CN102464635B - Separation and purification method of arctigenin - Google Patents
Separation and purification method of arctigenin Download PDFInfo
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- CN102464635B CN102464635B CN201010540480.2A CN201010540480A CN102464635B CN 102464635 B CN102464635 B CN 102464635B CN 201010540480 A CN201010540480 A CN 201010540480A CN 102464635 B CN102464635 B CN 102464635B
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Abstract
Belonging to the technical field of medicines, the invention specifically provides a separation and purification method of arctigenin. The method comprises the steps of: dissolution, filtration, separation and purification through high efficiency preparative liquid chromatography, product recovery, etc. Specifically, the separation and purification adopts reversed-phase high efficiency preparative liquid chromatography, a dynamic axial compression column is employed as a chromatographic column, an acetonitrile water solution is taken as a mobile phase, and an ultraviolet detector is used for on-line detection. The method of the invention has the advantages of simple operation, short production period, high separation efficiency, stable technology, low cost, and can realize high-purity separation and preparation of a lot of arctigenin.
Description
Technical field
The present invention relates to biochemical field, be specifically related to a kind of separation purification method of l-arctigenin.
Background technology
L-arctigenin (arctigenin), chemical name (3R, 4R)-4-[(3,4-dimethoxyphenyl) methyl) dihydro-3-[(4-hydroxy-3-methoxyphenyl) methyl]-2 (3H)-furanone; No. CAS: 7770-78-7; Molecular formula: C
21h
24o
6; Molecular weight 372.41; Structural formula is as follows:
L-arctigenin is the effective constituent that can directly be absorbed by the body, be used for the treatment of and preventing chronic renal failure and renal fibrosis, antiviral, antitumor, skin whitening, antianaphylaxis, anti-inflammatory, treatment diabetes and complication, diabetic nephropathy, also for killing aquatic animal vermin etc.
Natural l-arctigenin is mainly present in composite family (Compasitate) burdock burdock (Arctiumlappal).The method preparing l-arctigenin at present mainly contains two large classes: one is extracting directly from plant materials; Two is obtained through hydrolysis by the arctinin that content in plant materials is higher, the l-arctigenin crude product of this two class methods gained all needs purifying, and current purification process mainly adopts chromatography, recrystallization method, and the combination of two kinds of methods, wherein chromatography comprises silica gel column chromatography and liquid phase method.
Adopt the purification process of l-arctigenin to be silica gel H post decompression post in CN200710135648.X and CN200710135646., chloroform-methanol gradient elution, the purity of final gained l-arctigenin is 98.2%.Gradient elution requires higher, and chloroform toxicity is comparatively large, and solvent for use amount is large, and pollute large, the purity of last gained l-arctigenin is lower.
The purification process of l-arctigenin is adopted to be by l-arctigenin crude product 60 order silica gel mixed samples in CN200810094377.2, thick silica gel volume is 1-2 times of l-arctigenin crude product, water-bath is dried, dry method loading, through 200-300 order silica gel column chromatography, chloroform-methanol 98: 1 wash-out, elutriant concentrates merging and namely obtains l-arctigenin, and it is more than 90% that HPLC records purity.
With silica gel column chromatography in CN200410015465, solvent recrystallization or the sub-aglycon of method purified edible burdock that both combine, wherein silica gel column chromatography need through one or many, with chloroform/methanol (100: 0-95: 5) for elutriant, indicate with TLC (chloroform/methanol (97: 3)), again through recrystallizing methanol, obtaining l-arctigenin through HPLC detection purity is 99%.Detect after first chromatography, detect delayed, efficiency is low, complex steps, and quality is wayward, and yield is low.
" chromatogram " 2003,31 (1): 52-55), Liu Shiming etc.; The Isolation and ldentification of micro-lignan arctinin and l-arctigenin in Herba Arctii leaf, adopt the arctinin in reverse phase liquid chromatography separation and purification burdock leaf trace lignan and l-arctigenin, the chromatographic column of employing is common analytical column, and applied sample amount is little; Gradient elution is higher to equipment requirements; Except containing except first alcohol and water in moving phase, also containing phosphoric acid, cause and be difficult to removing in Product recycling, the purity of the finished product is not high, and more than 95%.
Summary of the invention
For above-mentioned the deficiencies in the prior art, the invention provides a kind of method adopting anti-phase preparative liquid chromatography separation and purification l-arctigenin, the inventive method adopts preparative high-performance liquid chromatographic system to carry out separation and purification to l-arctigenin, by suitable pre-treating process and chromatographic condition, reach good separating effect, and UV-detector on-line monitoring, process intuitive operation is easy, specific aim collects l-arctigenin, with clearly defined objective, avoid conventional column chromatography and be first separated the wasting of resources that rear detection causes, and be easy to control quality product.Products obtained therefrom purity, yield are high.The reference substance that can be used as assay uses, and also can directly be used for making its preparation.
In high performance preparative liquid chromatography sepn process, the selection of chromatographic condition is extremely important, and it plays conclusive effect to the peak sequence, peak shape, separating effect etc. of material each in sample solution; Chromatographic condition mainly comprises chromatographic column (comprising filler, column length and internal diameter etc.), moving phase (comprise composition and flow velocity etc.), column temperature, determined wavelength, detector etc., the selection of each chromatographic condition and combine most important.
The present inventor is by a large amount of experimental studies and comparative analysis, determine each chromatographic condition as above, make the optimizations such as the appearance time of each material in sample solution, peak shape, separating effect, being conducive to l-arctigenin monomer with each impurity is well separated, and shortens the production cycle.
In order to realize foregoing invention object, the technical solution used in the present invention is as follows:
L-arctigenin crude product acetonitrile-aqueous solution is dissolved, filter, filtrate is diluted to the l-arctigenin solution of 8-15mg/ml further by purified water, gained solution high performance preparative liquid chromatography wash-out is separated, use UV-detector on-line checkingi, specific aim collects the preparation component of l-arctigenin, obtains l-arctigenin solution, obtained l-arctigenin solution is removed acetonitrile and water with underpressure distillation and freeze-drying successively, obtains l-arctigenin sterling.
Specifically comprise the following steps:
(1) dissolve, filter:
According to solids quality: the ratio of solvent volume=1Kg: 50L, with acetonitrile-water (volume ratio 9: (0-3)) solution, by solid l-arctigenin dissolving crude product, with organic filtering with microporous membrane of 0.45 μm after dissolving, filtrate is diluted to the solution of 8-15mg (being equivalent to crude product quality)/ml further by purified water, prepare the raw material of the sub-aglycon of purified edible burdock as next step.
The acetonitrile solution dissolution filter of high density is first used in this step, remove outside conventional insolubles and bacterium, can also the larger impurity of remove portion polarity, effect preferably concentration range is acetonitrile-water (volume ratio 9: (0-3)), and then dilute further, in order to be applicable to chromatographic system better.
(2) separation and purification:
By the solution 50-100ml after dilution in step (1), inject reversed phase high efficiency preparative liquid chromatograph, isocratic elution, UV-detector on-line checkingi, specific aim collects the preparation component of l-arctigenin, obtains l-arctigenin solution.
Wherein reversed phase high efficiency preparative liquid chromatography is separated aretigenin chromatographic column used is dynamic axial compression column, diameter 80mm, packed height 250mm; Filler is octadecylsilane chemically bonded silica microballoon, particle diameter 10 μm; Moving phase is made up of acetonitrile-water, and the volume ratio of acetonitrile-water is 1: (1-4) isocratic elution; Flow velocity 260ml/min-330ml/min; Sample size 50-100ml; Sample concentration 8-15mg/ml; UV-detector on-line checkingi, determined wavelength 280nm.
Before carrying out reversed phase high efficiency and preparing liquid phase separation, by appearance time and the peak shape of l-arctigenin in LC-MS, reference substance location or the method determination high performance liquid chromatography commonly used of other the art.
For LC-MS method, can be identical with above-mentioned preparative separation liquid phase chromatogram condition, adopt the chromatographic column that filler is identical, form identical moving phase, identical determined wavelength etc., column temperature is room temperature (having no special requirements, room temperature); Get the filtrate sample introduction of step (1) gained, carry out the high performance liquid phase separation and purification of l-arctigenin, according to mass spectrometric detection result, the peak that l-arctigenin is corresponding in liquid chromatography can be determined.
(3) Product recycling
Step (2) high performance preparative liquid chromatography is separated acetonitrile and water in the l-arctigenin solution obtained and, successively through underpressure distillation (0.08MPa-0.09MPa, 35 DEG C-65 DEG C) and lyophilization removing, obtains l-arctigenin product.
Compared with prior art, excellent effect of the present invention is as follows:
The detection of the purity of finished product l-arctigenin adopts analysis mode reversed-phased high performace liquid chromatographic, and analyzing and testing purity is more than 99.8%, and single mixing all is less than 0.1%; Yield is more than 98%.(chromatographic condition: chromatographic column: C18 post (4.6mm × 150mm, 5um); Moving phase: methanol-water volume ratio is 60: 40; Flow velocity: 1.0ml/min; Sample size: 10 μ l; Determined wavelength: 280nm; Column temperature: 25 DEG C.)
The inventive method technique is simple, all recyclable recycling of organic solvent used, and solvent consumption is few; On-line checkingi, efficiency is high, and quality product easily controls; System is closed system, and operating environment is polluted little; Isocratic elution is simple to equipment requirements; Moving phase adopts volatile organic solvent and water, easily removes in Product recycling.
Accompanying drawing explanation
The high-efficient liquid phase analysis collection of illustrative plates of Fig. 1 l-arctigenin crude product
Fig. 2 prepares the HPLC collection of illustrative plates of gained l-arctigenin by the condition of embodiment 1
Fig. 3 prepares the HPLC collection of illustrative plates of gained l-arctigenin by the condition of embodiment 2
Fig. 4 prepares the HRLC collection of illustrative plates of gained l-arctigenin by the condition of embodiment 3
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail, but scope is not limited to following embodiment.
Embodiment 1
(1) dissolve, filter:
Get l-arctigenin crude product 2KG, be dissolved in acetonitrile-aqueous solution 100L (volume ratio of acetonitrile and water is 9: 3), by solid l-arctigenin dissolving crude product, with organic filtering with microporous membrane of 0.45 μm after dissolving, filtrate is diluted with water to the solution of 8mg/ml further, prepares the raw material of l-arctigenin as next step.
(2) separation and purification:
By the solution 100ml after dilution in step (1), inject reversed phase high efficiency preparative liquid chromatograph, moving phase is made up of acetonitrile-water, the volume ratio of acetonitrile-water is 35: 65, isocratic elution, flow velocity 320ml/min, UV-detector on-line checkingi, determined wavelength 280nm, specific aim collects the preparation component of l-arctigenin, obtains l-arctigenin solution.
(3) Product recycling:
Step (2) high performance preparative liquid chromatography is separated acetonitrile and water in the l-arctigenin solution obtained and, successively through underpressure distillation (0.08-0.09MPa, 40-50 DEG C) and freeze-drying removing, obtains l-arctigenin product.Total recovery can reach 98.2%, and product measures through HPLC, and purity is 99.89%.
Embodiment 2
(1) dissolve, filter:
Get l-arctigenin crude product 2KG, be dissolved in acetonitrile-aqueous solution 100L (volume ratio of acetonitrile and water is 9: 2), by solid l-arctigenin dissolving crude product, with organic filtering with microporous membrane of 0.45 μm after dissolving, filtrate is diluted with water to the solution of 10mg/ml further, prepares the raw material of l-arctigenin as next step.
(2) separation and purification:
By the solution 80ml after dilution in step (1), inject reversed phase high efficiency preparative liquid chromatograph, moving phase is made up of acetonitrile-water, the portion rate of acetonitrile-water is 45: 55, isocratic elution, flow velocity 290ml/min, UV-detector on-line checkingi, determined wavelength 280nm, specific aim collects the preparation component of l-arctigenin, obtains l-arctigenin solution.
(3) Product recycling:
Step (2) high performance preparative liquid chromatography is separated acetonitrile and water in the l-arctigenin solution obtained and, through underpressure distillation (0.08-0.09MPa, 40-50 DEG C) and lyophilization removing, obtains l-arctigenin product.Through the preparation that repeatedly circulates, yield can reach 98.3%, and product measures through HPLC, and purity is 99.82%.
Embodiment 3
(1) dissolve, filter:
Get l-arctigenin crude product 2KG, be dissolved in acetonitrile-aqueous solution 100L (volume ratio of acetonitrile and water is 9: 1), by solid l-arctigenin dissolving crude product, with organic filtering with microporous membrane of 0.45 μm after dissolving, filtrate is diluted with water to the solution of 15mg/ml further, prepares the raw material of l-arctigenin as next step.
(2) separation and purification:
By the solution 50ml after dilution in step (1), inject reversed phase high efficiency preparative liquid chromatograph, moving phase is made up of acetonitrile-water, the portion rate of acetonitrile-water is 50: 50, isocratic elution, flow velocity 290ml/min, UV-detector on-line checkingi, determined wavelength 280nm, specific aim collects the preparation component of l-arctigenin.
(3) Product recycling:
Step (2) high performance preparative liquid chromatography to be separated in the l-arctigenin solution obtained acetonitrile and water through underpressure distillation (0.08-0.09MPa, 40-50 DEG C) and lyophilization removing, obtain l-arctigenin product, yield can reach 98.5%, product measures through HPLC, and purity is 99.86%.
Claims (1)
1. a separation purification method for l-arctigenin, comprises the following steps:
(1) dissolution filter: dissolved by l-arctigenin crude product acetonitrile-aqueous solution, filters, and filtrate is the l-arctigenin solution of 8-15mg/ml further with purified water dilution;
(2) separation and purification: be separated by step (1) gained solution high performance preparative liquid chromatography wash-out, use UV-detector on-line checkingi, determined wavelength is 280nm, collects the preparation component of l-arctigenin, obtains l-arctigenin solution;
(3) Product recycling: the l-arctigenin solution that step (2) is obtained removes acetonitrile and water by underpressure distillation and lyophilization successively, obtains l-arctigenin sterling;
Wherein, in step (1), the volume ratio of acetonitrile and the aqueous solution is 9:0-3; The chromatographic column of the high performance preparative liquid chromatography in step (2) is dynamic axial compression column, and dynamic axial compression column diameter is 80mm, and packed height is 250mm; Filler is octadecylsilane chemically bonded silica microballoon; Particle diameter is 10 μm;
High performance preparative liquid chromatography wash-out moving phase used is acetonitrile-aqueous solution, and the volume ratio of acetonitrile and water is 1:1-4; Type of elution is isocratic elution; Elution speed is 260ml/min-330ml/min.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1560265A (en) * | 2004-02-26 | 2005-01-05 | 广州中医药大学 | Preparation process of burduck protoglucoside of anti virus and antiptmour natural medicine |
| CN101392279A (en) * | 2008-10-21 | 2009-03-25 | 浙江工业大学 | Method for preparing fructus arctii aglycone |
| CN101736050A (en) * | 2009-12-08 | 2010-06-16 | 辽宁中医药大学 | Preparation method of arctigenin |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1560265A (en) * | 2004-02-26 | 2005-01-05 | 广州中医药大学 | Preparation process of burduck protoglucoside of anti virus and antiptmour natural medicine |
| CN101392279A (en) * | 2008-10-21 | 2009-03-25 | 浙江工业大学 | Method for preparing fructus arctii aglycone |
| CN101736050A (en) * | 2009-12-08 | 2010-06-16 | 辽宁中医药大学 | Preparation method of arctigenin |
Non-Patent Citations (2)
| Title |
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| HPLC梯度洗脱同时测定牛蒡子液体制剂中牛蒡子苷和牛蒡子苷元的含量;许润春 等;《现代生物医学进展》;20070731;第7卷(第7期);第1084-1085页 * |
| 牛蒡叶中微量木脂体牛蒡子甙和牛蒡子甙元的分离与鉴定;刘世名 等;《色谱》;20030131;第21卷(第1期);第52-55页 * |
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