bark of red nanmu.
Summary of the invention
The method of purification that the purpose of this invention is to provide a kind of nanmu A of lignanoid, this method are carried the rate height, cost is low, are fit to large-scale production.
The objective of the invention is to realize through following technical scheme:
The method of purification of a kind of nanmu A of lignanoid is characterized in that may further comprise the steps:
(1) enzymolysis: get the dry bark of the red nanmu of raw material,, soak with enzyme solution through pulverization process, water enzyme digestion 2-5 hour, and fully stir;
(2) extract: above-mentioned enzymolysis solution is filtered, get filter residue and extract 2-3 time with the aqueous ethanolic solution of 5-6 times of medicinal material weight, united extraction liquid, concentrating under reduced pressure gets medicinal extract;
(3) enrichment: medicinal extract is crossed macroporous adsorptive resins, and pure water elution is collected elutriant;
(4) purifying: with above-mentioned elutriant decompression recycling ethanol, last silicagel column (the silica gel granularity is the 100-200 order), the ethyl acetate-ethanol gradient elution, the TLC tracking monitor is collected the nanmu A of lignanoid flow point, concentrating under reduced pressure, lyophilize gets bullion;
(5) high speed adverse current chromatogram separates: bullion adopts high speed adverse current chromatogram to separate, and is solvent systems with chloroform-methanol-water, and ratio is (1-5): (5-9): (3-7), collect target component, concentrate drying promptly gets the high purity nanmu A of lignanoid.
Described in the step (1) in the optional cellulase of enzyme, LSD and the polygalacturonase one or more, enzyme dosage are 2-3.5 ‰, and pH is 4-4.6, and hydrolysis temperature is 45-50 ℃.
Described in the step (2) in the aqueous ethanolic solution alcoholic acid concentration of volume percent be 80-90%.
Macroporous resin described in the step (3) is selected from a kind of in AB-8, XAD-16, XDA-8 or the HZ806 type.
Pure water elution described in the step (3) is 3-4BV10%-20% aqueous ethanolic solution → 4-6BV80%-90% aqueous ethanolic solution.
The ethyl acetate-ethanol volume ratio is 3-10:1 described in the step (4).
The present invention adopts enzymolysis and extraction, and effective constituent stripping has more easily improved extraction yield; Adopt macroporous resin and silicagel column rough segmentation, bigger than extraction process treatment capacity, content is high, operate more easily; The employing high speed adverse current chromatogram separates, and is simple to operate, and preparation cycle is short, and the product free of losses can obtain the high-load nanmu A of lignanoid, and product purity is higher.
Embodiment:
Embodiment 1:
Get the dry bark of red nanmu and pulverized 60 mesh sieves, take by weighing 5kg, the cellulase aqueous solution (every kg crude drug adds the 2.2g cellulase) that adds 15LpH4.2 soaks half a hour, then 42 ℃ of following water enzyme digestions 3 hours; And fully stir, filter, get filter residue with the 80% alcohol heating reflux extraction of 5 times of medicinal material weight 1 hour, extract 3 times; United extraction liquid, concentrating under reduced pressure gets medicinal extract, and medicinal extract is heated water-dispersion, adds the XAD-16 macroporous adsorptive resins; Get 3BV10% ethanol elution impurity earlier, get 4BV80% ethanol elution effective constituent again, decompression recycling ethanol; Last silica gel column chromatography (the silica gel granularity is 100 orders) is pressed 10:1,5:1,3:1 gradient elution, TLC tracking monitor with ethyl acetate-ethanol; Collect the nanmu A of lignanoid flow point, concentrating under reduced pressure, lyophilize gets the A of nanmu lignanoid bullion; Get chloroform, methyl alcohol, water, 1:5:3 thorough mixing in separating funnel leaves standstill in proportion, takes off and fills with the high speed adverse current chromatogram post mutually, opens main frame; Rotating speed 900rpm is set, pumps into and do moving phase mutually, set up running balance after, the adjustment flow velocity is 3ml/min; Dissolve crude extract with moving phase, by the sampling valve sample introduction, UV-detector detects, and collects target component; Preparation merges flow point continuously, gets the A1.3g of nanmu lignanoid, detects content 98.2% through HPLC.
Embodiment 2:
Get the dry bark of red nanmu and pulverized 60 mesh sieves, take by weighing 5kg, the cellulase aqueous solution (every kg crude drug adds the 3.1g cellulase) that adds 12LpH4.2 soaks half a hour, then 44 ℃ of following water enzyme digestions 2 hours; And fully stir, filter, get filter residue with the 90% alcohol heating reflux extraction of 6 times of medicinal material weight 1 hour, extract 2 times; United extraction liquid, concentrating under reduced pressure gets medicinal extract, and medicinal extract is heated water-dispersion, adds the AB-8 macroporous adsorptive resins; Get 4BV20% ethanol elution impurity earlier, get 4BV90% ethanol elution effective constituent again, decompression recycling ethanol; Last silica gel column chromatography (the silica gel granularity is 200 orders) is pressed 10:1,5:1,3:1 gradient elution, TLC tracking monitor with ethyl acetate-ethanol; Collect the nanmu A of lignanoid flow point, concentrating under reduced pressure, lyophilize must get the A of nanmu lignanoid bullion; Get chloroform, methyl alcohol, water, 5:9:7 thorough mixing in separating funnel leaves standstill in proportion, takes off and fills with the high speed adverse current chromatogram post mutually, opens main frame; Rotating speed 850rpm is set, pumps into and do moving phase mutually, set up running balance after, the adjustment flow velocity is 3ml/min; Dissolve crude extract with moving phase, by the sampling valve sample introduction, UV-detector detects, and collects target component; Preparation merges flow point continuously, gets the A1.6g of nanmu lignanoid, detects content 98.1% through HPLC.
Embodiment 3:
Get the dry bark of red nanmu and pulverized 60 mesh sieves, take by weighing 5kg, the cellulase aqueous solution (every kg crude drug adds 2g cellulase and polygalacturonase mixing prozyme, and the mass ratio of cellulase and polygalacturonase is 1:1) that adds 15LpH4.2 soaks half a hour; Then 50 ℃ of following water enzyme digestions 5 hours, and fully stir, filter; Get filter residue and extracted 1 hour, extract united extraction liquid 2 times with 90% alcohol heating reflux of 5 times of medicinal material weight; Concentrating under reduced pressure gets medicinal extract, and medicinal extract is heated water-dispersion, adds the XDA-8 macroporous adsorptive resins; Get 3BV15% ethanol elution impurity earlier, get 6BV90% ethanol elution effective constituent again, decompression recycling ethanol; Last silica gel column chromatography (the silica gel granularity is 200 orders) is pressed 10:1,5:1,3:1 gradient elution, TLC tracking monitor with ethyl acetate-ethanol; Collect the nanmu A of lignanoid flow point, concentrating under reduced pressure, lyophilize must get the A of nanmu lignanoid bullion; Get chloroform, methyl alcohol, water, 4:7:3 thorough mixing in separating funnel leaves standstill in proportion, takes off and fills with the high speed adverse current chromatogram post mutually, opens main frame; Rotating speed 800rpm is set, pumps into and do moving phase mutually, set up running balance after, the adjustment flow velocity is 3ml/min; Dissolve crude extract with moving phase, by the sampling valve sample introduction, UV-detector detects, and collects target component; Preparation merges flow point continuously, gets the A1.5g of nanmu lignanoid, detects content 98.3% through HPLC.
Embodiment 4:
Get the dry bark of red nanmu and pulverized 60 mesh sieves, take by weighing 5kg, the cellulase aqueous solution (every kg crude drug adds the 3.5g polygalacturonase) that adds 12LpH4.6 soaks half a hour, then 45 ℃ of following water enzyme digestions 4 hours; And fully stir, filter, get filter residue with the 80% alcohol heating reflux extraction of 10 times of medicinal material weight 1 hour, extract 3 times; United extraction liquid, concentrating under reduced pressure gets medicinal extract, and medicinal extract is heated water-dispersion, adds the HZ806 macroporous adsorptive resins; Get 4BV15% ethanol elution impurity earlier, get 6BV85% ethanol elution effective constituent again, decompression recycling ethanol; Last silica gel column chromatography (the silica gel granularity is 100 orders) is pressed 10:1,5:1,3:1 gradient elution, TLC tracking monitor with ethyl acetate-ethanol; Collect the nanmu A of lignanoid flow point, concentrating under reduced pressure, lyophilize must get the A of nanmu lignanoid bullion; Get chloroform, methyl alcohol, water, 3:7:6 thorough mixing in separating funnel leaves standstill in proportion, takes off and fills with the high speed adverse current chromatogram post mutually, opens main frame; Rotating speed 900rpm is set, pumps into and do moving phase mutually, set up running balance after, the adjustment flow velocity is 3ml/min; Dissolve crude extract with moving phase, by the sampling valve sample introduction, UV-detector detects, and collects target component; Preparation merges flow point continuously, gets the A1.5g of nanmu lignanoid, detects content 98.7% through HPLC.
Embodiment 5:
Get the dry bark of red nanmu and pulverized 60 mesh sieves, take by weighing 5kg, the cellulase aqueous solution (every kg crude drug adds the mixing prozyme of 2.6g LSD and polygalacturonase, and the mass ratio of LSD and polygalacturonase is 1:3) that adds 13LpH4 soaks half a hour; Then 48 ℃ of following water enzyme digestions 2 hours, and fully stir, filter; Get filter residue and extracted 1 hour, extract united extraction liquid 2 times with 85% alcohol heating reflux of 8 times of medicinal material weight; Concentrating under reduced pressure gets medicinal extract, and medicinal extract is heated water-dispersion, adds the AB-8 macroporous adsorptive resins; Get 3BV20% ethanol elution impurity earlier, get 5BV80% ethanol elution effective constituent again, decompression recycling ethanol; Last silica gel column chromatography (the silica gel granularity is 200 orders) is pressed 10:1,5:1,3:1 gradient elution, TLC tracking monitor with ethyl acetate-ethanol; Collect the nanmu A of lignanoid flow point, concentrating under reduced pressure, lyophilize must get the A of nanmu lignanoid bullion; Get chloroform, methyl alcohol, water, 3:9:7 thorough mixing in separating funnel leaves standstill in proportion, takes off and fills with the high speed adverse current chromatogram post mutually, opens main frame; Rotating speed 900rpm is set, pumps into and do moving phase mutually, set up running balance after, the adjustment flow velocity is 3ml/min; Dissolve crude extract with moving phase, by the sampling valve sample introduction, UV-detector detects, and collects target component; Preparation merges flow point continuously, gets the A1.3g of nanmu lignanoid, detects content 98.5% through HPLC.
Embodiment 6:
Get the dry bark of red nanmu and pulverized 60 mesh sieves, take by weighing 5kg, the cellulase aqueous solution (every kg crude drug adds the 3.3g cellulase) that adds 12LpH4.5 soaks half a hour, then 40 ℃ of following water enzyme digestions 5 hours; And fully stir, filter, get filter residue with the 85% alcohol heating reflux extraction of 8 times of medicinal material weight 1 hour, extract 3 times; United extraction liquid, concentrating under reduced pressure gets medicinal extract, and medicinal extract is heated water-dispersion, adds the HZ806 macroporous adsorptive resins; Get 4BV20% ethanol elution impurity earlier, get 5BV85% ethanol elution effective constituent again, decompression recycling ethanol; Last silica gel column chromatography (the silica gel granularity is 100 orders) is pressed 10:1,5:1,3:1 gradient elution, TLC tracking monitor with ethyl acetate-ethanol; Collect the nanmu A of lignanoid flow point, concentrating under reduced pressure, lyophilize must get the A of nanmu lignanoid bullion; Get chloroform, methyl alcohol, water, 2:5:3 thorough mixing in separating funnel leaves standstill in proportion, takes off and fills with the high speed adverse current chromatogram post mutually, opens main frame; Rotating speed 850rpm is set, pumps into and do moving phase mutually, set up running balance after, the adjustment flow velocity is 3ml/min; Dissolve crude extract with moving phase, by the sampling valve sample introduction, UV-detector detects, and collects target component; Preparation merges flow point continuously, gets the A1.7g of nanmu lignanoid, detects content 98.6% through HPLC.
Claims (6)
1. the method for purification of the A of nanmu lignanoid is characterized in that may further comprise the steps:
(1) enzymolysis: get the dry bark of the red nanmu of raw material,, soak with enzyme solution through pulverization process, water enzyme digestion 2-5 hour, and fully stir;
(2) extract: above-mentioned enzymolysis solution is filtered, get filter residue and extract 2-3 time with the aqueous ethanolic solution of 5-6 times of medicinal material weight, united extraction liquid, concentrating under reduced pressure gets medicinal extract;
(3) enrichment: medicinal extract is crossed macroporous adsorptive resins, and pure water elution is collected elutriant;
(4) purifying: with above-mentioned elutriant decompression recycling ethanol, last silicagel column (the silica gel granularity is the 100-200 order), the ethyl acetate-ethanol gradient elution, the TLC tracking monitor is collected the nanmu A of lignanoid flow point, concentrating under reduced pressure, lyophilize gets bullion;
(5) high speed adverse current chromatogram separates: bullion adopts high speed adverse current chromatogram to separate, and is solvent systems with chloroform-methanol-water, and ratio is (1-5): (5-9): (3-7), collect target component, concentrate drying promptly gets the high purity nanmu A of lignanoid.
2. method of purification according to claim 1 is characterized in that: one or more described in the step (1) in the optional cellulase of enzyme, LSD and the polygalacturonase, enzyme dosage are 2-3.5 ‰, and pH is 4-4.6, and hydrolysis temperature is 45-50 ℃.
3. method of purification according to claim 1 is characterized in that: described in the step (2) in the aqueous ethanolic solution alcoholic acid concentration of volume percent be 80-90%.
4. method of purification according to claim 1 is characterized in that: macroporous resin described in the step (3) is selected from a kind of in AB-8, XAD-16, XDA-8 or the HZ806 type.
5. method of purification according to claim 1 is characterized in that: pure water elution described in the step (3) is 3-4BV10%-20% aqueous ethanolic solution → 4-6BV80%-90% aqueous ethanolic solution.
6. method of purification according to claim 1 is characterized in that: the ethyl acetate-ethanol volume ratio is 3-10:1 described in the step (4).
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|---|---|---|---|
| CN 201210371887 CN102827137A (en) | 2012-09-29 | 2012-09-29 | Purification method of Machilin A |
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|---|---|---|---|
| CN 201210371887 CN102827137A (en) | 2012-09-29 | 2012-09-29 | Purification method of Machilin A |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105249531A (en) * | 2015-08-06 | 2016-01-20 | 云南中烟工业有限责任公司 | Method for preparing phoebe zhennan extracts and application thereof |
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2012
- 2012-09-29 CN CN 201210371887 patent/CN102827137A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105249531A (en) * | 2015-08-06 | 2016-01-20 | 云南中烟工业有限责任公司 | Method for preparing phoebe zhennan extracts and application thereof |
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Application publication date: 20121219 |