CN103509835B - A kind of method preparing indigo red for raw material with fresh and alive folium isatidis - Google Patents
A kind of method preparing indigo red for raw material with fresh and alive folium isatidis Download PDFInfo
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- CN103509835B CN103509835B CN201310451101.6A CN201310451101A CN103509835B CN 103509835 B CN103509835 B CN 103509835B CN 201310451101 A CN201310451101 A CN 201310451101A CN 103509835 B CN103509835 B CN 103509835B
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- folium isatidis
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- indigo red
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Abstract
The invention provides a kind of method preparing indigo red for raw material with fresh and alive folium isatidis, it is characterised in that: in pulverizing fresh and alive folium isatidis woaded blue raw material, add istain；Body of the present invention utilizes the generation of indoxyl free radical by the dissolved oxygen oxygen supply in former soak, therefore, makes reaction develop to indigo red direction by the control of system pH with enzymolysis time control oxygen；Method is easy, controlled；The present invention directly utilizes the raw material of folium isatidis as reaction raw materials, effectively reduces production cost；The present invention is controlled when still entering the generation of indoxyl free radical oxidation step, it is to avoid generates istain, but directly adds istain in reaction raw materials, is that reaction is to forward balance.
Description
Technical field
The present invention relates to the preparation of a kind of Chinese medicine active component, extracting method, a kind of with fresh and alive folium isatidis
The method preparing indigo red for raw material.
Technical background
Folium isatidis is the traditional Chinese medicine of China, for the dry leaf of cruciferae isatis Isatis indigotica Fortune,
Simultaneously also frequently with polygonaceae plant knotweed smalt Polygonum tinctirium Lour in commodity；Acanthaceous vegetable acanthaceous indigo
Baphicacanthus cusia fNees) Bremek. and Verbenaceae yellow avens Clerodendron cyrtophyllum
Turcz. as the substitute of folium isatidis.The root of woaded blue and leaf, have blood clearing heat and detoxicating, cool, effect of ecchymose removing, cure mainly warm disease
Heating, send out spot, dermexanthesis, anemopyretic cold, abscess of throat, meningococal meningitis, encephalitis B, hepatitis, parotitis,
The diseases such as erysipelas, pain are swollen, are to treat the bulk medicinal materials that flu is conventional.Main component in folium isatidis (woaded blue leaf) has woaded blue glycosides (isatan
B), woaded blue glycosides facile hydrolysis be indigo red (indirubin) and indigo (indigo), couroupitine A (trptanthrin), β
-paddy stay alcohol, nonacosane, benzoic acid, face bit amino benzoic acid, in the complete stool of folium isatidis possibly together with mustard glycosides
(glucobrassicin), new mustard glycosides (neoglucobrassicin), 1-sulfo group glycosides (1-sulpho-3-indolylmethyl
Glucosinolate), indanol, 1-thiocyanic acid-2-hydroxyl-3 butylene (1-thiocy a no-2-hydroxy-3-butene),
Adenosine (adenosine), palmitic acid, clionasterol etc., asparatate, threonine, proline, glycine, the third ammonia
The iron of acid, methionine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine and high-load and manganese.
It is reported, all containing indigo, indigo red in the four strains of folium isatidis, wherein, in woaded blue leaf, (isatanB has woaded blue glycosides B
Also referred to as rheum emodin B and isatan B) content is the highest, it was reported that high-load reaches 1%.
Indigo red has another name called couroupitine B, and molecular weight is: 262.26；Molecular structural formula is:
Indigo red is a kind of bisindole alkaloid；Multiple transplanted animal tumor there is is inhibitory action, leukaemia can be destroyed；
Under it acts on, the cell of sex change many in swelling, lytic necrosis.Experimental studies have found that, indigo red can also strengthen the list of animal
The phagocytic activity of core macrophage system, indigo red is the anti-leukemia medicine of the new structure that China initiates.The most domestic for
The indigo red of research has in 3 originates: the natural products i.e. extracted from Chinese medicine folium isatidis, indigo naturalis, semi-synthetic, fully synthetic indigo
Rubine.
Summary of the invention
In order to improve the yield extracting indigo red from folium isatidis (woaded blue), the invention provides a kind of at folium isatidis fresh goods raw material
In the method for direct semi-synthetic indigo red；When extracting, the indigo red existed in woaded blue crude drug is extracted in the lump simultaneously
Method.
The principle that woaded blue glycosides (isatan B) is converted in the present invention indigo red is:
1) woaded blue glycosides unstability in a heated condition；
2) woaded blue glycosides belongs to esters.The most easily hydrolyze；
3) free indoxyl cation out under anaerobic will not be oxidized to indoxyl free radical；
On the basis of above-mentioned principle, the present invention directly adds istain in pulverizing fresh and alive folium isatidis woaded blue raw material raw material, makes Yin
Diindyl phenol and istain reaction condensation become indigo red.Chemical equation is as follows:
But there is a side reaction, because isatan B belongs to esters, facile hydrolysis under the conditions of alkalescence in this reaction.It is in endogenous enzymes
Effect issue raw hydrolysis, produce indoxyl cation, under conditions of aerobic, indoxyl cation can be oxidized to indoles
Phenol free radical, generating by the dissolved oxygen oxygen supply in former soak of indoxyl free radical, indoxyl free radical may be condensed into
For indigo；But the isatin that oxidation generates further then needs extraneous extra oxygen supply.Chemical equation is as follows:
Therefore, lead to whether oxygen is the key making reaction generate which kind of indigo when the control of system pH and enzymolysis.If making product indigo beautiful towards indigo
Red is to carrying out, then, in enzymolysis process, promoted the generation of isatin by means such as logical oxygen, heightening pH, if towards
Indigo direction is carried out, then should control oxygen content and system pH in enzymolysis process, prevents the generation of isatin, therefore system
Oxygen content and which kind of indigo of pH and generation are closely related.
The particular content of the method preparing indigo red for raw material with fresh and alive folium isatidis is:
1) gather and the fresh and alive folium isatidis in 12h added the water of medicinal material weight 2-5 times, pulverize, the slurries of pulverizing can mistake mesh sieve,
Prepare extraction magma；
2) adding such as istain in extracting magma, be sufficiently stirred for, adjust pH value more than 7, described tune pH value more than 7 is
Limewash is added in reactant liquor；Heating, after reaction is more than 30min, filters, collects water liquid, in this step can be to former
Slurry is filled with inert gas, gets rid of oxygen therein；
3) with the mixture of one or more in chloroform, monochloro methane, dichloromethane, tetrachloromethane or ethyl acetate, heating,
Extraction water liquid, collects and merges organic phase；
4) organic phase is reclaimed, upper alumina column, wash-out, purify and obtain indigo red crude product.Described upper alumina column, elutes and is:
It is recycled to do by organic phase, by the one in chloroform, monochloro methane, dichloromethane, tetrachloromethane or ethyl acetate or several
The mixture heating for dissolving planted, in raw material: the ratio of aluminum oxide (1:2-4) is mixed, dry method wet method dress post, dry method loading；Collect indigo
The chromatographic band of rubine, reclaims, and crystallization obtains indigo red sterling.
The method have the benefit that: 1) body of the present invention utilize indoxyl free radical generate by the dissolved oxygen in former soak
Oxygen supply, therefore, makes reaction develop to indigo red direction by the control of system pH with enzymolysis time control oxygen；Method is easy,
Controlled；
2) present invention directly utilizes the raw material of folium isatidis as reaction raw materials, effectively reduces production cost；
3) present invention is controlled when still entering the generation of indoxyl free radical oxidation step, it is to avoid generation istain, but directly
It is connected in reaction raw materials interpolation istain, is to react to balance to forward.
Detailed description of the invention
The impact of woaded blue glycosides content in crude drug is investigated by embodiment 1 different disposal method
Plantation is gathered folium isatidis (woaded blue) fresh goods month next year 7-10,5-25 DEG C, and lucifuge stores standby；12h after gathering
In, separately sampled 5 groups, often group folium isatidis fresh goods 1kg；Give entitled 60 DEG C dry group, 120 DEG C dry group, backflow sample preparation respectively
Group, cold soaking sample preparation group；Dry in the shade group；Purchase commercially available folium isatidis as commercially available group.
Wherein, 60 DEG C are dried group and 120 DEG C of dried ingredients not for dry to be dried prepare dry product at 60 DEG C and 120 DEG C of air blast；
Backflow sample preparation group is with the water refluxing extraction sample of medicinal material weight 5 times；Group of drying in the shade is under normal temperature condition, and aeration-drying is dried
About 1 week time；Cold soaking sample preparation group is by the methyl alcohol co-grinding of fresh and alive folium isatidis medicinal material weight 3 times, adjust pH value to 4-6,
Prepare sample.
Extract cold soaking sample preparation group respectively and backflow sample preparation sample preparation group detects, measure woaded blue glycosides, indigo red and indigo containing respectively
Amount；Respectively 60 DEG C dry group, 120 DEG C dry group, group of drying in the shade and commercially available group are got it filled material in right amount with the methyl alcohol of medicinal material weight 5 times
Ultrasonic extraction, woaded blue glycosides, indigo red and the indigo content in H detection medicinal material.Particular content is as shown in table 1.
The comparison of folium isatidis active ingredient in table 1 different disposal
Note: in addition to commercially available group, the crude drug weight of each group calculates with fresh goods.
Data from table 1 understand, and gathering 12 hours in, process folium isatidis, all can detect woaded blue glycosides；And city
Sell and folium isatidis dry product can not detect woaded blue glycosides；It is reported, after the oxidation of this Substance P hydrolysis of woaded blue glycosides, become indigo, indigo red and furan
Mutter wood sugar ketone acid, use folium isatidis dry product to extract isatan B yield the lowest, be because doing folium isatidis according to analyzing its main cause
Product are dried or oxidized during storage, it is also possible to be because the enzyme that contains in fresh goods or other factors cause isatan B to be sent out
Unboiled water solution.
From data above, we will be consequently realised that, during folium isatidis is dried, woaded blue glycosides can lose；But from difference system
The contrast of Preparation Method understands, and after woaded blue glycosides loses, in medicinal material, indigo red and the increase of indigo not equal proportion, study carefully it
Reason is probably the oxidized destruction of woaded blue glycosides or generates other material；This and folium isatidis only process is beaten indigo and could be generated indigo
Blue reason is agreed with.In the present invention reacting precursor indole glycoside fresh and alive folium isatidis under conditions of the enzyme of source, the most unstable,
It is difficult to be separately separated out the reaction intermediate such as indoxyl cation and indoxyl free radical out carry out quantifying to investigate.But from reaction
The increase of target product amount, and reacting phenomenon proved response direction is correct.
The impact of embodiment 2 various indigo red extraction process many indigo reds productivity is investigated
1, tradition beats indigo technique
Using embodiment 1 to prepare the folium isatidis fresh goods gathered, root according to the literature traditional beats indigo technique, and main operation is:
Weigh folium isatidis fresh goods 10kg, pulverize, filter, obtain folium isatidis immersion liquid；Select fat lime block 2kg, add clear water and boil in right amount
After dissolving, cross 100 mesh sieves and filter, to remove its impurity and lime mud, lime liquor matter is spilled into equably the immersion liquid of folium isatidis
In, it is continuously agitated with wooden stick, makes infusion seethe the most up and down.Immersion liquid is red → grey purple by dark green → light green → grayish green → ash
→ cyan → purple, stops smashing punching and stirring.At this moment upper strata immersion liquid crossfades into light yellow, and indigo is deposited on cylinder or pond at leisure
The end.We do not use traditional handicraft to incline upper liquid, but the indigo red in this step directly whole cylinder of detection, indigo and ancient name for Chinese cabbage
The content of blue glycosides；Obtain beating indigo legal system sample group.
2, indigo red preparation method under aerobic conditions
Use embodiment 1 to prepare the folium isatidis fresh goods gathered, weigh folium isatidis fresh goods 10kg, add the water of medicinal material weight 3 times,
Pulverize, prepare folium isatidis magma, magma is added istain 100g, mixing mixing；It is heated to 90 DEG C；Select high-quality
Lime block 0.5kg, adds after clear water boils in right amount and dissolve, crosses 100 mesh sieves and filter, to remove its impurity and lime mud, by lime
Liquid matter is spilled in folium isatidis magma equably, is sufficiently stirred for 10h；Obtain aerobic indigo red and prepare group.
3, the preparation method of indigo red under oxygen free condition
Use embodiment 1 to prepare the folium isatidis fresh goods gathered, weigh folium isatidis fresh goods 10kg, add the water of medicinal material weight 3 times,
Pulverize, prepare folium isatidis magma, folium isatidis magma is added istain 100g, mixing mixing；With air compressor pair
Folium isatidis magma is filled with inert gas (nitrogen), is warming up to 90 DEG C；Select fat lime block 0.5kg, add clear water and boil in right amount and dissolve
After, cross 100 mesh sieves and filter, to remove its impurity and lime mud, lime liquor matter is spilled in folium isatidis magma equably, fills
Divide stirring 10h；Obtain anaerobic indigo red and prepare group.
The method that relatively above-mentioned three kinds of methods prepare indigo red, the most as shown in table 2.
The comparison of active ingredient in the method for indigo red prepared by table 2
Note: the crude drug weight of each group calculates with fresh goods.
Under anaerobic, the reason of indigo increase, it may be possible to have oxygen to enter during pulverizing folium isatidis, result in Yin
Diindyl phenol free radical is oxidized generate indigo.Simultaneously from the point of view of the data of this example, produce that each group leader is raw indigo and indigo red mole
Number has been above indigo red in crude drug, isatan B, indigo summation, and tracing it to its cause be extract before other indigo red
The matter such as material such as isatan A and isatan B take part in reaction.
The proposition of embodiment 3 indigo red purifies
Use in embodiment 2 preparation method of indigo red under oxygen free condition, take be equivalent to crude drug amount 1kg reactant liquor, mistake
Filter residue, with the centrifuge removal of impurities of 15000rpm, take supernatant, be concentrated into 800ml, be heated to 60 DEG C；Add
500ml dichloromethane extracts 3 times, merges organic phase；Reclaiming and have the medicinal extract being more than 1.1 to density, medicinal extract weight is 71.22g；
Above-mentioned medicinal extract is mixed sample in 200g aluminum oxide, be dried, make powder, prepare loading sample；Weigh aluminum oxide
1500g, with dichloromethane mixing dress post, dry method loading, respectively with chloroform, chloroform: ethyl acetate (9:1), chloroform: acetic acid second
Ester (4:1), chloroform: ethyl acetate (2:1), chloroform: ethyl acetate (1:1) isopolarity gradient elution, the replacement of eluting solvent,
Adjust with the change of wash-out colour band.Obtain indigo red colour band, crystallization purifying by TLC detection confirmation, obtain indigo red purity
98.5%(internal standard method) use extraction to separate the method combined with aluminum oxide column chromatography, relative to the content of indigo red in reactant liquor,
The yield of indigo red reaches 87.5%.
Claims (5)
1. the method preparing indigo red for raw material with fresh and alive folium isatidis, it is characterised in that: in pulverizing fresh and alive folium isatidis woaded blue raw material, add istain；
Said method comprising the steps of:
1) the fresh and alive folium isatidis in the 12h that gathers is added water, pulverize, prepare extraction magma；
2) according to the ratio interpolation istain of fresh and alive folium isatidis 10kg istain 100g in extraction magma, it is sufficiently stirred for, adjusts pH value more than 7, be heated to 90 DEG C, after reaction is more than 30min, filtration, collect water liquid；
3) with one or both the mixture in chloroform and ethyl acetate, heating, extraction water liquid, collect and merge organic phase；
4) organic phase is reclaimed, upper alumina column, wash-out, purify and obtain indigo red crude product.
The method preparing indigo red for raw material with fresh and alive folium isatidis the most according to claim 1, it is characterized in that: described step 1) is: the fresh and alive folium isatidis in the 12h that gathers is added the water of medicinal material weight 2-5 times, pulverizing, the slurries of pulverizing are crossed 100 mesh sieves, are prepared extraction magma.
The method preparing indigo red for raw material with fresh and alive folium isatidis the most according to claim 1, it is characterised in that: adding described in step 3), is filled with inert gas in thermal response, get rid of the oxygen in reaction liquid.
The method preparing indigo red for raw material with fresh and alive folium isatidis the most according to claim 1, it is characterised in that: step 2) described in tune pH value more than 7 be in reactant liquor add limewash.
The method preparing indigo red for raw material with fresh and alive folium isatidis the most according to claim 1, it is characterized in that: the upper alumina column described in step 4), wash-out is: be recycled to do by organic phase, by the mixture heating for dissolving of one or both in chloroform or ethyl acetate, in raw material: the alumina weight ratio than 1:2-4 mixes sample, wet method dress post, dry method loading；Collecting the chromatographic band of indigo red, reclaim, crystallization obtains indigo red sterling.
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| CN103980182B (en) * | 2014-05-28 | 2016-06-15 | 西北大学 | A kind of method of bionical synthetic indigo red and derivative thereof |
| CN103992260A (en) * | 2014-05-29 | 2014-08-20 | 哈尔滨医科大学 | Method for extracting indirubin from folium isatidis |
| CN112824458B (en) * | 2019-11-20 | 2022-11-01 | 哈工大机器人南昌智能制造研究院 | Method for preparing indigo and indirubin by using fresh leaves of kale |
| CN115819482A (en) * | 2022-09-20 | 2023-03-21 | 江苏本草新萃生物科技有限公司 | Processing method for making kalanchoe leaf as water-soluble indole glycoside extraction raw material |
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Patent Citations (2)
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| CN101648901A (en) * | 2009-05-26 | 2010-02-17 | 苏州派腾生物医药科技有限公司 | Method for preparing indirubin |
| CN101817778A (en) * | 2009-12-23 | 2010-09-01 | 南京泽朗医药科技有限公司 | Method for preparing indirubin from indigowoad leaves |
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