CN101747392B - Method for separating and preparing Cepimedin C chemical reference substance - Google Patents
Method for separating and preparing Cepimedin C chemical reference substance Download PDFInfo
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- CN101747392B CN101747392B CN 200810229887 CN200810229887A CN101747392B CN 101747392 B CN101747392 B CN 101747392B CN 200810229887 CN200810229887 CN 200810229887 CN 200810229887 A CN200810229887 A CN 200810229887A CN 101747392 B CN101747392 B CN 101747392B
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Abstract
The invention relates to a method for separating and preparing a Cepimedin C chemical reference substance. In the method, 10 to 50 percent icariin extract is used as a raw material, and is subjected to primary column separation; a preparation high-efficiency liquid chromatogram is taken as a main separation mode; and extracting solution is directly concentrated to dryness under reduced pressure to prepare the Cepimedin C chemical reference substance with the purity of over 98 percent through two-step high-efficiency liquid chromatogram preparation. Compared with the reported methods for separating and preparing the Cepimedin C, the method of the invention has the advantages of simple process steps, high purity, good color, and suitability for mass production.
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% epimedin chemical reference substance.Comprise that mainly post separates enrichment method and fast high performance liquid phase preparation method removal of impurities and refining.
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.Modern study shows that Herba Epimedii has many-sided pharmacological actions such as osteoporosis, sexual function improving, increase cardiovascular and cerebrovascular volume of blood flow.Epimedin be in the epimedium herb except icarin the higher composition of content, epimedin content even be higher than icarin and (thank to beautiful equality, herbal medicine, 2007,38 (4): 613-614) in some kind.Epimedin is the rhamnosyl substituent of icarin, along with its pharmacologically active illustrate gradually and increasing enterprise adopts icarin and epimedin jointly as the quality control index composition of Herba Epimedii and preparation thereof, it becomes the inevitable direction that the quality control index composition of accepting extensively should be future development.
The structural formula of epimedin is as follows:
The method for separating and preparing of epimedin mainly contains: (the Chinese patent CN2007100002610.5 such as Guo Baolin, 2007 are open) adopt the techniques such as ethyl acetate-methanol extraction, silicagel column rough segmentation, the anti-phase preparation of C18 (40-60% methanol-water), Sephadex LH 40 gel chromatographies, the anti-phase preparation of C18 (40-60% acetonitrile-water) to separate epimedin, although can make purity greater than 98% epimedin, main technological steps had more than five steps.(the Chinese patent CN200610101222.5 such as Wang Xiaochun, 2007 are open) adopt sherwood oil, chloroform, ethyl acetate, propyl carbinol solvent extraction to obtain the epimedin crude extract of n-butanol portion, then cross successively silica gel, polyamide column acquisition epimedin reference substance.The latter's technique is relatively simple, but a large amount of noxious solvents that use high volatilities in system's solvent extraction and the column separation process (such as chloroform etc.), be unfavorable for its large-scale promotion application.At present, the high performance liquid preparative chromatography method of simple process yet there are no report for the work of extensive (more than the gram level) preparation 98% content epimedin.
Summary of the invention
The present invention is integrating on the various epimedin preparation methods basis, many-sided researchs such as, target reference substance enriching method research preferred by bulk drug and the exploitation of reference substance refined highly effective liquid phase process, invented a kind of low cost, high performance liquid preparative chromatography method fast, preparative-scale can reach the 5g/ month and guarantee purity (＞98%) and product color.
The objective of the invention is to develop a kind of grade scale that restrains and purity of can obtaining in the novel method of the epimedin glucoside chemical reference substance more than 98%.
For achieving the above object, the technical solution used in the present invention:
1. the method for separating and preparing of an epimedin chemical reference substance, it is characterized in that: the icarin extract take mass content as 10-50% is raw material, separate through preliminary post, take preparative high performance liquid chromatography as main separation means, by two step high performance liquid phase preparations, extracting solution directly is evaporated to do and can obtains the 5 gram level scales of producing per month and purity greater than 98% epimedin chemical reference substance; Concrete steps are as follows:
1) preliminary post separates:
Icarin extract water or the dissolving of volumetric concentration 40-80% aqueous ethanolic solution, resin column or polyamide column separate, the ethanol-water solution gradient elution, and wherein the volumetric concentration of ethanol changes from 0%-80%, collect 20%-70% ethanol elution thing, concentrate drying gets yellow powder I;
2) yellow powder I is configured to the need testing solution that concentration is 50～150mg/ml, through 0.45 μ m filtering with microporous membrane with the dissolving of volumetric concentration 40-80% aqueous ethanolic solution; Adopt the high performance liquid preparative chromatography post of column length 10-50cm, diameter 1-10cm to separate, six-way valve or pump sample introduction, sampling volume is 1-50ml, methanol-water take volumetric concentration as 45-60% or volumetric concentration are eluent system as the acetonitrile-aqueous solution of 20-40%, flow rate control is at 10-400ml/min, online ultraviolet detection is collected the elutriant that contains epimedin, the dry yellow powder II that gets of concentrating under reduced pressure;
3) second step high performance liquid phase preparation
The contain acetonitrile solution of yellow powder II take volumetric concentration as 20-40% carries out the high performance liquid phase preparation second time as eluent system, and other condition is with step 2), collect purity greater than stream part of containing epimedin of 98%;
4) acquisition of epimedin chemical reference substance
Above-mentioned stream part directly is concentrated into to do and can obtains purity greater than 98% epimedin reference substance, and it is the glassy yellow powder.
In the aforesaid method, preliminary post separates with resin better with nonpolar macroporous adsorption resin.The moving phase that the high performance liquid phase preparation process adopts is that volumetric concentration is the acetonitrile solution that contains of 20-40%; 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 epimedin signal the overload situation and decide.
From Herba Epimedii extract, separate the epimedin chemical reference substance with the present invention and have following advantage and progress:
1. the present invention has avoided a large amount of use toxic volatile moving phases and opening column take preparation type high performance liquid phase as separation means when making epimedin obtain high efficiency separation.
2. the present invention carries out preliminary enrichment by the post separation to the target reference substance, then adopts two step high performance liquid phase preparation methods to carry out respectively removal of impurities and refining, and technique is simpler.
3, 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 10-30 minute; Add simultaneously the recycling of preparation solvent, can realize the in batches low cost of preparation.
4. the present invention adopts UV-detector to detect online, and the separation case of epimedin can direct-detection, direct selective collection high purity sample, thus realize the high-recovery of purity under guaranteeing.
In a word, processing step of the present invention is simple, purity is high, color and luster is good, and can be mass-produced.
Description of drawings
Fig. 1 is that the HPLC of epimedin analyzes collection of illustrative plates (270nm), and its integration is reported as follows:
| Name | Retention Time | Area | ％Area | |
| 1 | 1.240 | 2250 | 0.00 | |
| 2 | 1.731 | 7018 | 0.01 | |
| 3 | 2.574 | 410099 | 0.80 | |
| 4 | 3.411 | 4854 | 0.01 | |
| 5 | 3.640 | 51609 | 0.10 | |
| 6 | 4.626 | 20123 | 0.04 | |
| 7 | Epimedin | 8.667 | 50592362 | 99.03 |
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, take the icarin extract of content as 20% as raw material, preliminary post separates and adopts the HPD post, and high performance liquid phase prepare take 10 μ mC18 bonded phase packings as sorbent material, goes on foot high performance liquid phase through two and prepares the epimedin reference substance.The concrete technology step is as follows:
1) preliminary post separates
The icarin extract dissolves with the 40-80% aqueous ethanolic solution, and the HPD-100 resin column separates, and the ethanol-water solution gradient elution is collected 20%-70% ethanol elution thing, and concentrate drying gets yellow powder I.
2) the first step high performance liquid phase preparation
Yellow powder I is configured to the need testing solution that concentration is 150mg/ml, through 0.45 μ m filtering with microporous membrane with the dissolving of 40-80% aqueous ethanolic solution; Preparation type high performance liquid phase column packing brand is Chromatorex C18 bonded phase packings; Particle diameter is 10 μ m, column length 22cm, diameter 7.5cm; Six-way valve sample introduction, sampling volume are 10ml, and take 60% methanol-water solution as eluent system, flow rate control is at 160ml/min, and online ultraviolet detection is collected the elutriant that contains epimedin, the dry yellow powder II that gets of concentrating under reduced pressure.
3) second step high performance liquid phase preparation
Yellow powder II carries out the high performance liquid phase preparation second time take the acetonitrile solution that contains of volumetric concentration 28% as eluent system, and all the other conditions are with step 2), collect high-purity stream part of containing epimedin.
4) acquisition of epimedin chemical reference substance
Above-mentioned high-purity stream part directly is concentrated into to do and can obtains purity greater than 98% epimedin reference substance (glassy yellow powder) (Fig. 1).
Embodiment 2
In the present embodiment, take the icarin extract of content as 20% as raw material, preliminary post separates and adopts the HPD post, and high performance liquid phase prepare take 10 μ mC18 bonded phase packings as sorbent material, goes on foot high performance liquid phase through two and prepares the epimedin reference substance.The concrete technology step is as follows:
1) preliminary post separates
The icarin extract dissolves with the aqueous solution, and the HPD-100 resin column separates, and the ethanol-water solution gradient elution is collected 20%-70% ethanol elution thing, and concentrate drying gets yellow powder I.
2) the first step high performance liquid phase preparation
Yellow powder I is configured to the need testing solution that concentration is 100mg/ml, through 0.45 μ m filtering with microporous membrane with the dissolving of 40-80% aqueous ethanolic solution; Preparation type high performance liquid phase column packing brand is Chromatorex C18 bonded phase packings; Particle diameter is 10 μ m, column length 19, diameter 7.5cm; Pump sample introduction, sampling volume are 20ml, and take 50% methanol-water solution as eluent system, flow rate control is at 140ml/min, and online ultraviolet detection is collected the elutriant that contains epimedin, the dry yellow powder II that gets of concentrating under reduced pressure.
3) second step high performance liquid phase preparation
Yellow powder II carries out the high performance liquid phase preparation second time take the acetonitrile solution that contains of volumetric concentration 25% as eluent system, and condition is with step 2), collect high-purity stream part of containing epimedin.
4) acquisition of epimedin chemical reference substance
Above-mentioned high-purity stream part directly is concentrated into to do and can obtains purity greater than 98% epimedin reference substance (glassy yellow powder).
Embodiment 3
In the present embodiment, take the icarin extract of content as 15% as raw material, preliminary post separates and adopts polyamide column, and high performance liquid phase prepare take 5 μ mC18 bonded phase packings as sorbent material, goes on foot high performance liquid phase through two and prepares the epimedin reference substance.The concrete technology step is as follows:
1) preliminary post separates
The icarin extract dissolves with the 40-80% aqueous ethanolic solution, and polyamide column separates, and the ethanol-water solution gradient elution is collected 20%-70% ethanol elution thing, and concentrate drying gets yellow powder I.
2) the first step high performance liquid phase preparation
Yellow powder I is configured to the need testing solution that concentration is 120mg/ml, through 0.45 μ m filtering with microporous membrane with the dissolving of 40-80% aqueous ethanolic solution; 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 8ml, and take 55% methanol-water solution as eluent system, flow rate control is at 180ml/min, and online ultraviolet detection is collected the elutriant that contains epimedin, the dry yellow powder II that gets of concentrating under reduced pressure.
3) second step high performance liquid phase preparation
Yellow powder II carries out the high performance liquid phase preparation second time take the acetonitrile solution that contains of volumetric concentration 30% as eluent system, and condition is with step 2), collect high-purity stream part of containing epimedin.
4) acquisition of epimedin chemical reference substance
Above-mentioned high-purity stream part directly is concentrated into to do and can obtains purity greater than 98% epimedin reference substance.
Embodiment 4
In the present embodiment, take the icarin extract of content as 25% as raw material, preliminary post separates and adopts polyamide column, and high performance liquid phase prepare take 5 μ mC18 bonded phase packings as sorbent material, goes on foot high performance liquid phase through two and prepares the epimedin reference substance.The concrete technology step is as follows:
1) preliminary post separates
The icarin extract dissolves with the 40-80% aqueous ethanolic solution, and polyamide column separates, and the ethanol-water solution gradient elution is collected 20%-80% ethanol elution thing, and concentrate drying gets yellow powder I.
2) the first step high performance liquid phase preparation
Yellow powder I is configured to the need testing solution that concentration is 80mg/ml, through 0.45 μ m filtering with microporous membrane with the dissolving of 40-80% aqueous ethanolic solution; 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; Pump sample introduction, sampling volume are 20ml, and take 48% methanol-water solution as eluent system, flow rate control is at 180ml/min, and online ultraviolet detection is collected the elutriant that contains epimedin, the dry yellow powder II that gets of concentrating under reduced pressure.
3) second step high performance liquid phase preparation
Yellow powder II carries out the high performance liquid phase preparation second time take the acetonitrile solution that contains of volumetric concentration 32% as eluent system, and condition is with step 2), collect high-purity stream part of containing epimedin.
4) acquisition of epimedin chemical reference substance
Above-mentioned high-purity stream part directly is concentrated into to do and can obtains purity greater than 98% epimedin reference substance.
Claims (4)
1. the method for separating and preparing of an epimedin chemical reference substance, it is characterized in that: the icarin extract take mass content as 10-50% is raw material, separate through preliminary post, take preparative high performance liquid chromatography as main separation means, by two step high performance liquid phase preparations, extracting solution directly is evaporated to do and can obtains purity greater than 98% epimedin chemical reference substance; Concrete steps are as follows:
1) preliminary post separates:
Icarin extract water or the dissolving of volumetric concentration 40-80% aqueous ethanolic solution, resin column separates, the ethanol-water solution gradient elution, wherein the volumetric concentration of ethanol changes from 0%-80%, collects 20%-70% ethanol elution thing, and concentrate drying gets the yellow powder I;
2) the first step high performance liquid phase preparation
The yellow powder I is configured to the need testing solution that concentration is 50～150mg/ml, through 0.45 μ m filtering with microporous membrane with the dissolving of volumetric concentration 40-80% aqueous ethanolic solution; Adopt the high performance liquid preparative chromatography post of column length 10-50cm, diameter 1-10cm to separate, six-way valve or pump sample introduction, sampling volume is 1-50ml, methanol-water take volumetric concentration as 45-60% or volumetric concentration are eluent system as the acetonitrile-aqueous solution of 20-40%, flow rate control is at 10-400ml/min, online ultraviolet detection is collected the elutriant that contains epimedin, the dry yellow powder II that gets of concentrating under reduced pressure;
3) second step high performance liquid phase preparation
The contain acetonitrile solution of yellow powder II take volumetric concentration as 20-40% carries out the high performance liquid phase preparation second time as eluent system, and other condition is with step 2), collect purity greater than stream part of containing epimedin of 98%;
4) acquisition of epimedin chemical reference substance
Above-mentioned stream part directly is concentrated into to do and can obtains purity greater than 98% epimedin reference substance, and it is the glassy yellow powder,
Wherein the resin column of column separation process employing is the non-polar resin post.
2. method for separating and preparing according to claim 1 is characterized in that: the filler of the preparative column that the first step or second step high performance liquid phase preparation process adopt is C6, C8 or C18 bonded phase packings, and particle diameter is 5-20 μ m.
3. method for separating and preparing according to claim 1 is characterized in that: the moving phase that the high performance liquid phase preparation process adopts is that volumetric concentration is the acetonitrile solution that contains of 22-35%.
4. 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 wavelength 270nm, 318nm or 349nm.
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Non-Patent Citations (3)
| Title |
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| 杨振寰 等.制备高效液相色谱在天然产物分离中的应用.《中成药》.2005,第27卷(第12期),1444-1448. * |
| 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》.2005,第33卷(第01期),106-108. * |
| 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01期),22-25. * |
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