CN104447642B - The preparation of industrialization chromatographic separation and purification method of andrographolide - Google Patents
The preparation of industrialization chromatographic separation and purification method of andrographolide Download PDFInfo
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- CN104447642B CN104447642B CN201410795234.XA CN201410795234A CN104447642B CN 104447642 B CN104447642 B CN 104447642B CN 201410795234 A CN201410795234 A CN 201410795234A CN 104447642 B CN104447642 B CN 104447642B
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
- C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
- C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
- C07D307/56—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D307/60—Two oxygen atoms, e.g. succinic anhydride
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Abstract
The present invention relates to the preparation of industrialization chromatographic separation and purification method of a kind of andrographolide.Comprise the following steps: (1) is dissolved in solvent after being pulverized by Folium Andrographis, raw material is extracted, after solid-liquid separation, obtains extracting solution；(2) said extracted liquid drying means is removed liquid, filter and obtain andrographolide crude product；(3) andrographolide crude product is configured to solution, is filtered to remove insoluble matter, it is thus achieved that andrographolide solution；(4) above-mentioned andrographolide solution is pumped into dynamic axial compression column preparing chromatography system, through twice gradient elution, with the New UV Spectrophotometric detector that detection wavelength is 254mm, collect the retention time distillate at 15 20min.Solvent for use of the present invention is cheap, safety, and isolated and purified process one step completes, and can on-line real time monitoring, substantially increase safety, saved raw material and time cost, it is adaptable to large-scale industrial production.
Description
Technical field
The present invention relates to field of natural product extraction, the preparation of industrialization chromatograph particularly relating to a kind of andrographolide is divided
From purification process.
Background technology
Herba Andrographis is Acanthaceae Herba Andrographis platymiscium, is just used for treating gastrointestinal tract and respiratory tract before the several century of Asia
Infection, heating, herpes, throat pain and various bacterial infection.In the traditional Chinese medical science, Herba Andrographis is a kind of important grass treating flu
Medicine, it is used for bringing down a fever, healing, and cheap.
Owing to andrographolide is the principle active component of Herba Andrographis, compared with stem, fruit, containing in Herba Andrographis in Folium Andrographis
Ester is the highest；Andrographolide is esters structure, in aqueous facile hydrolysis, open loop, isomerization, therefore affects medicine stability.?
Find have in different temperatures, different pH environment, different biological samples and difference in the stability study of andrographolide
In machine solvent, andrographolide stability has obvious difference.Temperature is the lowest, and the stability of andrographolide is the best；?
Instability under the conditions of alkalescence, and along with the increase of base strength, its unstability strengthens；It is the most stable, but
And nonacid the strongest, stability is the best；Its most stable of pH value is 3～5.Have now been found that andrographolide has following
Pharmacological action: anti-inflammation, refrigeration function；Anticancer and immunoregulation effect；Virus and immunological enhancement；Cardiovascular；
The liver protecting.
It is more that relevant andrographolide extracts the method report separated, and has decoction and alcohol sedimentation technique, alkali to carry ethyl alcohol recrystallization method, water
Put forward macroporous adsorbent resin elution method etc..Chinese patent " andrographolide and the preparation side thereof of Patent No. 201310096203.0
Method " describe a kind of method that recrystallization prepares andrographolide, the method pre-treatment step is relatively complicated, need to use a large amount of second
Alcohol, and gained purity is the highest.Chinese patent " the preparation of high-quality andrographolide component of Patent No. 201310577892.7
Method " describe the extracting method carrying out a small amount of high-quality andrographolide with reversed phase chromatography and hydrophilic chromatographic combination, the method
Compared to previous piece patent, there has been bigger raising, but it is longer to still suffer from disengaging time on separation method, product fractional dose has
Limit, and the problem being difficult to monitor separation progress in real time.
Summary of the invention
The technical problem to be solved is numerous for technique existing during current Herba Andrographis crude product refining
Miscellaneous, purity is the highest, and disengaging time is longer, and product fractional dose is limited, and is difficult to monitor the problems such as separation progress in real time.
In order to solve above-mentioned technical problem, the present invention provides a kind of and uses preparation of industrialization chromatographic fractionation system one step online
Separate the new method of preparation high-purity andrographolide.Technical scheme is as follows: the industrialization of a kind of andrographolide
Preparative hplc isolation and purification method, comprises the following steps:
(1) it is dissolved in solvent after Folium Andrographis being pulverized, under assosting effect, raw material is extracted, obtain after solid-liquid separation
Extracting solution；
(2) said extracted liquid drying means is removed liquid, filter and obtain andrographolide crude product；
(3) andrographolide crude product is configured to solution, is filtered to remove insoluble matter, it is thus achieved that andrographolide solution；
(4) above-mentioned andrographolide solution is pumped into dynamic axial compression column preparing chromatography system, through twice gradient elution,
With the New UV Spectrophotometric detector that detection wavelength is 254mm, collect the retention time distillate at 15 20min；
Wherein:
Dynamic axial compression column preparing chromatography system described in step (4), column dimension is50*250mm, filler is 10
The 50 anti-phase spherical silica gels of μm, flowing is the aqueous solution of organic solvent mutually, and concentration is 10 90wt%.
Further, flowing described in step (4) is second alcohol and water mutually, and gradient scope is 30:70 80:20 for the first time,
Flow velocity is 100ml/min, and elution time is 30min, and gradient scope is 90:10 for the second time, and elution time is 20min.
Further, flowing described in step (4) is first alcohol and water mutually, and gradient scope is 40:60 20:80 for the first time,
Flow velocity be 120ml/min elution time be 50min, gradient scope is 90:10 for the second time, and elution time is 10min.
Further, flowing described in step (4) is acetonitrile and water mutually, and gradient scope is 20:80 30:70 for the first time,
Flow velocity is 80ml/min, and elution time is 30min, and gradient scope is 50:50 for the second time, and elution time is 20min.
Further, anti-phase spherical silica gel described in step (4) is anti-phase n-octadecane base silica gel.
Further, described in step (4), anti-phase n-octadecane base silica gel particle diameter is 10 μm or 22 μm.
Further, solvent described in step (1) is water or organic solvent or water and the mixture of organic solvent.
Further, assosting effect described in step (1) is heating, uses microwave or ultrasound wave heating.
Further, drying means described in step (2) steams for spraying or rotation.
The invention has the beneficial effects as follows the Herba Andrographis extracting solution after directly using crude product to extract to prepare andrographolide, nothing
Needing the pretreatment process that large amount of complex is loaded down with trivial details, solvent for use is cheap, safety, and isolated and purified process one step completes, and can be real online
Time monitoring, substantially increase safety, saved raw material and time cost, it is adaptable to large-scale industrial production.Efficiently liquid phase
Chromatograph finally measures display, and andrographolide purity is more than 97%, the most miscellaneous is less than 3%.
Accompanying drawing explanation
Fig. 1 is the high-performance liquid chromatogram determination collection of illustrative plates of original andrographolide crude product.
Fig. 2 is by the isolated and purified punching of dynamic axial compression column being filled with 10 μm n-octadecane base reverse phase silica gel fillers
The high-performance liquid chromatogram determination collection of illustrative plates of lotus lactone, sample introduction 400mg.
Fig. 3 is with the isolated and purified Herba Andrographis of dynamic axial compression column being filled with 10 μm n-octadecane base reverse phase silica gel fillers
The high-performance liquid chromatogram determination collection of illustrative plates of lactone, sample introduction 1g.
Fig. 4 is with the isolated and purified Herba Andrographis of dynamic axial compression column being filled with 10 μm n-octadecane base reverse phase silica gel fillers
The high-performance liquid chromatogram determination collection of illustrative plates of lactone, sample introduction 3g.
Fig. 5 is with the isolated and purified Herba Andrographis of dynamic axial compression column being filled with 10 μm n-octadecane base reverse phase silica gel fillers
Lactone prepare collection of illustrative plates, sample introduction 3g.
Detailed description of the invention
Below by specific embodiment, the present invention will be further described.
After Folium Andrographis is pulverized with water or organic solvent or water/organic solvent mixed solvent under assosting effect to raw material
Extracting, wherein assosting effect includes heating, uses microwave or ultrasonic etc.；Obtain blackish green Herba Andrographis after solid-liquid separation to extract
Liquid；Using the means of being dried to remove liquid again, the means that are wherein dried include spraying or rotation steaming etc., it is thus achieved that andrographolide crude product (is worn
Heart lotus lactone content is between 10-50%), now, the high-performance liquid chromatogram determination collection of illustrative plates of andrographolide crude product is as shown in Figure 1.
High performance liquid chromatography (HPLC) is used to study and optimize the optimal separation purification condition of andrographolide.Thereafter by it
Equal proportion is amplified, and separates, online in the dynamic axial compression column (DAC) being filled with anti-phase n-octadecane base silica filler
Collect the eluent of andrographolide correspondence chromatographic peak, a step separation purity > 95%, reach as high as more than 99%；Yield typically exists
In the range of 70% ~ 90%, it is inversely proportional to purity.
Using filler is 10-50 micron reverse phase spherical silica gel, the preferably 10 or 22 microns anti-phase spherical silica gels of C18；
Flowing is the aqueous solution of organic solvent mutually, and organic solvent includes but not limited to methanol, ethanol, acetonitrile etc., and concentration exists
Between 10-90wt%, for cost consideration, preferably methanol, ethanol.
Embodiment 1
1. take 400mg andrographolide crude product and be configured to solution, be filtered to remove insoluble matter；
2. andrographolide solution being pumped into dynamic axial compression column preparing chromatography system, column dimension is Φ 50 × 250mm,
N-octadecane base reverse phase silica gel packing material size is 10 μm, and applied sample amount is 400mg, and flowing is ethanol and water mutually, and gradient scope is 30:
70 ~ 80:20, flow velocity is 100ml/min, elution time 30min.Afterwards gradient is adjusted to 90:10, continuation eluting 20min, one
Individual separation cycle terminates.The detection wavelength of the New UV Spectrophotometric detector used is 254nm, collects retention time 15 ~ 20min's
Distillate.As in figure 2 it is shown, through HPLC purity assay be 99.0%.
Embodiment 2
1. take 1g andrographolide crude product and be configured to solution, be filtered to remove insoluble matter；
2. andrographolide solution being pumped into dynamic axial compression column preparing chromatography system, column dimension is Φ 50 × 250mm,
N-octadecane base reverse phase silica gel packing material size is 10 μm, and applied sample amount is 1g, flowing be ethanol and water mutually, gradient scope be 30:70 ~
80:20, flow velocity is 100ml/min, elution time 30min.Afterwards gradient is adjusted to 90:10, continues eluting 20min, one
Separation cycle terminates.The detection wavelength of the New UV Spectrophotometric detector used is 254nm, collects retention time evaporating at 15 ~ 20min
Go out liquid.As it is shown on figure 3, through HPLC purity assay be 99.3%.
Embodiment 3
1. take 3g andrographolide crude product and be configured to strength solution, be filtered to remove insoluble matter；
2. andrographolide solution is pumped into dynamic axial compression column preparing chromatography system, column dimension be Φ 50 ×
250mm, n-octadecane base reverse phase silica gel packing material size is 10 μm, and applied sample amount is 3g, and flowing is ethanol and water mutually, and gradient scope is
30:70 ~ 80:20, flow velocity is 100ml/min, elution time 30min.Afterwards gradient is adjusted to 90:10, continues eluting
20min, a separation cycle terminates.The detection wavelength of the New UV Spectrophotometric detector used is 254nm, collects retention time 15
The distillate of ~ 20min.As shown in Figure 4, through HPLC purity assay be 99.90%.Packing material size is that 10 μm n-octadecane bases are anti-phase
The isolated and purified andrographolide of dynamic axial compression column of silica filler prepare collection of illustrative plates, as shown in Figure 5.
Embodiment 4
1. take 3g andrographolide crude product and be configured to strength solution, be filtered to remove insoluble matter；
2. andrographolide solution is pumped into dynamic axial compression column preparing chromatography system, column dimension be Φ 50 ×
250mm, filler uses 22 micron reverse phase spherical silica gels, and applied sample amount is 3g, flowing be ethanol and water mutually, gradient scope be 30:70 ~
80:20, flow velocity is 100ml/min, elution time 30min.Afterwards gradient is adjusted to 90:10, continues eluting 20min, one
Separation cycle terminates.The detection wavelength of the New UV Spectrophotometric detector used is 254nm, collects retention time evaporating at 15 ~ 20min
Go out liquid.It is 98.3% through HPLC purity assay.
Embodiment 5
1. take 3g andrographolide crude product and be configured to strength solution, be filtered to remove insoluble matter；
2. andrographolide solution is pumped into dynamic axial compression column preparing chromatography system, column dimension be Φ 50 ×
250mm, n-octadecane base reverse phase silica gel packing material size is 10 μm, and applied sample amount is 3g, and using flowing is methanol and water mutually, gradient model
Enclosing for 40:60 ~ 20:80, flow velocity is 120ml/min, elution time 50min.Afterwards gradient is adjusted to 90:10, continues eluting
10min, a separation cycle terminates.The detection wavelength of the New UV Spectrophotometric detector used is 254nm, collects retention time 15
The distillate of ~ 20min.It is 99.5% through HPLC purity assay.
Embodiment 6
1. take 3g andrographolide crude product and be configured to strength solution, be filtered to remove insoluble matter；
2. andrographolide solution is pumped into dynamic axial compression column preparing chromatography system, column dimension be Φ 50 ×
250mm, n-octadecane base reverse phase silica gel packing material size is 10 μm, and applied sample amount is 3g, and using flowing is acetonitrile and water mutually, gradient model
Enclosing for 20:80 ~ 30:70, flow velocity is 80ml/min, elution time 30min.Afterwards gradient is adjusted to 50:50, continues eluting
20min, a separation cycle terminates.The detection wavelength of the New UV Spectrophotometric detector used is 254nm, collects retention time 15
The distillate of ~ 20min.It is 98.8% through HPLC purity assay.
The foregoing is only to explain the preferred embodiments of the present invention, the most according to this present invention is made any pro forma
Limit, therefore every any form made on the basis of the creation spirit of the present invention is modified or change, all should belong to the present invention's
Protection category.
Claims (3)
1. the preparation of industrialization chromatographic separation and purification method of an andrographolide, it is characterised in that: comprise the following steps；
(1) it is dissolved in solvent after Folium Andrographis being pulverized, raw material is extracted, after solid-liquid separation under microwave or ultrasound wave heat
Obtain extracting solution；
(2) said extracted liquid drying means is removed liquid, filter and obtain andrographolide crude product；
(3) andrographolide crude product is configured to solution, is filtered to remove insoluble matter, it is thus achieved that andrographolide solution；
(4) above-mentioned andrographolide solution is pumped into dynamic axial compression column preparing chromatography system, through twice gradient elution, with inspection
Survey the New UV Spectrophotometric detector that wavelength is 254mm, collect the retention time distillate at 15 20min；
Wherein, dynamic axial compression column preparing chromatography system described in step (4), column dimension is 50*250mm, and filler is particle diameter
The anti-phase n-octadecane base silica gel of 10 μm, flowing is ethanol and water mutually, and concentration is 10 90wt%, and gradient scope is 30 for the first time:
70 80:20, flow velocity is 100ml/min, and elution time is 30min, and gradient scope is 90:10 for the second time, and elution time is
20min；Or, filler is the anti-phase n-octadecane base silica gel of particle diameter 10 μm, and flowing is acetonitrile and water mutually, and concentration is 10
90wt%, gradient scope is 20:80 30:70 for the first time, and flow velocity is 80ml/min, and elution time is 30min, for the second time gradient
Scope is 50:50, and elution time is 20min；Or, filler is the anti-phase n-octadecane base silica gel of particle diameter 22 μm, and flowing is mutually
Ethanol and water, concentration is 10 90wt%, and gradient scope is 30:70 80:20 for the first time, and flow velocity is 100ml/min, during eluting
Between be 30min, gradient scope is 90:10 for the second time, and elution time is 20min.
Method the most according to claim 1, it is characterised in that: solvent described in step (1) is water or organic solvent or water
And organic solvent.
Method the most according to claim 1, it is characterised in that: drying means described in step (2) steams for spraying or rotation.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101347473A (en) * | 2007-07-20 | 2009-01-21 | 天津天士力制药股份有限公司 | Effective component of creat and preparation and use thereof |
| CN103585208A (en) * | 2013-11-18 | 2014-02-19 | 华东理工大学 | Preparation method of high-quality andrographolide component |
| CN103664842A (en) * | 2013-12-16 | 2014-03-26 | 无锡济民可信山禾药业股份有限公司 | Continuous chromatographic separation method for andrographolide |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101347473A (en) * | 2007-07-20 | 2009-01-21 | 天津天士力制药股份有限公司 | Effective component of creat and preparation and use thereof |
| CN103585208A (en) * | 2013-11-18 | 2014-02-19 | 华东理工大学 | Preparation method of high-quality andrographolide component |
| CN103664842A (en) * | 2013-12-16 | 2014-03-26 | 无锡济民可信山禾药业股份有限公司 | Continuous chromatographic separation method for andrographolide |
Non-Patent Citations (1)
| Title |
|---|
| 工业制备色谱在中药分离制备中的应用;闫海全等;《广东微量元素科学》;20091231;第16卷(第2期);第14-18页 * |
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Effective date of registration: 20150324 Address after: 213000 Changzhou science and Education City, No. 801 middle Wu Road, Wujin District, Jiangsu, Changzhou, Nanjing University, Changzhou Hi Tech Research Institute, Room 202 Applicant after: Huang Xi Applicant after: Huang Genliang Address before: 213000 Changzhou science and Education City, No. 801 middle Wu Road, Wujin District, Jiangsu, Changzhou, Nanjing University, Changzhou Hi Tech Research Institute, Room 202 Applicant before: Huang Xi |
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