CN110016062A - A kind of typhaneoside preparative liquid chromatography separation method - Google Patents
A kind of typhaneoside preparative liquid chromatography separation method Download PDFInfo
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- CN110016062A CN110016062A CN201811406252.9A CN201811406252A CN110016062A CN 110016062 A CN110016062 A CN 110016062A CN 201811406252 A CN201811406252 A CN 201811406252A CN 110016062 A CN110016062 A CN 110016062A
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- typhaneoside
- preparative liquid
- liquid chromatography
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
- C07H1/08—Separation; Purification from natural products
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
- C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
- C07H17/06—Benzopyran radicals
- C07H17/065—Benzo[b]pyrans
- C07H17/07—Benzo[b]pyran-4-ones
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Abstract
It include that alcohol extracting is concentrated the invention discloses providing a kind of, macroporous resin column chromatography, the typhaneoside preparative liquid chromatography separation method of preparative liquid chromatography purifying, then typhaneoside fine work is dissolved with methanol, filtering, evaporated under reduced pressure, then it is placed with solvent recrystallization to room temperature, finally filter, high-purity typhaneoside monomer is obtained after drying, high-purity typhaneoside monomer is obtained from cattail pollen is greater than 99.0% by HPLC detection purity, this method is controllable, it can continuous large-scale production, and overcome in conventional method normal phase silica gel chromatography column and solvent-extracted cumbersome, separation cycle is long, the shortcomings that environmental pollution is serious, reduce the use of easy volatile solvent, to more environment-friendly, it is more efficient.
Description
Technical field
The invention belongs to active ingredient of autonomic drug extractive technique fields, and in particular to a kind of typhaneoside preparative liquid chromatography
Separation method.
Background technique
Cattail platymiscium is longer than in pond, waterside or banados, and the world shares 18 kinds, and China is there are about 10 kinds, and resource is extremely
It is abundant.Cattail pollen, alias cattail's spike pollen, the stem or leaf of cattail Huang system Typhaceae (Typhaceae) plant raupo cattail (Typha
AngustifoliaL.), the dry pollen of typha orientalis (Typha orientalisPresl.) or congener has only
Blood changes glue, treating stranguria function, for spitting blood, metrorrhagia and metrostaxis, traumatic hemorrhage, amenorrhea and algomenorrhea, and blood strangury and dry pain.Cattail pollen mainly contain fierce class,
Huang reward class, long-chain fat nephew's class compound, acid ingredient, amino acid, inorganic constituents etc..Wherein yellow reward constituents have improvement micro-
Circulation reduces heart and brain tissues oxygen demand, expansion blood vessel, promotees blood coagulation, is anti-inflammatory, reducing the effects of blood lipid and antiatherosclerosis.
Typhaneoside (Typhaneoside) is one of the main yellow reward ingredient in cattail pollen, molecular formula C34H42O20, molecule
Amount 770.The typhaneoside monomer of high-purity is needed to use in real work as reference substance, especially in the drug containing cattail pollen
In formulation development process, how quickly, easily obtain high-purity typhaneoside monomer be formulation method research and development in pass
One of key link.Most of main extraction separation method uses the separation means such as extraction, normal phase silica gel column chromatography, method road at present
Line is cumbersome, and yield is lower, and reagent consumption is big in production process, it is difficult to which the purity of industrialized production, final products is not high, so that fragrant
Pu Xin glycosides is difficult to meet the market demand with reference substance as quality control.
Summary of the invention
The present invention solves the deficiencies in the prior art, and providing a kind of includes alcohol extracting concentration, macroporous resin column chromatography, preparation solution
The typhaneoside preparative liquid chromatography separation method of phase chromatogram purification overcomes conventional method normal phase silica gel chromatography column and solvent extraction
Take it is cumbersome, separation cycle is long, environmental pollution is serious the shortcomings that.
The technical scheme adopted by the invention is that: a kind of typhaneoside preparative liquid chromatography separation method, this method include
Following steps:
Step 1: alcohol extracting concentration
Using cattail pollen as raw material, Pollen typhae is concentrated to get through alcohol extracting；
Step 2: macroporous resin column chromatography
Pollen typhae described in step 1 is added in macroporous resin column and is chromatographed, typhaneoside crude product is obtained after eluting；
Step 3: preparative liquid chromatography purifying
Typhaneoside crude product described in step 2 is purified by preparative liquid chromatograph, that is, uses reverse phase C18 chromatography
Column is through mobile phase isocratic elution, then post-treated obtains typhaneoside fine work；
Step 4:
By typhaneoside fine work described in step 3 through methanol dissolution, filtering, evaporated under reduced pressure, then recrystallized with solvent, most
High-purity typhaneoside monomer is obtained by filtering, drying.
Preferably, in step 1, extraction is mixed with alcoholic solution after dry cattail pollen is crushed, wherein cattail pollen and alcoholic solution
Solid-to-liquid ratio be 1:3~1:10, it is to be extracted completely after by extracting solution using centrifuge carry out centrifugal treating, then will be after centrifugation
Solution carries out rotary evaporation in vacuo except alcohol at 40~60 DEG C, obtains Pollen typhae.
It is furthermore preferred that the alcoholic solution is one or both of methanol aqueous solution or ethanol water, and the alcohol is molten
Liquid is 50~100% alcohol-water system of concentration expressed in percentage by volume.
Even more preferably, the revolving speed of the centrifuge is 10000~25000r/min.
Even more preferably, in step 2, the Pollen typhae is injected in macroporous resin column, deionization is first used
Water rinses macroporous resin column, then the alcoholic solution gradient elution for being 20~90% with concentration expressed in percentage by volume, then collected volume percentage
The alcoholic solution eluent that concentration is 60~70%, then rotary evaporation in vacuo removes alcohol at 50~80 DEG C of temperature, obtains typhaneoside
Crude product.
Even more preferably, the macroporous resin column is AB-8 or D101.
Even more preferably, in step 3, after typhaneoside crude product described in step 2 is redissolved with deionized water, note
Enter in preparative liquid chromatograph and purified, the reverse phase C18 chromatographic column specification is 250*200mm, and silica gel partial size is 5~20 μ
M, flow velocity are 300~800ml/min, and the mobile phase is mixed by A phase with B, and wherein A phase is acetonitrile, and B phase is volume hundred
Dividing concentration is 0.1~1% sour water system, and the isocratic elution ratio is 15%A phase.
Even more preferably, the acid in the sour water system is one of formic acid, acetic acid, phosphoric acid, trifluoroacetic acid.
Even more preferably, in step 4, the recrystallization solvent are as follows: methanol, ethyl alcohol, acetone, in acetonitrile at least
It is a kind of.
Compared to the prior art, the invention has the benefit that being examined to high-purity typhaneoside monomer by HPLC
Purity, according to document and nuclear magnetic spectrum parsing result, obtained component is typhaneoside, is divided from cattail pollen with method of the invention
From typhaneoside monomer is obtained, purity may be up to 99.0%, and the separation method quantity of sample handling is big, and reproducible, product is pure
Degree is high, is suitable for laboratory and industrial-scale production, and this method is controllable, can continuous large-scale production, and overcome
Conventional method normal phase silica gel chromatography column and it is solvent-extracted cumbersome, separation cycle is long, environmental pollution is serious the shortcomings that, reduce
The use of easy volatile solvent, to more environment-friendly, more efficient.
Detailed description of the invention
Fig. 1 is that 1 step 1 Pollen typhae analyzes liquid chromatogram in embodiment；
Fig. 2 is 1 step 3 preparative liquid chromatography figure in embodiment.
Specific embodiment
The following describes the present invention in detail with reference to the accompanying drawings and specific embodiments.
Embodiment one
A kind of typhaneoside preparative liquid chromatography separation method, method includes the following steps:
Step 1: alcohol extracting concentration
5kg dry cattail pollen is crushed and is placed in 50L extractor, is that 50% methanol mixes with concentration expressed in percentage by volume,
The solid-to-liquid ratio of middle cattail pollen and methanol solution is 1:4, is extracted twice, each 180min, and completely rear combined extract to be extracted carries out
Centrifugal treating, centrifuge speed is 10000r/min when centrifugal treating, and the solution after centrifugation is then carried out vacuum at 40 DEG C
Rotary evaporation removes alcohol, obtains Pollen typhae；
Step 2: macroporous resin column chromatography
Pollen typhae liquid described in step 1 is injected in the AB-8 type large pore resin absorption column handled well and is chromatographed,
It is first eluted with the deionized water of 3 times of column volumes, then the methanol aqueous solution elution for being 30% with concentration expressed in percentage by volume, then with volume hundred
The methanol aqueous solution that concentration is 50% is divided to elute, then the methanol aqueous solution for being 70% with concentration expressed in percentage by volume elutes, and then collects
The alcoholic solution eluent that concentration expressed in percentage by volume is 70%, then rotary evaporation in vacuo removes alcohol at 55 DEG C, obtains typhaneoside crude product；
Step 3: preparative liquid chromatography purifying
Typhaneoside crude product described in step 2 is redissolved with deionized water, is carried out in injection preparative liquid chromatograph pure
Change, monitored in real time by UV detector, chromatographic column is reverse phase C18, and specification 250*200mm, silica gel partial size is 5 μm, flow velocity
300ml/min, the mobile phase are mixed by A phase with B, and wherein A phase is acetonitrile, and B phase is that concentration expressed in percentage by volume is 0.2 first
Acid, the isocratic elution ratio are 15%A phase, elute ratio are as follows:
| Time (min) | Acetonitrile (%) | Water (%) | Formic acid (%) |
| 0 | 15 | 84.8 | 0.2 |
| 100 | 15 | 84.8 | 0.2 |
Then 7.8-9.2min fraction is collected, typhaneoside fine work is concentrated to get；
Step 4:
The methanol of typhaneoside fine work described in step 3 is dissolved, filtering, evaporated under reduced pressure, then methanol heating for dissolving is put
It sets to room temperature, finally filters, obtains high-purity typhaneoside monomer after drying.
Embodiment two
A kind of typhaneoside preparative liquid chromatography separation method, method includes the following steps:
Step 1: alcohol extracting concentration
5kg dry cattail pollen is crushed and is placed in 50L extractor, is that 70% ethyl alcohol mixes with concentration expressed in percentage by volume,
The solid-to-liquid ratio of middle cattail pollen and ethanol solution is 1:5, is extracted twice, each 180min, and completely rear combined extract to be extracted carries out
Centrifugal treating, centrifuge speed is 20000r/min when centrifugal treating, and the solution after centrifugation is then carried out vacuum at 50 DEG C
Rotary evaporation removes alcohol, obtains Pollen typhae；
Step 2: macroporous resin column chromatography
Pollen typhae liquid described in step 1 is injected in the AB-8 type large pore resin absorption column handled well and is chromatographed,
It is first eluted with the deionized water of 3 times of column volumes, then the ethanol water elution for being 40% with concentration expressed in percentage by volume, then with volume hundred
The ethanol water that concentration is 50% is divided to elute, then the ethanol water for being 60% with concentration expressed in percentage by volume elutes, and then collects
The alcoholic solution eluent that concentration expressed in percentage by volume is 60%, then rotary evaporation in vacuo removes alcohol at 65 DEG C, obtains typhaneoside crude product；
Step 3: preparative liquid chromatography purifying
Typhaneoside crude product described in step 2 is redissolved with deionized water, injects in preparative liquid chromatograph and is purified,
It is monitored in real time by UV detector, chromatographic column is reverse phase C18, and specification 250*200mm, silica gel partial size is 10 μm, flow velocity
600ml/min, the mobile phase are mixed by A phase with B, and wherein A phase is acetonitrile, and B phase is that concentration expressed in percentage by volume is 0.5%
Acetic acid, the isocratic elution ratio are 15%A phase, elute ratio are as follows:
| Time (min) | Acetonitrile (%) | Water (%) | Acetic acid (%) |
| 0 | 15 | 84.5 | 0.5 |
| 100 | 15 | 84.5 | 0.5 |
Then 7.8-9.2min fraction is collected, typhaneoside fine work is concentrated to get；
Step 4:
The methanol of typhaneoside fine work described in step 3 is dissolved, filtering, evaporated under reduced pressure, is then dissolved by heating with acetonitrile
It places to room temperature, finally filters, obtains high-purity typhaneoside monomer after drying.
Embodiment three
A kind of typhaneoside preparative liquid chromatography separation method, method includes the following steps:
Step 1: alcohol extracting concentration
5kg dry cattail pollen is crushed and is placed in 50L extractor, is that 90% ethyl alcohol mixes with concentration expressed in percentage by volume,
The solid-to-liquid ratio of middle cattail pollen and ethanol solution is 1:8, is extracted twice, each 180min, and completely rear combined extract to be extracted carries out
Centrifugal treating, centrifuge speed is 25000r/min when centrifugal treating, and the solution after centrifugation is then carried out vacuum at 60 DEG C
Rotary evaporation removes alcohol, obtains Pollen typhae；
Step 2: macroporous resin column chromatography
Chromatographic column in the D101 type macroporous absorbent resin that the injection of Pollen typhae liquid described in step 1 has been handled well,
It is first eluted with the deionized water of 3 times of column volumes, then the ethanol water elution for being 50% with concentration expressed in percentage by volume, then with volume hundred
The ethanol water that concentration is 60% is divided to elute, then the ethanol water for being 70% with concentration expressed in percentage by volume elutes, and then collects
The alcoholic solution eluent that concentration expressed in percentage by volume is 70%, then rotary evaporation in vacuo removes alcohol at 80 DEG C, obtains typhaneoside crude product；
Step 3: preparative liquid chromatography purifying
Typhaneoside crude product described in step 2 is redissolved with deionized water, is carried out in injection preparative liquid chromatograph pure
Change, monitored in real time by UV detector, chromatographic column is reverse phase C18, and specification 250mm*200mm, silica gel partial size is 20 μm, stream
Fast 800ml/min, the mobile phase are mixed by A phase with B, and wherein A phase is acetonitrile, and B phase is for concentration expressed in percentage by volume
0.9% phosphoric acid, the isocratic elution ratio are 15%A phase, elute ratio are as follows:
| Time (min) | Acetonitrile (%) | Water (%) | Phosphoric acid (%) |
| 0 | 15 | 84.1 | 0.9 |
| 100 | 15 | 84.1 | 0.9 |
Then 7.8-9.2min fraction is collected, typhaneoside fine work is concentrated to get；
Step 4:
The methanol of typhaneoside fine work described in step 3 is dissolved, filtering, evaporated under reduced pressure, is then dissolved by heating with ethyl alcohol
It places to room temperature, finally filters, obtains high-purity typhaneoside monomer after drying.
High-purity typhaneoside monomer is carried out to examine purity through HPLC, HPLC analysis condition are as follows: C18 (5 μm of * 250mm*
4.6mm), mobile phase acetonitrile: water: phosphoric acid=15:84.8:0.2, Detection wavelength 254nm, flow velocity 1.0ml/min, column temperature are
35 DEG C, according to document and nuclear magnetic spectrum parsing result, obtained component is typhaneoside, is divided from cattail pollen by means of the present invention
From typhaneoside monomer is obtained, purity may be up to 99.0%, and the separation method quantity of sample handling is big, and reproducible, product is pure
Degree is high, is suitable for laboratory and industrial-scale production, this method can continuous large-scale production, overcome in conventional method
Normal phase silica gel chromatography column and it is solvent-extracted cumbersome, separation cycle is long, environmental pollution is serious the shortcomings that, reduce volatile
The use of solvent, it is more efficient to more environment-friendly.
Above-described embodiment, only presently preferred embodiments of the present invention, the practical range being not intended to limit the invention, thus it is all with
The equivalent variations that content described in the claims in the present invention is done should all be included within scope of the invention as claimed.
Claims (9)
1. a kind of typhaneoside preparative liquid chromatography separation method, which is characterized in that method includes the following steps:
Step 1: alcohol extracting concentration
Using cattail pollen as raw material, Pollen typhae is concentrated to get through alcohol extracting；
Step 2: macroporous resin column chromatography
Pollen typhae described in step 1 is added in macroporous resin column and is chromatographed, typhaneoside crude product is obtained after eluting；
Step 3: preparative liquid chromatography purifying
Typhaneoside crude product described in step 2 is purified by preparative liquid chromatograph, i.e., is passed through using reverse phase C18 chromatographic column
Mobile phase isocratic elution, then post-treated obtain typhaneoside fine work；
Step 4:
By typhaneoside fine work described in step 3 through methanol dissolution, filtering, evaporated under reduced pressure, is then recrystallized with solvent, most passed through afterwards
Filtering, drying obtain high-purity typhaneoside monomer.
2. a kind of typhaneoside preparative liquid chromatography separation method as described in claim 1, which is characterized in that in step 1
In, extraction is mixed with alcoholic solution after dry cattail pollen is crushed, wherein the solid-to-liquid ratio of cattail pollen and alcoholic solution is 1:3~1:10, to
Extracting solution is subjected to centrifugal treating using centrifuge after extracting completely, is then carried out the solution after centrifugation at 40~60 DEG C true
Empty rotary evaporation removes alcohol, obtains Pollen typhae.
3. a kind of typhaneoside preparative liquid chromatography separation method as claimed in claim 2, which is characterized in that the alcoholic solution
For one or both of methanol aqueous solution or ethanol water, and it is 50~100% that the alcoholic solution, which is concentration expressed in percentage by volume,
Alcohol-water system.
4. a kind of typhaneoside preparative liquid chromatography separation method as claimed in claim 2, which is characterized in that the centrifuge
Revolving speed be 10000~25000r/min.
5. a kind of typhaneoside preparative liquid chromatography separation method as described in claim 1, which is characterized in that in step 2
In, the Pollen typhae is injected in macroporous resin column, first rinses macroporous resin column with deionized water, then dense with volume basis
The alcoholic solution gradient elution that degree is 20~90%, the alcoholic solution eluent that then collected volume percentage concentration is 60~70%, then
Rotary evaporation in vacuo removes alcohol at 50~80 DEG C of temperature, obtains typhaneoside crude product.
6. a kind of typhaneoside preparative liquid chromatography separation method as claimed in claim 5, which is characterized in that the macropore tree
Rouge column is AB-8 or D101.
7. a kind of typhaneoside preparative liquid chromatography separation method as described in claim 1, which is characterized in that in step 3
In, after typhaneoside crude product described in step 2 is redissolved with deionized water, injects in preparative liquid chromatograph and purified, institute
Stating reverse phase C18 chromatographic column specification is 250*200mm, and silica gel partial size is 5~20 μm, and flow velocity is 300~800ml/min, the stream
Dynamic mutually to be mixed by A phase with B, wherein A phase is acetonitrile, and B phase is that concentration expressed in percentage by volume is 0.1~1% sour water system, described
Isocratic elution ratio is 15%A phase.
8. a kind of typhaneoside preparative liquid chromatography separation method as claimed in claim 7, which is characterized in that the sour water body
Acid in system is one of formic acid, acetic acid, phosphoric acid, trifluoroacetic acid.
9. a kind of typhaneoside preparative liquid chromatography separation method as described in claim 1, which is characterized in that in step 4
In, the recrystallization solvent is at least one of methanol, ethyl alcohol, acetone, acetonitrile.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1468859A (en) * | 2002-07-19 | 2004-01-21 | 复旦大学 | Longbract cattail general flavone extractive and its prepn and use |
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2018
- 2018-11-23 CN CN201811406252.9A patent/CN110016062A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1468859A (en) * | 2002-07-19 | 2004-01-21 | 复旦大学 | Longbract cattail general flavone extractive and its prepn and use |
Non-Patent Citations (2)
| Title |
|---|
| 冯绪强，等: "水烛香蒲花粉镇痛活性部位化学成分研究", 《中南药学》 * |
| 吕紫薇，等: "不同成熟度蒲黄中异鼠李素-3-O-新橙皮苷、香蒲新苷的含量研究", 《临床医药文献杂志》 * |
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