CN101747391B - Method for separating and preparing chemical reference substance of Icariin - Google Patents
Method for separating and preparing chemical reference substance of Icariin Download PDFInfo
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- CN101747391B CN101747391B CN 200810229886 CN200810229886A CN101747391B CN 101747391 B CN101747391 B CN 101747391B CN 200810229886 CN200810229886 CN 200810229886 CN 200810229886 A CN200810229886 A CN 200810229886A CN 101747391 B CN101747391 B CN 101747391B
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Abstract
The invention relates to a method for separating and preparing chemical reference substance of Icariin. Icariin extract with content no less than 20 percent is used as raw material and is preliminarily separated through a column or settled through alcohol to remove impurities and colors. By means of the preparative high-efficiency liquid-phase chromatographic separation, by taking aqueous solution containing acetonitrile of certain proportion as an elution system, the obtained liquid extract is directly concentrated under reduced pressure to the dry state, so chemical reference substance of Icariin with purity more than 98 percent is obtained. Compared with the reported method for separating and preparing Icariin, the invention has the advantages the technological steps are simple, the cycle is short, the purity is high, the color is good and the mass production can be realized.
Description
Technical field
The present invention relates to a kind of purity that from Herba Epimedii extract, separate to obtain greater than the preparation method of 98% icarin chemical reference substance, comprise that mainly preliminary post separates or alcohol precipitation removal of impurities decolouring and fast high performance liquid phase preparation method mass-producing preparation.
Background technology
Herba Epimedii (Herba Epimedii) is that China uses one of the longest Chinese medicine, has the effect of kidney-replenishing, strengthening the bones and muscles, wind-damp dispelling.Many-sided pharmacological actions such as modern study shows, Herba Epimedii has osteoporosis, sexual function improving, increase cardiovascular and cerebrovascular volume of blood flow, promote hemopoietic function, strengthen immune, anti-ageing, anti-inflammatory, antitumor.Icarin is the main active ingredient of Herba Epimedii, also is the assay composition of epimedium herb in 2005 editions state-promulgated pharmacopoeia of China, and stipulates that its content in medicinal material should reach more than 0.5%.The structural formula of icarin is as follows:
The method for separating and preparing of icarin mainly contains: Hu Yunfeng etc. (Chinese patent CN200510061251.1,2007 open) adopt lixiviate, removing impurities, extraction, resin absorption, dry five large operations to obtain the icarin (5%-50%) of different content; Tian Jing (the journal .2004 of Shandong Traditional Chinese Medicine University, 28 (6): 456-458) can obtain the icarin of 60.30% content with Flavonoids by Macroporous Adsorption Resin such as shake; (the northwest agricultural journal 2005 such as Liang Ran, 14 (2): 141 ~ 14) technique of employing 30% ethanol ultrasonic-assisted extraction, polyamide column and ethyl acetate extraction can obtain the icarin of 60% content, and further recrystallization can obtain the icarin of 90.94% content.Document (Liu Renmin, et al.Journal of Chromatography A, 1064,2005,53-57) reported that also high-speed countercurrent chromatography prepares the icarin of 98% content, but only be laboratory scale and use special equipment and expensive poisonous moving phase.At present, high performance liquid preparative chromatography yet there are no report for the work of the icarin of extensive (more than the gram level) preparation 98% content.
Summary of the invention
The present invention is integrating on the various icarin preparation methods basis, the optimization of the selection by sample pretreatment research, efficient liquid phase chromatographic stuffing, exploitation, sampling volume and the concentration of flash chromatography condition, and the many-sided researchs such as selection that detect wavelength, invented a kind of low cost, high performance liquid preparative chromatography method fast, preparative-scale can reach the 20g/ month and guarantee purity (＞98%) and product color.The objective of the invention is to develop a kind of ten gram level scales and purity of can obtaining at the novel preparation method of the icarin chemical reference substance more than 98%.
For achieving the above object, the technical solution used in the present invention:
A kind of method for separating and preparing of icarin chemical reference substance, with mass content 〉=20%, preferred 50% above icarin extract is raw material, separate or alcohol precipitation removal of impurities decolouring through preliminary post, take preparative high performance liquid chromatography as separation means, take a certain proportion of acetonitrile solution that contains as eluent system, obtain monthly output 20g scale and purity greater than 98% icarin chemical reference substance; Concrete steps are:
1) preliminary post separates or alcohol precipitation removal of impurities decolouring:
Post separation, impurity removal decolouring: icarin extract water or the dissolving of volumetric concentration 40-80% aqueous ethanolic solution, upper resin column or polyamide column separate, with the ethanol-water solution gradient elution, wherein the volumetric concentration of ethanol changes from 0%-70%, collected volume concentration 50-70% ethanol elution thing, concentrate drying gets yellow powder;
Or alcohol precipitation removal of impurities decolouring: with the dissolving of 40-80% aqueous ethanolic solution, centrifugal throw out or leave standstill 〉=4 hours filtering precipitates can get yellow powder after the throw out oven dry with the icarin extract;
2) high performance liquid phase preparation:
Yellow powder is configured to the need testing solution that concentration is 50～150mg/ml, through 0.45 μ m filtering with microporous membrane with the aqueous ethanolic solution dissolving of volumetric concentration 40-80%; The employing column length is that 10-50cm, diameter are that the high performance liquid preparative chromatography post of 1-10cm separates, six-way valve or pump sample introduction, sampling volume is 1-50ml, acetonitrile solution take volumetric concentration as 15-55% is eluent system, flow rate control is at 10-400ml/min, online ultraviolet detection, collection purity flows part greater than 98% icarin;
3) acquisition of icarin chemical reference substance:
Above-mentioned stream part directly is concentrated into dried or can obtains purity greater than 98% yellow-white icarin reference substance with methyl alcohol or ethyl alcohol recrystallization.
In the aforesaid method, preliminary post separates with resin better with nonpolar macroporous adsorption resin.The volumetric concentration of acetonitrile is 20-30% in the moving phase that the high performance liquid phase preparation process adopts; The filler of high performance liquid phase preparative column is C6, C8 or C18 bonded phase packings, and its particle diameter is 5～20 μ m, the preferred 270nm of ultraviolet detection wavelength, 318nm or 349nm, specifically detect wavelength per sample in the icarin signal the overload situation and decide.
Have following advantage and progress with the present invention's separating icariin chemical reference substance from Herba Epimedii extract:
1. the present invention makes icarin obtain high efficiency separation take preparation type high performance liquid phase as separation means, can obtain purity at the icarin chemical reference substance more than 98%.
2. the high performance liquid phase preparation method of the present invention's development is fast preparation method, and the single needle disengaging time was fit to the preparation of chemical reference substance in enormous quantities very much between 10-20 minute; Add simultaneously the recycling of preparation solvent, can realize the in batches low cost of preparation.
3. the present invention adopts UV-detector to detect online, and the separation case of icarin can direct-detection, direct selective collection high purity flow part, thus realize the high-recovery of purity under guaranteeing.
4. the present invention had increased removal of impurities decolouring step before the preparation high performance liquid phase, can effectively prolong the work-ing life of chromatographic column on the one hand, had guaranteed that on the other hand product has good color.
In a word, processing step of the present invention is simple, the cycle is short, purity is high, color and luster is good, and can be mass-produced.
Description of drawings
Fig. 1 is that the HPLC of icarin before the recrystallization analyzes collection of illustrative plates (270nm), and its integration is reported as follows:
| Name | Retention Time | Area | ％Area | |
| 1 | 1.661 | 15090 | 0.05 | |
| 2 | 2.522 | 1909 | 0.01 | |
| 3 | 2.722 | 10334 | 0.03 | |
| 4 | 3.390 | 8783 | 0.03 | |
| 5 | 4.709 | 53338 | 0.17 | |
| 6 | 8.354 | 11070 | 0.04 | |
| 7 | 9.097 | 59791 | 0.19 | |
| 8 | Icarin | 11.261 | 30832346 | 99.48 |
Fig. 2 is that the HPLC of icarin behind the recrystallization analyzes collection of illustrative plates (270nm), and its integration is reported as follows:
| Name | Retention Time | Area | ％Area | |
| 1 | 1.666 | 12113 | 0.03 | |
| 2 | 2.635 | 77282 | 0.21 | |
| 3 | 3.396 | 4958 | 0.01 | |
| 4 | 4.385 | 4795 | 0.01 | |
| 5 | 4.613 | 12192 | 0.03 | |
| 6 | 8.931 | 66448 | 0.18 | |
| 7 | Icarin | 11.119 | 36908028 | 99.52 |
Embodiment
Now with accompanying drawing the present invention is described in further details in conjunction with the embodiments, embodiment only limits to illustrate the present invention, but not limitation of the invention.
Embodiment 1:
In the present embodiment, the icarin extract take 50% take 10 μ mC18 bonded phase packings as sorbent material, is eluting solvent to contain acetonitrile-water (30: 70, volume ratio) solution as raw material, and the concrete technology step is as follows:
1) decolouring
Extract dissolves with 70% aqueous ethanolic solution, leaves standstill 12 hours, filters, and gets yellow powder after the throw out oven dry.
2) high performance liquid phase preparation
Above-mentioned yellow powder dissolves with 70% aqueous ethanolic solution, is configured to the need testing solution that concentration is 50mg/ml, through 0.45 μ m filtering with microporous membrane; Preparation type high performance liquid phase column packing brand is the ChromatorexC18 bonded phase packings, and particle diameter is 10 μ m; Column length 19cm, diameter 7.5cm; Six-way valve sample introduction, sampling volume are 10ml; Take 30% acetonitrile-aqueous solution as eluent system, flow rate control is at 160ml/min, and online ultraviolet detection is collected high purity flow part of containing icarin;
3) acquisition of icarin chemical reference substance
Above-mentioned stream part directly be concentrated into do can obtain purity be 99.48% icarin reference substance (yellow-white powder) (Fig. 1); The yellow-white powder with recrystallizing methanol obtain purity be 99.52% icarin reference substance (yellow-white crystallization) (Fig. 2).
Embodiment 2
In the present embodiment, take 50% icarin extract as raw material, take the C18 bonded phase packings of 5 μ m as sorbent material, be eluting solvent to contain acetonitrile-water (25: 75, volume ratio) solution, the concrete technology step is as follows:
1) decolouring
Extract dissolves with 70% aqueous ethanolic solution, and is centrifugal, filters, and gets yellow powder after the throw out oven dry.
2) high performance liquid phase preparation
Above-mentioned yellow powder dissolves with 60% aqueous ethanolic solution, is configured to the need testing solution that concentration is 80mg/ml, through 0.45 μ m filtering with microporous membrane; Preparation type high performance liquid phase column packing brand is the McirosorbC18 bonded phase packings, and particle diameter is 5 μ m; Column length 17cm, diameter 5cm; Six-way valve sample introduction, sampling volume are 2ml; Take 25% acetonitrile-aqueous solution as eluent system, flow rate control is at 60ml/min, online ultraviolet detection, and preferred detection wavelength 270nm collects high-purity stream part of containing icarin;
3) acquisition of icarin chemical reference substance
Above-mentioned stream part directly is concentrated into does that can to obtain purity be 98.84% icarin reference substance (yellow-white powder); It is 99.45% icarin reference substance (yellow-white crystallization) that the yellow-white powder obtains purity with recrystallizing methanol.
Embodiment 3:
In the present embodiment, the icarin extract take 20% take 10 μ m C18 bonded phase packings as sorbent material, is eluting solvent to contain acetonitrile-water (30: 70, volume ratio) solution as raw material, and the concrete technology step is as follows:
1) decolouring
Extract dissolves with 40% aqueous ethanolic solution, upper HPD resin column (I.D.6 * 68), and the alcohol-water gradient elution is collected 50% ethanol elution thing, and concentrate drying gets yellow powder.
2) high performance liquid phase preparation
Yellow powder dissolves with 70% aqueous ethanolic solution, is configured to the need testing solution that concentration is 100mg/ml, through 0.45 μ m filtering with microporous membrane; Preparation type high performance liquid phase column packing brand is the ChromatorexC18 bonded phase packings, and particle diameter is 10 μ m, column length 22cm, diameter 7.5cm; Pump sample introduction, sampling volume are 20ml; Take 30% acetonitrile-aqueous solution as eluent system, flow rate control is at 160ml/min, online ultraviolet detection, and preferred detection wavelength 318nm or 349nm collect high-purity stream part of containing icarin;
3) acquisition of icarin chemical reference substance
Above-mentioned stream part directly is concentrated into does that can to obtain purity be 98.16% icarin reference substance (yellow-white powder).It is 99.10% icarin reference substance (yellow-white crystallization) that the yellow-white powder obtains purity with recrystallizing methanol.
Embodiment 4
In the present embodiment, the icarin extract take 33% is as raw material, take the C18 bonded phase packings of 5 μ m as sorbent material, is eluting solvent to contain acetonitrile-water (28: 72, volume ratio) solution, and the concrete technology step is as follows:
1) decolouring
Extract dissolves with 60% aqueous ethanolic solution, and is centrifugal, filters, and gets yellow powder after the throw out oven dry.
2) high performance liquid phase preparation
Yellow powder dissolves with 70% aqueous ethanolic solution, is configured to the need testing solution that concentration is 60mg/ml, through 0.45 μ m filtering with microporous membrane; Preparation type high performance liquid phase column packing brand is Mcirosorb C18 bonded phase packings; Particle diameter is 5 μ m; Column length 17cm, diameter 5cm; Six-way valve sample introduction, sampling volume are 5ml; Take 28% acetonitrile-aqueous solution as eluent system, flow rate control is at 60ml/min, and online ultraviolet detection is collected high-purity stream part of containing icarin;
3) acquisition of icarin chemical reference substance
Above-mentioned stream part directly is concentrated into does that can to obtain purity be 98.84% icarin reference substance (yellow-white powder); It is 99.45% icarin reference substance (yellow-white crystallization) that the yellow-white powder obtains purity with recrystallizing methanol.
Claims (4)
1. the method for separating and preparing of an icarin chemical reference substance is characterized in that:
Take the icarin extract of mass content 〉=20% as raw material, separate or alcohol precipitation removal of impurities decolouring through preliminary post, take preparative high performance liquid chromatography as separation means, take a certain proportion of acetonitrile solution that contains as eluent system, obtain purity greater than 98% icarin chemical reference substance; Concrete steps are:
1) preliminary post separates or alcohol precipitation removal of impurities decolouring:
The decolouring of post separation, impurity removal: icarin extract water or volumetric concentration are the aqueous ethanolic solution dissolving of 40-80%, upper resin column separates, with the ethanol-water solution gradient elution, wherein the volumetric concentration of ethanol changes from 0%-70%, collected volume concentration is the ethanol elution thing of 50-70%, and concentrate drying gets yellow powder;
Or alcohol precipitation removal of impurities decolouring: with the dissolving of 40-80% aqueous ethanolic solution, centrifugal throw out or leave standstill 〉=filtered to get throw out in 4 hours can get yellow powder after the throw out oven dry with the icarin extract;
2) high performance liquid phase preparation:
Yellow powder is configured to the need testing solution that concentration is 50～150mg/ml, through 0.45 μ m filtering with microporous membrane with the aqueous ethanolic solution dissolving of volumetric concentration 40-80%; The employing column length is that 10-50cm, diameter are that the high performance liquid preparative chromatography post of 1-10cm separates, six-way valve or pump sample introduction, sampling volume is 1-50ml, acetonitrile solution take volumetric concentration as 15-55% is eluent system, flow rate control is at 10-400ml/min, online ultraviolet detection, collection purity flows part greater than 98% icarin;
3) acquisition of icarin chemical reference substance:
Above-mentioned stream part directly is concentrated into dried or can obtains purity greater than 98% yellow-white icarin reference substance with methyl alcohol or ethyl alcohol recrystallization;
The resin column that column separation process adopts is the non-polar resin post;
The filler of the preparative column that the high performance liquid phase preparation process adopts is C6, C8 or C18 bonded phase packings, and particle diameter is 5-20 μ m.
2. method for separating and preparing according to claim 1 is characterized in that: the volumetric concentration of acetonitrile is 20-30% in the moving phase that the high performance liquid phase preparation process adopts.
3. method for separating and preparing according to claim 1, it is characterized in that: the high performance liquid phase preparation process adopts online ultraviolet detection, and it detects the preferred 270nm of wavelength, 318nm or 349nm.
4. method for separating and preparing according to claim 1 is characterized in that: take mass content 〉=50% icarin extract as raw material.
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| CN109289939A (en) * | 2018-10-26 | 2019-02-01 | 劲牌生物医药有限公司 | A kind of method of heavy metal ion in removal Shorthorned Epimedium P.E |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1283627A (en) * | 2000-07-12 | 2001-02-14 | 徐绥绪 | Process for extracting icariin from epimedium |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1283627A (en) * | 2000-07-12 | 2001-02-14 | 徐绥绪 | Process for extracting icariin from epimedium |
Non-Patent Citations (3)
| Title |
|---|
| 王超展等.箭叶淫羊藿中5种黄酮类化合物的反相色谱分离制备.《分析化学研究简报》.2005,第33卷(第1期),106-108. * |
| 田景振等.淫羊藿苷的分离纯化工艺研究.《山东中医药大学学报》.2004,第28卷(第6期),456-458. * |
| 赵燕燕等.淫羊藿总黄酮提取工艺对总黄酮和淫羊藿苷含量的影响.《烟台大学学报(自然科学与工程版)》.2007,第20卷(第l期),22-25. * |
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