CN103509835A - Method for preparing indirubin with fresh folium isatidis as raw material - Google Patents
Method for preparing indirubin with fresh folium isatidis as raw material Download PDFInfo
- Publication number
- CN103509835A CN103509835A CN201310451101.6A CN201310451101A CN103509835A CN 103509835 A CN103509835 A CN 103509835A CN 201310451101 A CN201310451101 A CN 201310451101A CN 103509835 A CN103509835 A CN 103509835A
- Authority
- CN
- China
- Prior art keywords
- indirubin
- indigowoad
- raw material
- leaf
- fresh
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Indole Compounds (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention provides a method for preparing indirubin with fresh folium isatidis as a raw material. The method is characterized in that isatin is added in the smashed fresh folium isatidis woad raw material. The method can supply oxygen only through dissolved oxygen in an original soak solution by using the generated indoxyl free radicals, so that the reaction is developed to the indirubin direction through the control of pH of the system and the oxygen control in enzymolysis, and the method is easy, convenient and controllable. The method directly uses the folium isatidis raw material as the reaction raw material, so as to effectively reduce the production cost; the method implements the control for further oxidization generation of the indoxyl free radicals to prevent the generation of isatin, and directly adds the isatin in the reaction raw material, so that the reaction is in forward balance.
Description
Technical field
The present invention relates to a kind of preparation, extracting method of Chinese medicinal materials activeconstituents, relate in particular and a kind ofly take fresh and alive Leaf of Indigowoad and prepare the method for Indirubin as raw material.
Technical background
Leaf of Indigowoad is the traditional Chinese medicine of China, is the dry leave of cruciferae isatis Isatis indigotica Fortune, also often adopts polygonaceae plant knotweed smalt Polygonum tinctirium Lour in commodity simultaneously; Acanthaceous vegetable acanthaceous indigo Baphicacanthus cusia fNees) Bremek. and Verbenaceae Herba Diclipterae Chinensis Clerodendron cyrtophyllum Turcz. are as the surrogate of Leaf of Indigowoad.The root of woaded blue and leaf, the effect with clearing heat and detoxicating, cool blood, ecchymose removing, diseases such as curing mainly warm disease heating, send out spot, dermexanthesis, common cold due to wind-heat, swelling and pain in the throat, meningococcal meningitis, encephalitis B, hepatitis, parotitis, erysipelas, pain are swollen is the conventional bulk medicinal materials for the treatment of flu.Main component in Leaf of Indigowoad (woaded blue leaf) has woaded blue glycosides (isatan B), woaded blue glycosides facile hydrolysis is Indirubin (indirubin) and indigo (indigo), couroupitine A (trptanthrin), β-paddy stays alcohol, nonacosane, phenylformic acid, face bit amino phenylformic acid, in the complete stool of Leaf of Indigowoad, also contain 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), palmitinic acid, clionasterol etc., aspartic acid, Threonine, proline(Pro), glycine, L-Ala, methionine(Met), Isoleucine, leucine, tyrosine, phenylalanine, Histidine, the iron of Methionin and high-content and manganese.It is reported, all contain indigo, Indirubin in the four strains of Leaf of Indigowoad, wherein, woaded blue glycosides B in woaded blue leaf (isatanB, the also title Schuttgelb B and the isatan B that have) content is the highest, it is reported that high-content reaches 1%.
Indirubin has another name called couroupitine B, and molecular weight is: 262.26; Molecular structural formula is:
Indirubin is a kind of bisindole alkaloid; Multiple transplanted animal tumor is had to restraining effect, can destroy leukemia cell; Under its effect, the cell of sex change is swelling, lytic necrosis more.Experimental studies have found that, Indirubin can also strengthen the phagocytic activity of the mononuclear phagocyte system of animal, and Indirubin is the anti-leukemia medicine of the pioneering novel texture of China.At present domestic have 3 sources for the Indirubin of studying: the natural product extracting from Chinese medicine Leaf of Indigowoad, indigo naturalis, semi-synthetic, complete synthesis Indirubin.
Summary of the invention
In order to improve from Leaf of Indigowoad (woaded blue) yield that extracts Indirubin, the invention provides a kind of in Leaf of Indigowoad fresh goods raw material the method for direct semi-synthetic Indirubin; When extracting, the Indirubin having existed in woaded blue crude drug is extracted to method in the lump simultaneously.
The principle that in the present invention, woaded blue glycosides (isatan B) is converted into Indirubin is:
1) unstable of woaded blue glycosides under heating condition;
2) woaded blue glycosides belongs to ester class.Easily hydrolysis under alkaline condition;
3) free indolol positively charged ion out under anaerobic can not be oxidized to indolol free radical;
On the basis of above-mentioned principle, the present invention directly adds istain in pulverizing fresh and alive Leaf of Indigowoad woaded blue raw material raw material, makes indolol and istain reaction condensation become Indirubin.Chemical equation is as follows:
But there is a side reaction in this reaction, because isatan B belongs to ester class, facile hydrolysis under alkaline condition.Its effect in endogenous enzyme issues raw hydrolysis reaction, produce indolol positively charged ion, under the condition of aerobic, indolol positively charged ion can be oxidized to indolol free radical, the dissolved oxygen oxygen supply of the generation of indolol free radical in former soak solution, indolol free radical may condensation become indigo; But the isatin that further oxidation generates needs extraneous extra oxygen supply.Chemical equation is as follows:
When therefore, the control of system pH is with enzymolysis, whether logical oxygen is to make to react the key that generates which kind of indigo.If make, producing indigo carries out towards Indirubin direction, in enzymolysis process, by means such as logical oxygen, heightening pH, promote the generation of isatin, if carry out towards indigo direction, should in enzymolysis process, control oxygen content and system pH, prevent the generation of isatin, therefore which kind of indigo of system oxygen level and pH and generation is closely related.
The particular content that the fresh and alive Leaf of Indigowoad of take is prepared the method for Indirubin as raw material is:
1) in the fresh and alive Leaf of Indigowoad of gathering in 12h, add medicinal material weight 2-5 water doubly, pulverize, the slurries of pulverizing can mistake mesh sieve, prepares extraction magma;
2) in extracting magma, add as istain, fully stir, adjust pH is greater than 7, and described adjust pH is greater than 7 for to add liming in reaction solution; Heating, reaction is greater than after 30min, filters, and collects water liquid, in this step, can in magma, be filled with rare gas element, gets rid of oxygen wherein;
3) mixture of one or more in use chloroform, monochloro methane, methylene dichloride, tetrachloromethane or ethyl acetate, heating, extraction water liquid, collects and merges organic phase;
4) reclaim organic phase, upper alumina column, wash-out, purifying obtains Indirubin crude product.Described upper alumina column, wash-out is:
Organic phase is recycled to dry, by one or more the mixture heating for dissolving in chloroform, monochloro methane, methylene dichloride, tetrachloromethane or ethyl acetate, by raw material: the ratio of aluminum oxide (1:2-4) is mixed, and dry method wet method fills post, dry method loading; Collect the chromatographic band of Indirubin, reclaim, crystallization obtains Indirubin sterling.
Useful technique effect of the present invention is: the dissolved oxygen oxygen supply of the generation that 1) body of the present invention utilizes indolol free radical in former soak solution, and therefore, the control by system pH makes to react to Indirubin future development with enzymolysis time control oxygen; Method is easy, controlled;
2) the present invention directly utilizes the raw material of Leaf of Indigowoad as reaction raw materials, effectively reduces production cost;
3) the present invention, still entering when indolol free radical oxidation step generates to control, avoids generating istain, but directly in reaction raw materials, adds istain, is that reaction is to forward balance.
Embodiment
Embodiment 1 different treatment method is investigated the impact of woaded blue glycosides content in crude drug
Plant Leaf of Indigowoad (woaded blue) fresh goods of gathering the next year 7-10 month, 5-25 ℃, lucifuge, stores standby; After gathering, in 12h, sample respectively 5 groups, every group of Leaf of Indigowoad fresh goods 1kg; Give respectively 60 ℃ dry group, 120 ℃ dry group by name, backflow sample preparation group, cold soaking sample preparation group; The group of drying in the shade; Purchase commercially available Leaf of Indigowoad as commercially available group.
Wherein, 60 ℃ of dry groups and 120 ℃ of dried ingredients be not for to prepare dry product at 60 ℃ and 120 ℃ of air blast bake dryings; Backflow sample preparation group is the water refluxing extraction sample by 5 times of medicinal material weight; The group of drying in the shade is under normal temperature condition, air seasoning, approximately 1 week time of drying; Cold soaking sample preparation group is that fresh and alive Leaf of Indigowoad is pulverized by the methanol mixed of 3 times of medicinal material weight, and adjust pH, to 4-6, prepares sample.
Extract respectively cold soaking sample preparation group and backflow sample preparation sample preparation group detects, measure respectively woaded blue glycosides, Indirubin and indigo content; Respectively 60 ℃ dry group, 120 ℃ dry group, the group of drying in the shade and commercially available group of material of getting it filled are used in right amount to the methyl alcohol supersound extraction of 5 times of medicinal material weight, H detects woaded blue glycosides, Indirubin and the indigo content in medicinal material.Particular content is as shown in table 1.
The comparison of Leaf of Indigowoad effective constituent in table 1 different treatment
Note: except commercially available group, the crude drug weight of each group is calculated with fresh goods.
Data from table 1 are known, gathering 12 hours in, Leaf of Indigowoad processed, and all can detect woaded blue glycosides; And in commercially available Leaf of Indigowoad dry product, can not detect woaded blue glycosides; It is reported, after this material hydrolysis oxidation of woaded blue glycosides, become indigo, Indirubin and furyl xylose ketone acid, adopt Leaf of Indigowoad dry product to extract isatan B yield very low, its major cause is because of oxidized in the process of drying or preserving at Leaf of Indigowoad dry product according to one's analysis, is likely also because the enzyme or other factors that contain in fresh goods cause isatan B to be hydrolyzed.
From above data, we can recognize, in the dry process of Leaf of Indigowoad, woaded blue glycosides can lose; But from different preparation methods' contrast, after woaded blue glycosides loses, Indirubin and the indigo not increase of equal proportion in medicinal material, tracing it to its cause may be the oxidized destruction of woaded blue glycosides or the material that has generated other; This and Leaf of Indigowoad only have through beating indigo and could generate indigo reason and agree with.Reacting precursor indole glycoside is fresh and alive Leaf of Indigowoad under the condition of source enzyme in the present invention, and very unstable, the very difficult reaction intermediates such as indolol positively charged ion and indolol free radical of isolating separately out quantize to investigate.But from the increase of target product amount of reaction, and reacting phenomenon proved response direction is correct.
The impact of many Indirubins of the various Indirubin extraction process of embodiment 2 productive rate is investigated
1, tradition is beaten indigo technique
The Leaf of Indigowoad fresh goods that adopts embodiment 1 preparation to gather, according to traditional indigo technique of beating of bibliographical information, main is operating as: take Leaf of Indigowoad fresh goods 10kg, pulverize, filtration, obtains Leaf of Indigowoad immersion liquid; Select fat lime piece 2kg, after adding clear water and boiling in right amount and dissolve, cross 100 mesh sieves and filter, to remove its impurity and lime mud, liquor calcis matter is spilled into equably in the immersion liquid of Leaf of Indigowoad, with wooden stick, ceaselessly stir, infusion is ceaselessly seethed up and down.Immersion liquid, by dark green → light green → grayish green → red → grey purple → cyan → purple of ash, stops smashing punching and stirs.At this moment upper strata immersion liquid crossfades into light yellow, and at the bottom of indigo is deposited on Gang Huo pond at leisure.We do not adopt traditional technology Qing Qu upper strata liquid, but the content of Indirubin in this step direct-detection whole cylinder, indigo and woaded blue glycosides; Obtain beating indigo legal system sample group.
2, Indirubin preparation method under aerobic conditions
The Leaf of Indigowoad fresh goods that adopts embodiment 1 preparation to gather, takes Leaf of Indigowoad fresh goods 10kg, adds the water of 3 times of medicinal material weight, pulverizes, and prepares Leaf of Indigowoad magma, adds istain 100g in magma, and mixing mixes; Be heated to 90 ℃; Select fat lime piece 0.5kg, after adding clear water and boiling in right amount and dissolve, cross 100 mesh sieves and filter, to remove its impurity and lime mud, liquor calcis matter is spilled in Leaf of Indigowoad magma equably, fully stir 10h; Obtain aerobic Indirubin and prepare group.
3, the preparation method of Indirubin under oxygen free condition
The Leaf of Indigowoad fresh goods that adopts embodiment 1 preparation to gather, takes Leaf of Indigowoad fresh goods 10kg, adds the water of 3 times of medicinal material weight, pulverizes, and prepares Leaf of Indigowoad magma, adds istain 100g in Leaf of Indigowoad magma, and mixing mixes; With air compressor, Leaf of Indigowoad magma is filled with to rare gas element (nitrogen), is warming up to 90 ℃; Select fat lime piece 0.5kg, after adding clear water and boiling in right amount and dissolve, cross 100 mesh sieves and filter, to remove its impurity and lime mud, liquor calcis matter is spilled in Leaf of Indigowoad magma equably, fully stir 10h; Obtain anaerobic Indirubin and prepare group.
More above-mentioned three kinds of methods are prepared the method for Indirubin, specifically as shown in table 2.
Table 2 is prepared the comparison of effective constituent in the method for Indirubin
Note: the crude drug weight of each group is calculated with fresh goods.
Under anaerobic, the reason of indigo increase, may be to have oxygen to enter in pulverizing the process of Leaf of Indigowoad, caused indolol free radical oxidized generated indigo.Simultaneously from these routine data, produce each group leader raw indigo and mole number Indirubin and all surpassed Indirubin in crude drug, isatan B, indigo summation, tracing it to its cause be that other Indirubin prerequisite material has participated in reaction as materials such as isatan A and isatan Bs.
The proposition purifying of embodiment 3 Indirubins
Adopt in embodiment 2 preparation method of Indirubin under oxygen free condition, get be equivalent to crude drug amount 1kg reaction solution, remove by filter residue, with the whizzer removal of impurities of 15000rpm, get supernatant liquor, be concentrated into 800ml, be heated to 60 ℃; Add 500ml dichloromethane extraction 3 times, merge organic phase; Recovery has the medicinal extract that is greater than 1.1 to density, and medicinal extract weight is 71.22g;
Above-mentioned medicinal extract is mixed to sample in 200g aluminum oxide, dry, make powder, prepare loading sample; Take aluminum oxide 1500g, with methylene dichloride, mix dress post, dry method loading, use respectively chloroform, chloroform: ethyl acetate (9:1), chloroform: ethyl acetate (4:1), chloroform: ethyl acetate (2:1), chloroform: ethyl acetate (1:1) isopolarity gradient elution, the replacement of eluting solvent, adjusts with the variation of wash-out colour band.By TLC, detected and confirmed to obtain Indirubin colour band, crystallization purifying, obtains Indirubin purity 98.5%(marker method) adopt the extraction method that combine separated with aluminum oxide column chromatography, with respect to the content of Indirubin in reaction solution, the yield of Indirubin reaches 87.5%.
Claims (6)
1. the fresh and alive Leaf of Indigowoad of take is prepared the method for Indirubin as raw material, it is characterized in that: in pulverizing fresh and alive Leaf of Indigowoad woaded blue raw material, add istain.
2. according to claim 1ly take fresh and alive Leaf of Indigowoad and prepare the method for Indirubin as raw material, it is characterized in that: said method comprising the steps of:
1) in the fresh and alive Leaf of Indigowoad in the 12h that gathers, add water, pulverize, prepare extraction magma;
2) in extracting magma, add as istain, fully stir, adjust pH is greater than 7, heating, and reaction is greater than after 30min, filters, and collects water liquid;
3) mixture of one or more in use chloroform, monochloro methane, methylene dichloride, tetrachloromethane or ethyl acetate, heating, extraction water liquid, collects and merges organic phase;
4) reclaim organic phase, upper alumina column, wash-out, purifying obtains Indirubin crude product.
3. according to claim 2ly take fresh and alive Leaf of Indigowoad and prepare the method for Indirubin as raw material, it is characterized in that: described in step 1), be in the fresh and alive Leaf of Indigowoad in the 12h that gathers, to add medicinal material weight 2-5 water doubly, pulverize, the slurries of pulverizing can be crossed mesh sieve, prepare extraction magma.
4. according to claim 2ly take fresh and alive Leaf of Indigowoad and prepare the method for Indirubin as raw material, it is characterized in that: in the reacting by heating described in step 3), be filled with rare gas element, get rid of the oxygen in reaction liquid.
5. according to claim 2ly take fresh and alive Leaf of Indigowoad and prepare the method for Indirubin as raw material, it is characterized in that: step 2) described in adjust pH be greater than 7 for to add liming in reaction solution.
6. according to claim 2ly take fresh and alive Leaf of Indigowoad and prepare the method for Indirubin as raw material, it is characterized in that: the upper alumina column described in step 4), wash-out is: organic phase is recycled to dry, by one or more the mixture heating for dissolving in chloroform, monochloro methane, methylene dichloride, tetrachloromethane or ethyl acetate, in raw material: the ratio of aluminum oxide (1:2-4) is mixed, dry method wet method dress post, dry method loading; Collect the chromatographic band of Indirubin, reclaim, crystallization obtains Indirubin sterling.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310451101.6A CN103509835B (en) | 2013-09-29 | 2013-09-29 | A kind of method preparing indigo red for raw material with fresh and alive folium isatidis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310451101.6A CN103509835B (en) | 2013-09-29 | 2013-09-29 | A kind of method preparing indigo red for raw material with fresh and alive folium isatidis |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103509835A true CN103509835A (en) | 2014-01-15 |
| CN103509835B CN103509835B (en) | 2016-08-31 |
Family
ID=49893267
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310451101.6A Expired - Fee Related CN103509835B (en) | 2013-09-29 | 2013-09-29 | A kind of method preparing indigo red for raw material with fresh and alive folium isatidis |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103509835B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103980182A (en) * | 2014-05-28 | 2014-08-13 | 西北大学 | Biomimetic synthesis method of indirubin and derivatives thereof |
| CN103992260A (en) * | 2014-05-29 | 2014-08-20 | 哈尔滨医科大学 | Method for extracting indirubin from folium isatidis |
| CN112824458A (en) * | 2019-11-20 | 2021-05-21 | 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 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
-
2013
- 2013-09-29 CN CN201310451101.6A patent/CN103509835B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Non-Patent Citations (2)
| Title |
|---|
| 杨明 等: ""蓝"类植物中的前体物质转化为"靛"的机制探讨", 《中国中药杂志》, vol. 35, no. 7, 30 April 2010 (2010-04-30), pages 928 - 930 * |
| 陈雪梅: "青黛炮制过程基本原理研究", 《中国优秀硕士学位论文全文数据库医药卫生科技辑》, no. 7, 15 July 2009 (2009-07-15), pages 057 - 36 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103980182A (en) * | 2014-05-28 | 2014-08-13 | 西北大学 | Biomimetic synthesis method of indirubin and derivatives thereof |
| 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 |
| CN112824458A (en) * | 2019-11-20 | 2021-05-21 | 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 |
| 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 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103509835B (en) | 2016-08-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102669423B (en) | Moringa extract as well as preparation method of moringa extract and moringa feed additive | |
| CN102391549B (en) | Preparation method of abelmoschs esculentus water-soluble polysaccharide spray-dried powder | |
| CN100589813C (en) | Pomegranate rind extraction and its preparation method | |
| KR101277760B1 (en) | Process for production of urushiol free lacquer tree bark extract and its utilization | |
| CN103509835A (en) | Method for preparing indirubin with fresh folium isatidis as raw material | |
| CN101961371A (en) | Method for extracting and separating ginsenoside, flavone and polysaccharide from sweet gynostemma pentaphylla | |
| CN105648016A (en) | Herba cistanche small molecule peptide extract, preparation method and application thereof | |
| CN103468759B (en) | A kind of take leaf of Indigoplant as the method that Indirubin prepared by raw material | |
| CN112824386A (en) | Method for continuously preparing indigo naturalis, indigo and indirubin from fresh leaves of kale | |
| CN104026329A (en) | Extraction method of natural high-content selenium-source rice peptide | |
| CN103739650B (en) | A kind of preparation method of β monoammonium glycyrrhizinate | |
| CN105012778A (en) | Dendrobium officinale-coix seed capsules and preparation method thereof | |
| CN112608949A (en) | Preparation method and application of pseudo-ginseng flower extract | |
| CN101704703A (en) | Method for producing high-purity lycopene powder by utilizing plant products | |
| CN105901738A (en) | Fermentation grape extract, preparing method of fermentation grape extract and preparation containing fermentation grape extract | |
| CN109678834A (en) | A kind of preparation method for extracting anthocyanidin from mangosteen peel | |
| KR20010007847A (en) | The product method of Extract in Semissulcopira libertina and its foods | |
| CN102850463A (en) | Preparation method of allium chinense polysaccharide | |
| CN104026357B (en) | A kind of high protein pig feed easy to digest and preparation method thereof | |
| KR20130087769A (en) | Method for preparing boiled salt comprising red ginseng | |
| CN105594921A (en) | Cinnamomum kanehirai leaf finished product preparation method and cinnamomum kanehirai leaf concentrate preparation method and liver protection beverage | |
| CN105031290A (en) | Compound dendrobium officinale capsules and preparation method thereof | |
| CN105343468A (en) | Kidney-tonifying Banlong tablets and preparation method thereof | |
| CN104522429A (en) | Bamboo shoot shell fermentation compound duck feed and making method thereof | |
| CN103497222B (en) | A kind of method extracting woaded blue glycosides from fresh and alive Leaf of Indigowoad |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20160831 |