CN103585208B - Preparation method of high-quality andrographolide component - Google Patents
Preparation method of high-quality andrographolide component Download PDFInfo
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- CN103585208B CN103585208B CN201310577892.7A CN201310577892A CN103585208B CN 103585208 B CN103585208 B CN 103585208B CN 201310577892 A CN201310577892 A CN 201310577892A CN 103585208 B CN103585208 B CN 103585208B
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Abstract
The invention relates to a preparation method of a high-quality andrographolide component. The method comprises the steps that an andrographis paniculata medicinal material is smashed, and then subjected to methanol backflow extraction, water sedimentation, reversed phase chromatography and hydrophilic chromatography purification; and the andrographolide component is prepared. The andrographolide component is straw yellow, and the main ingredients of the andrographolide component is andrographolide, neoandrographolide, deoxyandrographolide and 14-deoxy-11,12-didehydroandrographolide. The content of andrographolide, neoandrographolide, deoxyandrographolide and 14-deoxy-11,12-didehydroandrographolide in the andrographolide component measured by a high performance liquid chromatography external standard method accounts for above 75% of the total mass, wherein andrographolide accounts for 41-44% of the total andrographolide component; neoandrographolide accounts for 14-21% of the total andrographolide component; deoxyandrographolide accounts for 18-25% of the total andrographolide component; and the balance is 14-deoxy-11,12-didehydroandrographolide. The method has the prominent advantages that the method is simple in technology, easy to operate and low in cost, and facilitates industrial production.
Description
[technical field]
The invention belongs to medical art, relate to a kind of method of extraction and isolation high-quality andrographolide component from Herba Andrographis medical material, particularly a kind of method preparing high-quality andrographolide component successively with reversed phase chromatography, hydrophilic chromatographic separation.
[background technology]
Herba Andrographis Andrographis paniculata (Burm.f) Nees. is herb or the leaf of acanthaceous plant Herba Andrographis, and dry aerial parts are used as medicine.Bitter in the mouth cold in nature, have antiphlogistic antibacterial, removing heat from blood subsides a swelling effect, cures mainly cold, fever, laryngopharynx swelling and pain, aphtha of the mouth and tongue, the puckery pain of pyretic stranguria etc. are important " Chinese medicine antibiotic ".The main chemical compositions of Herba Andrographis is diterpene ginkgolide and flavone compound, Folium Andrographis is mainly diterpene ginkgolide, that wherein content is maximum is andrographolide (Andrographolide), also comprise neoandrographolide (Neoandrographolide), desoxyandrographolide (Deoxyandrographolide) and dehydrorographolide (14-Deoxy-11,12-didehydroandrographolide) etc., the main active of andrographis root is flavone compound.
In recent years to the research of Herba Andrographis, mainly concentrate on the aspects such as the extraction and isolation of andrographolide and dehydrorographolide, refining, derivative, assay.What the extraction of Herba Andrographis active component was conventional has percolation, infusion process, potass extraction method, ultrasonic extraction and microwave―assisted extraction etc.; What the purification of Herba Andrographis active component was conventional has activated carbon decolorizing, positive solvent extraction, Sehadex LH-20 column chromatography, silica column purification etc.And rarely to study be Hydrolysis kinetics about andrographolide component.
Herba Andrographis product on sale in the market covers the multiple dosage forms such as tablet, capsule, drop pill, injection, and wherein andrographis tablet and CHUANXINLIAN JIAONANG are extracted by technique to be prepared into creat extract, makes corresponding dosage form; The main component of Herba Andrographitis dripping pills is andrographolide.Market analysis research shows, CHUANXINLIAN ZHUSHEYE occupies the maximum market share in Herba Andrographis product.The CHUANXINLIAN ZHUSHEYE such as Andrographolide, andrographolide, Lian Bizhi take andrographolide as raw material, be prepared from through certain technique, but clinical trial and research show, its unstability and some side effect cause the Zeng Huihuang CHUANXINLIAN ZHUSHEYE experience bottleneck phase for the moment.Xiyanping is the sulfonate injection that total andrographolides is obtained by sulfonation, because it is treating the unique curative effect in hand-foot-mouth disease, Xiyanping extensively obtains market accreditation, 2011 annual turnovers have broken through 8,000 ten thousand, again due to Xiyanping antiinflammatory, antiviral significant curative effect and safety reliably thereof, become the principal item of domestic Chinese medicine antiinflammatory, antiviral class medicine, space, future market is huge.
[summary of the invention]
The object of the invention is to overcome the deficiencies in the prior art, a kind of preparation method of high-quality andrographolide component is provided.
The object of the invention is to be achieved through the following technical solutions:
A preparation method for high-quality andrographolide component, its concrete steps are:
(1) reflux, extract: by methanol aqueous solution reflux, extract, twice after being pulverized by Folium Andrographis, merge extracted twice liquid;
The volumetric concentration of described methanol aqueous solution is 70 ~ 100%, and consumption is 10 ~ 15 times amount (v:m);
Described extraction time is each 1 ~ 3 hour;
(2) water precipitating: to be diluted to methanol volumetric concentration be 30 ~ 80% directly to adding pure water in step (1) gained extracting solution, leaves standstill after fully stirring, and filters, gets filtrate;
Described static conditions is: dwell temperature is 0 ~ 4 DEG C, and time of repose is 6 ~ 15 hours;
(3) Reverse phase chromatography: directly to adding the rear loading of pure water dilution in step (2) gained filtrate, carry out Reverse phase chromatography; The mass ratio of applied sample amount and filler is 1:20 ~ 1:80, carries out gradient elution successively with methanol aqueous solution, and each elution volume is 2 ~ 5 column volumes; Collect the effective meoh eluate of merging, be evaporated near doing and obtain thick paste;
The volumetric concentration of described methanol aqueous solution is 30 ~ 70%;
Described reversed phase chromatography packing material size is 5 ~ 60 μm;
(4) hydrophilic chromatographic purification: carry out hydrophilic chromatographic purification after step (3) gained thick paste ethyl acetate or acetonitrile being dissolved, applied sample amount and packing quality are than being 1:3 ~ 1:6, gradient elution is carried out successively with elute soln, each elution volume is 3 ~ 10 column volumes, collect and merge effective eluent concentrating under reduced pressure, dry faint yellow andrographolide component dry powder.
Wherein, the composition of faint yellow andrographolide component dry powder is mainly andrographolide, neoandrographolide, desoxyandrographolide and dehydrorographolide, four kinds of compositions are with quantified by external standard method, content accounts for more than 75% of gross mass, wherein andrographolide accounts for 41 ~ 44% of total lactones component, neoandrographolide accounts for 14 ~ 21% of total lactones component, and desoxyandrographolide accounts for 18 ~ 25% of total lactones component, and dehydrorographolide is surplus.
Described elute soln to be volumetric concentration be 1 ~ 20% methanol-ethyl acetate mixed solution or acetonitrile, 1% ~ 15% methanol-acetonitrile mixed solution.
Described hydrophilic chromatographic packing material size is 5 ~ 60 μ.
Compared with prior art, good effect of the present invention is:
(1) andrographolide component purity is high: because Herba Andrographis medical material total lactones content is up to 1% ~ 3%, the present invention is directed to lactone component characteristic, carry out extraction and isolation selectively, the content preparing gained total andrographolides component accounts for more than 75% of gross mass.
(2) solvent utilization rate is high, cost is low: the present invention is completed by FOUR EASY STEPS and prepares the extraction of high-quality andrographolide component, directly add water in solution in first three step operation and can carry out next step operation, there is not solvent and switch phenomenon, solvent utilization rate is high, can significantly reduce costs during commercial production.
(3) anti-phase and hydrophilic chromatographic technology coupling: utilize anti-phase and hydrophilic chromatographic technology to carry out separation and purification successively, make full use of filler and lactone component characteristic, reach highly purified separating resulting.
(4) method is simple, easy and simple to handle: the method preparing high-quality andrographolide component that the present invention relates to, automation equipment degree is high, simply easy to operate, is applicable to large-scale production.
[detailed description of the invention]
The detailed description of the invention of the preparation method of a kind of high-quality andrographolide component of the present invention is below provided.
Embodiment 1
After 10g Folium Andrographis is suitably pulverized, add 6 times amount 80% methanol solution reflux, extract, twice at every turn, each 2 hours, merge blackish green extracting solution.In gained extracting solution, directly adding certain water gaging, to be diluted to methanol volumetric concentration be 60%, leaves standstill after 10 hours and filter, obtain yellow green filtrate in 4 DEG C of refrigerators.
In gained filtrate, directly add water be diluted to methanol volumetric concentration and be 40% and be equally divided into two batches to carry out Reverse phase chromatography.Often pulling on sample solid masses is 0.96g, and reversed phase chromatography packing quality is 20g.Be first that the methanol of 40% is by strong for major part polar impurity and the removal of pigment eluting by 3VB volumetric concentration, then with the methanol that methanol, 2VB volumetric concentration that 5VB volumetric concentration is 55% are 60%, the main lactone component of Herba Andrographis is carried out eluting and collected successively, merge 55% methanol and 60% meoh eluate, be evaporated to closely to do and obtain thick paste, quality is 0.57g.Carry out hydrophilic chromatographic purification after gained thick paste 10mL acetic acid ethyl dissolution, hydrophilic filler quality is 3g.First use 8VB methanol-ethyl acetate (5:95v:v) to andrographolide, desoxyandrographolide and dehydrorographolide component carry out eluting and collect, 4V B methanol-ethyl acetate (10:90v:v) is used to carry out eluting to neoandrographolide and collect again, merge all eluents, the dry faint yellow dry powder of end product obtained after concentrating under reduced pressure, quality is 0.35g.Measure through high-performance liquid chromatography, in this component, total andrographolides content accounts for 80% of product gross mass, wherein andrographolide accounts for 43% of total lactones component, neoandrographolide accounts for 14% of total lactones component, desoxyandrographolide accounts for 23% of total lactones component, and dehydrorographolide accounts for 20% of total lactones component.
Embodiment 2
After 20g Folium Andrographis is suitably pulverized, add 5 times amount 85% methanol solution reflux, extract, twice, each 2.5 hours, merge blackish green extracting solution.In gained extracting solution, directly adding water, to be diluted to methanol volumetric concentration be 50%, leaves standstill after 9 hours and filter, obtain yellow green filtrate in 4 DEG C of refrigerators.
In gained filtrate, directly add water be diluted to methanol volumetric concentration and be 30% and gained filtrate is equally divided into four batches to carry out Reverse phase chromatography.Often pulling on sample solid masses is 0.98g, and reversed phase chromatography packing quality is 20g.Be first that the methanol of 30% is by strong for major part polar impurity and the removal of pigment eluting by 3VB volumetric concentration, then with the methanol that methanol, 3VB volumetric concentration that 3VB volumetric concentration is 55% are 60%, the main lactone component of Herba Andrographis is carried out eluting and collected successively, merge 55% methanol and 60% meoh eluate, be evaporated to closely to do and obtain thick paste, quality is 1.2g.Be divided into two batches after gained thick paste 20mL acetic acid ethyl dissolution and carry out hydrophilic chromatographic purification, hydrophilic chromatographic packing quality is 3g.First use 7VB methanol-ethyl acetate (5:95v:v) to andrographolide, desoxyandrographolide and dehydrorographolide component carry out eluting and collect, 5VB methanol-ethyl acetate (10:90v:v) is used to carry out eluting to neoandrographolide and collect again, merge all eluents, the dry faint yellow dry powder of end product obtained of concentrating under reduced pressure final vacuum, quality is 0.64g.Measure through high-performance liquid chromatography, in this component, total andrographolides content accounts for 76% of product gross mass, wherein andrographolide accounts for 43% of total lactones component, neoandrographolide accounts for 17% of total lactones component, desoxyandrographolide accounts for 21% of total lactones component, and dehydrorographolide accounts for 19% of total lactones component.
Embodiment 3
After 15g Folium Andrographis is suitably pulverized, add 7 times amount 80% methanol solution reflux, extract, twice, each 2 hours, merge blackish green extracting solution.In gained extracting solution, directly adding water, to be diluted to methanol volumetric concentration be 65%, leaves standstill after 12 hours and filter, obtain yellow green filtrate in 4 DEG C of refrigerators.
Directly add water in gained filtrate to be diluted to methanol volumetric concentration and to be 40% and to be equally divided into three batches to carry out Reverse phase chromatography.Often pulling on sample solid masses is 1.10g, and reversed phase chromatography packing quality is 20g.Be first that the methanol of 40% is by strong for major part polar impurity and the removal of pigment eluting by 3VB volumetric concentration, then with the methanol that methanol, 3VB volumetric concentration that 4VB volumetric concentration is 55% are 60%, the main lactone component of Herba Andrographis is carried out eluting and collected successively, merge the eluent of 55% methanol and 60% methanol, be evaporated to closely to do and obtain thick paste, quality is 0.86g.Gained thick paste 10mL acetonitrile carries out the purification of hydrophilic chromatographic after dissolving, hydrophilic chromatographic packing quality is 3g.First use 8VB acetonitrile to andrographolide, neoandrographolide, desoxyandrographolide and dehydrorographolide component carry out eluting and collect, 4VB methanol-acetonitrile (5:95v:v) is used to carry out eluting to neoandrographolide and collect again, merge all eluents, the dry faint yellow dry powder of the heaviest product obtained of concentrating under reduced pressure final vacuum, quality is 0.45g.Measure through high-performance liquid chromatography, in this component, total andrographolides content accounts for 75% of product gross mass, wherein andrographolide accounts for 44% of total lactones component, neoandrographolide accounts for 21% of total lactones component, desoxyandrographolide accounts for 18% of total lactones component, and dehydrorographolide accounts for 17% of total lactones component.
Embodiment 4
After 30g Folium Andrographis is suitably pulverized, add 5 times amount 90% methanol solution reflux, extract, twice, each 2 hours, merge blackish green extracting solution.In gained extracting solution, directly adding water, to be diluted to methanol volumetric concentration be 40%, leaves standstill after 6 hours and filter, obtain yellow green filtrate in 4 DEG C of refrigerators.
Gained filtrate is equally divided into six batches and carries out Reverse phase chromatography.Often pulling on sample solid masses is 1.20g, and reversed phase chromatography packing quality is 20g.Be first that the methanol of 40% is by strong for major part polar impurity and the removal of pigment eluting by 4VB volumetric concentration, then with the methanol that methanol, 3VB volumetric concentration that 2VB volumetric concentration is 55% are 60%, the main lactone component of Herba Andrographis is carried out eluting and collected successively, merge effective eluent, be evaporated to closely to do and obtain thick paste, quality is 1.77g.Gained thick paste 27mL acetonitrile is equally divided into three batches after dissolving and carries out hydrophilic chromatographic purification, and hydrophilic filler quality is 3g.First use 10VB acetonitrile to andrographolide, neoandrographolide, desoxyandrographolide and dehydrorographolide component carry out eluting and collect, 4VB methanol-acetonitrile (10:90v:v) is used to carry out eluting to neoandrographolide and collect again, merge all eluents, the dry faint yellow dry powder of end product obtained of concentrating under reduced pressure final vacuum, quality is 1.05g.Measure through high-performance liquid chromatography, in this component, total andrographolides content accounts for 75% of gross mass, wherein andrographolide accounts for 41% of total lactones component, neoandrographolide accounts for 15% of total lactones component, desoxyandrographolide accounts for 25% of total lactones component, and dehydrorographolide accounts for 19% of total lactones component.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, without departing from the inventive concept of the premise; can also make some improvements and modifications, these improvements and modifications also should be considered within the scope of protection of the present invention.
Claims (5)
1. a preparation method for high-quality andrographolide component, is characterized in that, its concrete steps are:
(1) reflux, extract: by methanol aqueous solution reflux, extract, twice after being pulverized by Folium Andrographis, merge extracted twice liquid;
The volumetric concentration of described methanol aqueous solution is 70 ~ 100%, and consumption is 10 ~ 15 times amount;
(2) water precipitating: to be diluted to methanol volumetric concentration be 30 ~ 80% directly to adding pure water in step (1) gained extracting solution, leaves standstill after fully stirring, and filters, gets filtrate;
Described static conditions is: dwell temperature is 0 ~ 4 DEG C, and time of repose is 6 ~ 15 hours;
(3) Reverse phase chromatography: directly to adding the rear loading of pure water dilution in step (2) gained filtrate, carry out Reverse phase chromatography; The mass ratio of applied sample amount and filler is 1: 20 ~ 1: 80, carries out gradient elution successively with methanol aqueous solution, and each elution volume is 2 ~ 5 column volumes; Collect the effective meoh eluate of merging, be evaporated near doing and obtain thick paste;
The volumetric concentration of described methanol aqueous solution is 30 ~ 70%;
Described reversed phase chromatography packing material size is 5 ~ 60 μm;
(4) hydrophilic chromatographic purification: carry out hydrophilic chromatographic purification after step (3) gained thick paste ethyl acetate or acetonitrile being dissolved, applied sample amount is 1: 3 ~ 1: 6 with packing quality ratio, gradient elution is carried out successively with elute soln, each elution volume is 3 ~ 10 column volumes, collect and merge effective eluent concentrating under reduced pressure, dry faint yellow andrographolide component dry powder;
Described elute soln to be volumetric concentration be 1 ~ 20% methanol-ethyl acetate mixed solution or acetonitrile, 1% ~ 15% methanol-acetonitrile mixed solution.
2. the preparation method of a kind of high-quality andrographolide component as claimed in claim 1, is characterized in that, in described step (1), described extraction time is each 1 ~ 3 hour.
3. the preparation method of a kind of high-quality andrographolide component as claimed in claim 1, it is characterized in that, in described step (4), the composition of described faint yellow andrographolide component dry powder is andrographolide, neoandrographolide, desoxyandrographolide and dehydrorographolide.
4. the preparation method of a kind of high-quality andrographolide component as claimed in claim 1, it is characterized in that, in described step (4), the composition of described faint yellow andrographolide component dry powder is andrographolide, neoandrographolide, desoxyandrographolide and dehydrorographolide; Andrographolide accounts for 41 ~ 44% of total lactones component, and neoandrographolide accounts for 14 ~ 21% of total lactones component, and desoxyandrographolide accounts for 18 ~ 25% of total lactones component, and dehydrorographolide is surplus.
5. the preparation method of a kind of high-quality andrographolide component as claimed in claim 1, is characterized in that, in described step (4), described hydrophilic chromatographic packing material size is 5 ~ 60 μm.
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| CN104382968B (en) * | 2014-10-27 | 2021-03-30 | 江苏康缘药业股份有限公司 | Extraction method of andrographolide, andrographolide pharmaceutical composition and application |
| CN104447642B (en) * | 2014-12-19 | 2016-07-13 | 黄曦 | The preparation of industrialization chromatographic separation and purification method of andrographolide |
| WO2018081959A1 (en) | 2016-11-02 | 2018-05-11 | Nutrition Science Partners Limited | Extracts of andrographis paniculata, methods for preparation and use thereof |
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| CN101347473B (en) * | 2007-07-20 | 2011-09-21 | 天津天士力制药股份有限公司 | Effective component of creat and preparation and use thereof |
| CN101559088B (en) * | 2009-05-21 | 2012-07-25 | 雷允上药业有限公司 | Production technique of andrographolide and neoandrographolide, dehydroanddrographolide, oxyandrographolide |
| CN101671322B (en) * | 2009-09-30 | 2012-02-29 | 合肥工业大学 | Method for separating and purifying andrograholide |
| CN102382083B (en) * | 2010-09-01 | 2015-07-15 | 天津天士力现代中药资源有限公司 | Preparation method of andrographolide |
| CN103275042A (en) * | 2013-04-23 | 2013-09-04 | 四川省裕通生物技术有限公司 | Extraction and purification method for andrographolide |
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