CN101817778A - Method for preparing indirubin from indigowoad leaves - Google Patents
Method for preparing indirubin from indigowoad leaves Download PDFInfo
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- CN101817778A CN101817778A CN200910264451A CN200910264451A CN101817778A CN 101817778 A CN101817778 A CN 101817778A CN 200910264451 A CN200910264451 A CN 200910264451A CN 200910264451 A CN200910264451 A CN 200910264451A CN 101817778 A CN101817778 A CN 101817778A
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- ethyl acetate
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- methylene dichloride
- indirubin
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Abstract
The invention discloses a novel process for preparing indirubin from indigowoad leaves. In the process, 2 to 3 percent solution of hydrochloric acid is used to wet raw materials, refluxing extraction in 60 to 80 percent methanol is performed 2 to 3 times, the extract of the first time or the previous two times is decolorized by active carbon, the pH value is adjusted by stronger ammonia water to 9 to 10, precipitation and crystallization are performed, the crystals are mixed with alumina in a ratio of 1:1-2 to make a sample, the sample is filled into a short wide column, mixed solution of dichloromethane and ethyl acetate is used for gradient elution, indirubin eluent is collected, a reagent is recovered till the volume is reduced to 1/4 to 1/5, the solution is stood for crystallization and recrystallization is performed. The process is simple and is easy in operation. The obtained product is high in content.
Description
Technical field:
The present invention relates to a kind of method for preparing Indirubin from Leaf of Indigowoad, this method is a kind of method that adopts acidifying alcohol extracting, alumina separation, recrystallization to prepare Indirubin.
Background technology:
Indirubin is couroupitine B again.
Molecular formula: C16H10N2O2
Molecular weight: 262.26
Proterties: garnet needle crystal, mp is dissolved in ethyl acetate, ethyl acetate, methylene dichloride, ether for 356～358 ℃, is slightly soluble in ethanol and acetic acid, and is water insoluble.
Indirubin is a bisindole alkaloid.Multiple transplanted animal tumor there is restraining effect, can destroys the leukemia cell; Under its effect, the cell of degeneration necrosis is swelling, lytic necrosis more.Experimental studies have found that Indirubin can also strengthen the phagocytic activity of the mononuclear phagocyte system of animal.Indirubin is the anti-leukemia medicine of the novel texture of China's initiative.
At present the domestic Indirubin that is used for studying has 3 kinds of sources: i.e. natural product, semi-synthetic, the complete synthesis Indirubin of extracting from Chinese medicine Leaf of Indigowoad and indigo naturalis.
The existing technical study for preparing Indirubin from Leaf of Indigowoad seldom, open method is not detailed yet." extraction and separation process of the Indirubin research in the Leaf of Indigowoad " delivered in " Chinese patent medicine " the 28th 2006 the 6th phases of volume as Xie Guoxiang etc., disclosed method is to adopt 95% ethanol in the document, be condensed into medicinal extract, add 3 times of water gaging dissolvings, the aqueous solution is crossed macroporous resin column.Get supernatant concentration to doing, alkali alumina is with chloroform one vinyl acetic monomer wash-out, elutriant through concentrate the red-purple end, use methyl alcohol. re-crystallizing in ethyl acetate.This process cycle is long, production cost is high, moreover Indirubin is water insoluble, is difficult to realize in the macroporous resin adsorption step, and alumina column separates dress post step, does not have detailed open operation.Mostly other documents are to introduce and utilize means such as ultrasonic, microwave, supercritical CO 2 extraction to improve extraction yield and shorten extraction time, " supercritical extraction process of Indirubin and the assay in the Leaf of Indigowoad " on " The Fourth Military Medical University's journal ", delivered as people such as Li Hui, the disclosed method of document is just introduced the step of supercritical extraction, extraction yield introduction in detail.
Summary of the invention:
The present invention provides a kind of novel method for preparing Indirubin from Leaf of Indigowoad.This method is simple to operation, improves yield, shortens the production cycle.
The object of the present invention is achieved like this:
A kind of method for preparing Indirubin from Leaf of Indigowoad is characterized in that comprising following steps:
1) extracts: with Leaf of Indigowoad raw material pulverizing 20-40 order, add the 2-3% hydrochloric acid soln and soak, add the 60-80% methanol solution and extract united extraction liquid 2-3 time;
2) with the 0.5-1% gac reflux decolour of the above-mentioned first time or its amount of preceding extracted twice liquid merging adding, filter, the adding strong aqua is regulated PH to 9-10, and the placement crystallization leaches crystallization, and hot wash neutrality gets coarse crystallization, and mother liquor transfers to neutrality, does and extracts solution usefulness;
3) post separates: with the dry water that divides of above-mentioned crystallisate, add aluminum oxide and mix sample, and the short wide column of packing into, methylene dichloride and ethyl acetate mixture gradient elution are collected the red-purple elutriant, and reclaim under reduced pressure reagent is placed crystallization to 1/4-1/5;
4) recrystallization: above-mentioned crystallisate is leached, use ethyl acetate methanol mixing solutions, re-crystallizing in ethyl acetate successively, be drying to obtain.
Described extraction conditions: the 6-10 that each ethanol adds raw material doubly measures refluxing extraction, extraction time 1-2 hour.
Described post separation condition: aluminum oxide is a 120-160 order neutral alumina, sample is mixed in coarse crystallization and aluminum oxide 1: 1-2 mixing, adorn post with pure aluminum oxide 1: 5-7 again, 3-4 times of column volume methylene dichloride of elder generation and 20: 1 wash-outs of ethyl acetate, use methylene dichloride and ethyl acetate to be eluted to red-purple look liquid at 10: 3 again and flow out, use methylene dichloride and ethyl acetate 1: 2-3 instead and be eluted to the outflow of redfree liquid.
Described recrystallization condition: the saturated dissolving of ethyl acetate backflow adds the 1/10-1/15 methanol crystallization of its volume again, and ethyl acetate backflow is saturated dissolving crystallized.
In sum, there is following advantage in the present invention: hydrochloric acid soln soaks raw material, and favourable raising extraction yield shortens extraction time; Ammoniacal liquor is regulated PH, helps the Indirubin crystallization, has reduced separating out of impurity, has reduced the consumption of postorder aluminum oxide and eluent, and mother liquor need not to concentrate the direct raw material that adds and does to extract solvent, cuts down the consumption of energy and extraction cost; Opposed polarity eluent wash-out, good impurity removing effect, different reagent crystallizations, product content height.
Further specify the present invention below in conjunction with embodiment, but the scope of protection of present invention is not limited to following embodiment.
Embodiment:
Embodiment 1:
Get Leaf of Indigowoad and pick inclusion-free, pulverize 20 orders, take by weighing 20kg and add the 500L extractor, soak the back with the 5L2% hydrochloric acid soln and add the 200L80% methanol solution, refluxing extraction 2 hours, suction filtration solution adds the 160L80% methanol solution again, refluxing extraction 1 hour, suction filtration solution merges, and gets extracting solution 320L, continued to add 120L80% methanol solution refluxing extraction 1 hour, and be inserted in to extract next time and use.Extracting solution added the 3.2kg activated carbon decolorizing 1 hour, filtered, and drips of solution adds strong aqua and regulates PH to 10, places crystallization 10 hours.Leach coarse crystallization, the hot wash crystallisate is to neutral, dry solid 500g pulverizes and adds 1kg120-160 order neutral alumina and mix, and adds the pure aluminum oxide of 2.5kg in diameter 40cm short wide column bottom earlier and adds mixed aluminium oxides again, and 200 order stainless steel meshs cover the top compacting.Earlier with 12L methylene dichloride and 20: 1 wash-outs of ethyl acetate, wash-out finishes, use methylene dichloride and ethyl acetate instead and be eluted to the outflow of red-purple look liquid at 10: 3, using methylene dichloride and ethyl acetate to be eluted to redfree liquid at 1: 2 again flows out, collect red liquid, reclaim reagent to 1/4 of original volume, place crystallization, above elute soln reclaims next time respectively and uses.Filter crystallisate 23g, 99% methyl alcohol that slowly adds its volume 1/10 with the saturated dissolving of ethyl acetate backflow is again placed crystallization, filters, and crystallisate is placed crystallization with the saturated dissolving of ethyl acetate backflow again, gets product 15g, content 98%.
Embodiment 2:
Get Leaf of Indigowoad and pick inclusion-free, pulverize 40 orders, take by weighing 20kg and add the 500L extractor, soak the back with the 5L3% hydrochloric acid soln and add the 200L60% methanol solution, refluxing extraction 2 hours, suction filtration solution adds the 120L60% methanol solution again, refluxing extraction 1 hour, suction filtration solution merges, and gets extracting solution 280L, continued to add 120L60% methanol solution refluxing extraction 1 hour, and be inserted in to extract next time and use.Extracting solution added the 1.4kg activated carbon decolorizing 0.5 hour, filtered, and drips of solution adds strong aqua and regulates PH to 9, places crystallization 8 hours.Leach coarse crystallization, the hot wash crystallisate is to neutral, dry solid 510g pulverizes and adds 510g120-160 order neutral alumina and mix, and adds the pure aluminum oxide of 3.5kg in diameter 40cm short wide column bottom earlier and adds mixed aluminium oxides again, and 200 order stainless steel meshs cover the top compacting.Earlier with 15L methylene dichloride and 20: 1 wash-outs of ethyl acetate, wash-out finishes, use methylene dichloride and ethyl acetate instead and be eluted to the outflow of red-purple look liquid at 10: 3, using methylene dichloride and ethyl acetate to be eluted to redfree liquid at 1: 3 again flows out, collect red liquid, reclaim reagent to 1/5 of original volume, place crystallization, above elute soln reclaims next time respectively and uses.Filter crystallisate 26g, 99% methyl alcohol that slowly adds its volume 1/15 with the saturated dissolving of ethyl acetate backflow is again placed crystallization, filters, and crystallisate is placed crystallization with the saturated dissolving of ethyl acetate backflow again, gets product 14g, content 98.1%.
Embodiment 3:
Get Leaf of Indigowoad and pick inclusion-free, pulverize 20 orders, take by weighing 20kg and add the 500L extractor, soak the back with the 5L3% hydrochloric acid soln and add the 200L80% methanol solution, refluxing extraction 2 hours, suction filtration solution gets extracting solution 160L, continued to add 120L80% methanol solution refluxing extraction 1 hour, and be inserted in to extract next time and use.Extracting solution added the 1.6kg activated carbon decolorizing 1 hour, filtered, and drips of solution adds strong aqua and regulates PH to 10, places crystallization 12 hours.Leach coarse crystallization, the hot wash crystallisate is to neutral, dry solid 420g pulverizes and adds 800g120-160 order neutral alumina and mix, and adds the pure neutral alumina of 2.8kg in diameter 40cm short wide column bottom earlier and adds mixed aluminium oxides again, and 200 order stainless steel meshs cover the top compacting.Earlier with 16L methylene dichloride and 20: 1 wash-outs of ethyl acetate, wash-out finishes, use methylene dichloride and ethyl acetate instead and be eluted to the outflow of red-purple look liquid at 10: 3, using methylene dichloride and ethyl acetate to be eluted to redfree liquid at 1: 2 again flows out, collect red liquid, reclaim reagent to 1/5 of original volume, place crystallization, above elute soln reclaims next time respectively and uses.Filter crystallisate 18g, 99% methyl alcohol that slowly adds its volume 1/10 with the saturated dissolving of ethyl acetate backflow is again placed crystallization, filters, and crystallisate is placed crystallization with the saturated dissolving of ethyl acetate backflow again, gets product 11g, content 98%.
Embodiment 4:
Get Leaf of Indigowoad and pick inclusion-free, pulverize 40 orders, take by weighing 20kg and add the 500L extractor, soak the back with the 5L2% hydrochloric acid soln and add the 200L60% methanol solution, refluxing extraction 2 hours, suction filtration solution gets extracting solution 160L, continued to add 120L60% methanol solution refluxing extraction 1 hour, and be inserted in to extract next time and use.Extracting solution added the 1kg activated carbon decolorizing 1 hour, filtered, and drips of solution adds strong aqua and regulates PH to 9, places crystallization 8 hours.Leach coarse crystallization, the hot wash crystallisate is to neutral, dry solid 430g pulverizes and adds 430g120-160 order neutral alumina and mix, and adds the pure neutral alumina of 3kg in diameter 40cm short wide column bottom earlier and adds mixed aluminium oxides again, and 200 order stainless steel meshs cover the top compacting.Earlier with 10L methylene dichloride and 20: 1 wash-outs of ethyl acetate, wash-out finishes, use methylene dichloride and ethyl acetate instead and be eluted to the outflow of red-purple look liquid at 10: 3, using methylene dichloride and ethyl acetate to be eluted to redfree liquid at 1: 2 again flows out, collect red liquid, reclaim reagent to 1/4 of original volume, place crystallization, above elute soln reclaims next time respectively and uses.Filter crystallisate 16g, 99% methyl alcohol that slowly adds its volume 1/10 with the saturated dissolving of ethyl acetate backflow is again placed crystallization, filters, and crystallisate is placed crystallization with the saturated dissolving of ethyl acetate backflow again, gets product 10g, content 98.2%.
Embodiment 5:
Get Leaf of Indigowoad and pick inclusion-free, pulverize 20 orders, take by weighing 10kg and add the 500L extractor, soak the back with the 2L2% hydrochloric acid soln and add the 100L80% methanol solution, refluxing extraction 2 hours, suction filtration solution adds the 80L80% methanol solution again, refluxing extraction 1 hour, suction filtration solution merges, and gets extracting solution 160L, continued to add 60L80% methanol solution refluxing extraction 1 hour, and be inserted in to extract next time and use.Extracting solution added the 800g activated carbon decolorizing 1 hour, filtered, and drips of solution adds strong aqua and regulates PH to 10, places crystallization 10 hours.Leach coarse crystallization, the hot wash crystallisate is to neutral, dry that solid 240g pulverizing 480g120-160 order neutral alumina mixes, add the pure neutral alumina of 1.6kg in diameter 20cm short wide column bottom earlier and add mixed aluminium oxides again, 200 order stainless steel meshs cover the top compacting.Earlier with 8L methylene dichloride and 20: 1 wash-outs of ethyl acetate, wash-out finishes, use methylene dichloride and ethyl acetate instead and be eluted to the outflow of red-purple look liquid at 10: 3, using methylene dichloride and ethyl acetate to be eluted to redfree liquid at 1: 2 again flows out, collect red liquid, reclaim reagent to 1/4 of original volume, place crystallization, above elute soln reclaims next time respectively and uses.Filter crystallisate 9g, 99% methyl alcohol that slowly adds its volume 1/15 with the saturated dissolving of ethyl acetate backflow is again placed crystallization, filters, and crystallisate is placed crystallization with the saturated dissolving of ethyl acetate backflow again, gets product 7g, content 98%.
Embodiment 6:
Get Leaf of Indigowoad and pick inclusion-free, pulverize 20 orders, take by weighing 10kg and add the 500L extractor, soak the back with the 2L3% hydrochloric acid soln and add the 100L60% methanol solution, refluxing extraction 2 hours, suction filtration solution gets extracting solution 80L, continued to add 100L60% methanol solution refluxing extraction 1 hour, and be inserted in to extract next time and use.Extracting solution added the 500g activated carbon decolorizing 1 hour, filtered, and drips of solution adds strong aqua and regulates PH to 10, places crystallization 15 hours.Leach coarse crystallization, the hot wash crystallisate is to neutral, dry solid 200g pulverizes and adds 400g120-160 order neutral alumina and mix, and adds the pure neutral alumina of 1kg in diameter 20cm short wide column bottom earlier and adds mixed aluminium oxides again, and 200 order stainless steel meshs cover the top compacting.Earlier with 5L methylene dichloride and 20: 1 wash-outs of ethyl acetate, wash-out finishes, use methylene dichloride and ethyl acetate instead and be eluted to the outflow of red-purple look liquid at 10: 3, using methylene dichloride and ethyl acetate to be eluted to redfree liquid at 1: 2 again flows out, collect red liquid, reclaim reagent to 1/4 of original volume, place crystallization, above elute soln reclaims next time respectively and uses.Filter crystallisate 7g, 99% methyl alcohol that slowly adds its volume 1/10 with the saturated dissolving of ethyl acetate backflow is again placed crystallization, filters, and crystallisate is placed crystallization with the saturated dissolving of ethyl acetate backflow again, gets product 5g, content 98%.
Claims (4)
1. method for preparing Indirubin from Leaf of Indigowoad is characterized in that comprising following steps:
1) extracts: with Leaf of Indigowoad raw material pulverizing 20-40 order, add the 2-3% hydrochloric acid soln and soak, add the 60-80% methanol solution and extract 2-3 time;
2) decolouring crystallization: the 0.5-1% gac reflux decolour with the above-mentioned first time or its amount of preceding extracted twice liquid merging adding, filter, add strong aqua and regulate PH to 9-10, the placement crystallization leaches crystallization, and hot wash neutrality gets coarse crystallization;
3) post separates: with the dry water that divides of above-mentioned crystallisate, add aluminum oxide and mix sample, and the short wide column of packing into, methylene dichloride and ethyl acetate mixture gradient elution are collected the red-purple elutriant, and reclaim under reduced pressure reagent is placed crystallization to 1/4-1/5;
4) recrystallization: above-mentioned crystallisate is leached, use ethyl acetate methanol mixing solutions, re-crystallizing in ethyl acetate successively, be drying to obtain.
2. preparation method as claimed in claim 1 is characterized in that described extraction conditions: the 6-10 that each ethanol adds raw material doubly measures refluxing extraction, extraction time 1-2 hour.
3. preparation method as claimed in claim 1, it is characterized in that described post separation condition: aluminum oxide is a 120-160 order neutral alumina, sample is mixed in coarse crystallization and aluminum oxide 1: 1-2 mixing, adorn post with pure aluminum oxide 1: 5-7 again, 3-4 times of column volume methylene dichloride of elder generation and 20: 1 wash-outs of ethyl acetate, use methylene dichloride and ethyl acetate to be eluted to red-purple look liquid at 10: 3 again and flow out, use methylene dichloride and ethyl acetate 1: 2-3 instead and be eluted to the outflow of redfree liquid.
4. preparation method as claimed in claim 1 is characterized in that described recrystallization condition: the saturated dissolving of ethyl acetate backflow adds the 1/10-1/15 methanol crystallization of its volume again, and ethyl acetate backflow is saturated dissolving crystallized.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910264451A CN101817778A (en) | 2009-12-23 | 2009-12-23 | Method for preparing indirubin from indigowoad leaves |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910264451A CN101817778A (en) | 2009-12-23 | 2009-12-23 | Method for preparing indirubin from indigowoad leaves |
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| CN101817778A true CN101817778A (en) | 2010-09-01 |
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| CN200910264451A Pending CN101817778A (en) | 2009-12-23 | 2009-12-23 | Method for preparing indirubin from indigowoad leaves |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102174009A (en) * | 2011-03-09 | 2011-09-07 | 中国科学院近代物理研究所 | Method for preparing indigo and indirubin from dyers woad leaf |
| CN103509835A (en) * | 2013-09-29 | 2014-01-15 | 重庆市中药研究院 | Method for preparing indirubin with fresh folium isatidis as raw material |
| CN103992260A (en) * | 2014-05-29 | 2014-08-20 | 哈尔滨医科大学 | Method for extracting indirubin from folium isatidis |
-
2009
- 2009-12-23 CN CN200910264451A patent/CN101817778A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102174009A (en) * | 2011-03-09 | 2011-09-07 | 中国科学院近代物理研究所 | Method for preparing indigo and indirubin from dyers woad leaf |
| CN102174009B (en) * | 2011-03-09 | 2013-08-21 | 中国科学院近代物理研究所 | Method for preparing indigo and indirubin from dyers woad leaf |
| CN103509835A (en) * | 2013-09-29 | 2014-01-15 | 重庆市中药研究院 | Method for preparing indirubin with fresh folium isatidis as raw material |
| CN103509835B (en) * | 2013-09-29 | 2016-08-31 | 重庆市中药研究院 | A kind of method preparing indigo red for raw material with fresh and alive folium isatidis |
| CN103992260A (en) * | 2014-05-29 | 2014-08-20 | 哈尔滨医科大学 | Method for extracting indirubin from folium isatidis |
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Application publication date: 20100901 |