CN103242333A - Method for purifying Chapa picrasin - Google Patents
Method for purifying Chapa picrasin Download PDFInfo
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- CN103242333A CN103242333A CN 201310196135 CN201310196135A CN103242333A CN 103242333 A CN103242333 A CN 103242333A CN 201310196135 CN201310196135 CN 201310196135 CN 201310196135 A CN201310196135 A CN 201310196135A CN 103242333 A CN103242333 A CN 103242333A
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- picrasmin
- chapa
- handkerchief
- extract
- picrasin
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Abstract
The invention discloses a method for purifying Chapa picrasin. The method comprises the following steps of: (1) taking raw materials to twist dry barks of a castela; pulverizing; soaking with an enzyme solution; and performing water-bath enzymolysis; (2) filtering enzymolysis raw materials: extracting filtering slag for 2-3 times by using an ethanol water solution of which the weight is 5-10 times that of the filter slag, merging extracting liquids, and reducing pressure and concentrating to obtain an extractum; (3) performing macroporous resin chromatography on the extractum, eluting with an alcohol-water solution and collecting eluant; (4) performing pressure reduction on the eluant and recovering ethanol, spreading the eluant on a silicagel column, eluting with ethyl acetate-methanol in a gradient mode, tracking and monitoring in a thin-layer chromatography (TLC) process, collecting Chapa picrasin fractions, and concentrating and drying to obtain high-purity Chapa picrasin. The method is easy to operate, and the obtained product is high in yield, low in cost, and suitable for scale production.
Description
Technical field
The invention belongs to technical field of plant extraction, relate to a kind of method that purifying is looked into the handkerchief picrasmin of from the dry bark of twisted fort tree, extracting.
Background technology
Look into handkerchief picrasmin (Chaparrin
)Be a kind of quassinoids compound, be mainly derived from the twisted fort tree of Simarubaceae (Simaroubaceae)
Castela tortuosaThe bark of Liemb.Modern pharmacological research shows, looks into the handkerchief picrasmin and has antitumous effect, and the T/C to P388 when 40mg/kg is 145%; Have cytotoxic activity, external ED50 to nasopharyngeal carcinoma 9KB cell is 6.5 μ g/mL; Having the antimalarial effect, is 0.180 μ g/mL (the reference substance chloroquine is 0.21 μ g/mL) to the IC50 of plasmodium falciparum vitro culture, is 0.25 μ g/mL to the IC50 of D6 strain, and the W2 strain is 0.35 μ g/mL; Have anti-amoeba activity, the amoeba of vitro culture is being added this product 100 μ M, 100% the inhibiting rate of having an appointment after 48 hours.
Looking into handkerchief picrasmin molecular formula is C
20H
28O
7, molecular weight is 380.44, structural formula is:
By literature search, still there is not the extracting and purifying method report that the high purity of being applicable to is looked into the handkerchief picrasmin.
Summary of the invention
The purpose of this invention is to provide a kind of method of purification of looking into the handkerchief picrasmin, this method is carried the rate height, cost is low, is fit to large-scale production.
The objective of the invention is to be achieved through the following technical solutions:
A kind of method of purification of looking into the handkerchief picrasmin is characterized in that may further comprise the steps:
(1) enzymolysis: get the dry bark of the twisted fort tree of raw material, through pulverization process, soak water enzyme digestion 2-5 hour with enzyme solution;
(2) extract: above-mentioned enzymolysis solution is filtered, get filter residue and extract 2-3 time with the aqueous ethanolic solution of 5-8 times of material weight, united extraction liquid, concentrating under reduced pressure gets medicinal extract;
(3) enrichment: medicinal extract is crossed macroporous resin column, and after absorption was finished, pure water elution was collected elutriant;
(4) purifying: with above-mentioned elutriant decompression recycling ethanol, last silicagel column (the silica gel granularity is the 100-200 order), ethyl acetate-methyl alcohol gradient elution, the TLC tracking monitor is collected and is looked into handkerchief picrasmin flow point, concentrating under reduced pressure, lyophilize gets crude product;
(5) high speed adverse current chromatogram separates: crude product adopts high speed adverse current chromatogram to separate, and is solvent systems with chloroform-methanol-water, and ratio is (2-5): (5-9): (4-7), collect target component, concentrate drying namely gets high purity and looks into the handkerchief picrasmin.
Described in the step (1) in the optional cellulase of enzyme, dextranase and the polygalacturonase one or more, enzyme dosage are 2.5-3.5 ‰, and pH is 4-4.6, and hydrolysis temperature is 45-50 ℃.
Described in the step (2) in the aqueous ethanolic solution concentration of volume percent of ethanol be 80-95%.
Macroporous resin described in the step (3) is selected from a kind of in AB-8, XDA-8, XAD-16 or the HZ806 type.
Pure water elution described in the step (3) is 3-4BV20%-30% aqueous ethanolic solution → 4-6BV80%-90% aqueous ethanolic solution.
Ethyl acetate described in the step (4)-methyl alcohol volume ratio is 3-11:1.
The present invention adopts enzymolysis and extraction, and the easier stripping of effective constituent has improved extraction yield; Adopt to adopt the macroporous resin column rough segmentation, bigger than extraction process treatment capacity, content is high, easier operation; The employing high speed adverse current chromatogram separates, and is simple to operate, and preparation cycle is short, the product free of losses, and what can obtain high-content looks into the handkerchief picrasmin, and product purity is higher.
Embodiment:
Embodiment 1:
Get the dry bark of twisted fort tree 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 an hour, then 42 ℃ of following water enzyme digestions 3 hours, filter, get filter residue with 80% ethanol ultrasonic extraction of 5 times of medicinal material weight 0.5 hour, extract united extraction liquid 3 times, concentrating under reduced pressure gets medicinal extract, medicinal extract is heated water-dispersion, add the AB-8 macroporous adsorptive resins, get 3BV20% ethanol elution impurity earlier, get 4BV90% ethanol elution effective constituent again, decompression recycling ethanol, last silica gel column chromatography (the silica gel granularity is 100 orders) is pressed 10:1 with ethyl acetate-methyl alcohol, 5:1, the 3:1 gradient elution, the TLC tracking monitor, handkerchief picrasmin flow point is looked in collection, concentrating under reduced pressure, lyophilize De Chapa picrasmin crude product; Get chloroform, methyl alcohol, water, 2:5:4 fully mixes in separating funnel in proportion, leaves standstill, take off and fill with the high speed adverse current chromatogram post mutually, open main frame, rotating speed 950rpm is set, pump into and do moving phase mutually, set up running balance after, the adjustment flow velocity is 2.5ml/min, dissolve crude extract with moving phase, by the sampling valve sample introduction, UV-detector detects, collect target component, preparation merges flow point, De Chapa picrasmin 1.3g continuously, detect content 98.2% through HPLC.
Embodiment 2:
Get the dry bark of twisted fort tree and pulverized 60 mesh sieves, take by weighing 5kg, the cellulase aqueous solution (every kg crude drug adds the 3.1g cellulase) that adds 14LpH4.2 soaks half an hour, then 44 ℃ of following water enzyme digestions 2 hours, filter, get filter residue with 90% ethanol ultrasonic extraction of 6 times of medicinal material weight 1 hour, extract united extraction liquid 2 times, concentrating under reduced pressure gets medicinal extract, medicinal extract is heated water-dispersion, add the XAD-16 macroporous adsorptive resins, get 4BV25% 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 with ethyl acetate-methyl alcohol, 5:1, the 3:1 gradient elution, the TLC tracking monitor, handkerchief picrasmin flow point is looked in collection, concentrating under reduced pressure, lyophilize De Dechapa picrasmin crude product; Get chloroform, methyl alcohol, water, 5:9:7 fully mixes in separating funnel in proportion, leaves standstill, take off and fill with the high speed adverse current chromatogram post mutually, open main frame, rotating speed 850rpm is set, pump 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, collect target component, preparation merges flow point, De Chapa picrasmin 1.6g continuously, detect content 98.1% through HPLC.
Embodiment 3:
Get the dry bark of twisted fort tree and pulverized 60 mesh sieves, take by weighing 5kg, the cellulase aqueous solution (every kg crude drug adding 2g cellulase and the polygalacturonase mixing prozyme that add 12.5LpH4.2, the mass ratio of cellulase and polygalacturonase is 1:1) soak half an hour, then 50 ℃ of following water enzyme digestions 5 hours, filter, get filter residue with 95% ethanol ultrasonic extraction of 5 times of medicinal material weight 0.5 hour, extract 2 times, united extraction liquid, concentrating under reduced pressure gets medicinal extract, and medicinal extract is heated water-dispersion, add the XDA-8 macroporous adsorptive resins, get 3BV25% 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), press 11:1 with ethyl acetate-methyl alcohol, 5:1, the 3:1 gradient elution, the TLC tracking monitor is collected and is looked into handkerchief picrasmin flow point, concentrating under reduced pressure, lyophilize De Dechapa picrasmin crude product; Get chloroform, methyl alcohol, water, 4:7:4 fully mixes in separating funnel in proportion, leaves standstill, take off and fill with the high speed adverse current chromatogram post mutually, open main frame, rotating speed 800rpm is set, pump 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, collect target component, preparation merges flow point, De Chapa picrasmin 1.5g continuously, detect content 98.3% through HPLC.
Embodiment 4:
Get the dry bark of twisted fort tree and pulverized 60 mesh sieves, take by weighing 5kg, the cellulase aqueous solution (every kg crude drug adds the 3.5g polygalacturonase) that adds 15LpH4.6 soaks half an hour, then 45 ℃ of following water enzyme digestions 4 hours, filter, get filter residue with 80% ethanol ultrasonic extraction of 10 times of medicinal material weight 0.5 hour, extract united extraction liquid 3 times, concentrating under reduced pressure gets medicinal extract, medicinal extract is heated water-dispersion, add the HZ806 macroporous adsorptive resins, get 4BV25% 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 11:1 with ethyl acetate-methyl alcohol, 5:1, the 3:1 gradient elution, the TLC tracking monitor, handkerchief picrasmin flow point is looked in collection, concentrating under reduced pressure, lyophilize De Dechapa picrasmin crude product; Get chloroform, methyl alcohol, water, 3:7:6 fully mixes in separating funnel in proportion, leaves standstill, take off and fill with the high speed adverse current chromatogram post mutually, open main frame, rotating speed 900rpm is set, pump 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, collect target component, preparation merges flow point, De Chapa picrasmin 1.8g continuously, detect content 98.7% through HPLC.
Embodiment 5:
Get the dry bark of twisted fort tree and pulverized 60 mesh sieves, take by weighing 5kg, the cellulase aqueous solution (the mixing prozyme of every kg crude drug adding 2.6g dextranase and polygalacturonase that adds 13LpH4, the mass ratio of dextranase and polygalacturonase is 1:3) soak half an hour, then 48 ℃ of following water enzyme digestions 2 hours, filter, get filter residue with 85% ethanol ultrasonic extraction of 8 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, add the AB-8 macroporous adsorptive resins, get 3BV30% 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), press 11:1 with ethyl acetate-methyl alcohol, 6:1, the 3:1 gradient elution, the TLC tracking monitor is collected and is looked into handkerchief picrasmin flow point, concentrating under reduced pressure, lyophilize De Dechapa picrasmin crude product; Get chloroform, methyl alcohol, water, 3:8:7 fully mixes in separating funnel in proportion, leaves standstill, take off and fill with the high speed adverse current chromatogram post mutually, open main frame, rotating speed 900rpm is set, pump 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, collect target component, preparation merges flow point, De Chapa picrasmin 2.3g continuously, detect content 98.5% through HPLC.
Embodiment 6:
Get the dry bark of twisted fort tree and pulverized 60 mesh sieves, take by weighing 5kg, the cellulase aqueous solution (every kg crude drug adds the 3.3g cellulase) that adds 13LpH4.5 soaks half an hour, then 40 ℃ of following water enzyme digestions 5 hours, filter, get filter residue with 85% ethanol ultrasonic extraction of 8 times of medicinal material weight 1 hour, extract united extraction liquid 3 times, concentrating under reduced pressure gets medicinal extract, medicinal extract is heated water-dispersion, add 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 with ethyl acetate-methyl alcohol, 5:1, the 3:1 gradient elution, the TLC tracking monitor, handkerchief picrasmin flow point is looked in collection, concentrating under reduced pressure, lyophilize De Dechapa picrasmin crude product; Get chloroform, methyl alcohol, water, 2:5:3 fully mixes in separating funnel in proportion, leaves standstill, take off and fill with the high speed adverse current chromatogram post mutually, open main frame, rotating speed 1000rpm is set, pump into and do moving phase mutually, set up running balance after, the adjustment flow velocity is 3.5ml/min, dissolve crude extract with moving phase, by the sampling valve sample introduction, UV-detector detects, collect target component, preparation merges flow point, De Chapa picrasmin 1.7g continuously, detect content 98.6% through HPLC.
Claims (6)
1. method of purification of looking into the handkerchief picrasmin is characterized in that may further comprise the steps:
(1) enzymolysis: get the dry bark of the twisted fort tree of raw material, through pulverization process, soak water enzyme digestion 2-5 hour with enzyme solution;
(2) extract: above-mentioned enzymolysis solution is filtered, get filter residue and extract 2-3 time with the aqueous ethanolic solution of 5-8 times of material weight, united extraction liquid, concentrating under reduced pressure gets medicinal extract;
(3) enrichment: medicinal extract is crossed macroporous resin column, and after absorption was finished, pure water elution was collected elutriant;
(4) purifying: with above-mentioned elutriant decompression recycling ethanol, last silicagel column (the silica gel granularity is the 100-200 order), ethyl acetate-methyl alcohol gradient elution, the TLC tracking monitor is collected and is looked into handkerchief picrasmin flow point, concentrating under reduced pressure, lyophilize gets crude product;
(5) high speed adverse current chromatogram separates: crude product adopts high speed adverse current chromatogram to separate, and is solvent systems with chloroform-methanol-water, and ratio is (2-5): (5-9): (4-7), collect target component, concentrate drying namely gets high purity and looks into the handkerchief picrasmin.
2. method of purification according to claim 1, it is characterized in that: one or more described in the step (1) in the optional cellulase of enzyme, dextranase and the polygalacturonase, enzyme dosage are 2.5-3.5 ‰, 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 concentration of volume percent of ethanol be 80-95%.
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, XDA-8, XAD-16 or the HZ806 type.
5. method of purification according to claim 1, it is characterized in that: pure water elution described in the step (3) is 3-4BV20%-30% aqueous ethanolic solution → 4-6BV80%-90% aqueous ethanolic solution.
6. method of purification according to claim 1, it is characterized in that: ethyl acetate described in the step (4)-methyl alcohol volume ratio is 3-11:1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201310196135 CN103242333A (en) | 2013-05-24 | 2013-05-24 | Method for purifying Chapa picrasin |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201310196135 CN103242333A (en) | 2013-05-24 | 2013-05-24 | Method for purifying Chapa picrasin |
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| Publication Number | Publication Date |
|---|---|
| CN103242333A true CN103242333A (en) | 2013-08-14 |
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ID=48922238
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201310196135 Pending CN103242333A (en) | 2013-05-24 | 2013-05-24 | Method for purifying Chapa picrasin |
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|---|---|
| CN (1) | CN103242333A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108653395A (en) * | 2018-06-21 | 2018-10-16 | 南京林业大学 | A kind of artificial forest bark active constituent Coupled |
| CN108704002A (en) * | 2018-06-21 | 2018-10-26 | 南京林业大学 | A kind of artificial forest bark active constituent enzymatic treatment assists Coupled with ultrasonic wave |
-
2013
- 2013-05-24 CN CN 201310196135 patent/CN103242333A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108653395A (en) * | 2018-06-21 | 2018-10-16 | 南京林业大学 | A kind of artificial forest bark active constituent Coupled |
| CN108704002A (en) * | 2018-06-21 | 2018-10-26 | 南京林业大学 | A kind of artificial forest bark active constituent enzymatic treatment assists Coupled with ultrasonic wave |
| CN108653395B (en) * | 2018-06-21 | 2020-12-22 | 南京林业大学 | Coupling extraction method for active ingredients of bark of artificial forest |
| CN108704002B (en) * | 2018-06-21 | 2021-05-11 | 南京林业大学 | Artificial forest bark active ingredient enzyme treatment and ultrasonic-assisted coupling extraction method |
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| PB01 | Publication | ||
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Application publication date: 20130814 |
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| WD01 | Invention patent application deemed withdrawn after publication |