CN103432195A - Method for one step separation and purification of the total iridoid glycoside component in lamiophlomis rotata by using active carbon chromatograph column - Google Patents
Method for one step separation and purification of the total iridoid glycoside component in lamiophlomis rotata by using active carbon chromatograph column Download PDFInfo
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- CN103432195A CN103432195A CN2013102751180A CN201310275118A CN103432195A CN 103432195 A CN103432195 A CN 103432195A CN 2013102751180 A CN2013102751180 A CN 2013102751180A CN 201310275118 A CN201310275118 A CN 201310275118A CN 103432195 A CN103432195 A CN 103432195A
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Abstract
The present invention relates to a method for separating and purifying the total iridoid glycoside in lamiophlomis rotata by using an active carbon chromatograph column. The method is characterized by comprising: extracting the aerial part of lamiophlomis rotata with ethanol, concentrating the extraction solution into an extract, dissolving the extract, adding the dissolved extract to an active carbon chromatograph column, rinsing with distilled water until achieving a colorless state, then eluting with 70-90% ethanol, and collecting the fraction, wherein active carbon (0.42-0.5 mm) is adopted as a chromatograph column immobile phase, 70-90% ethanol is adopted as a mobile phase, the total iridoid glycoside component content is 80-90%, and a recovery rate can be 70-82%. According to the invention, the one step separation and purification method is adopted, and the obtained total iridoid glycoside component has advantages of high separation efficiency, good purity and high yield.
Description
Technical field
The present invention relates to the preparation method of separation and purification total iridoid glycoside in a kind of Radix Lamiophlomidis Rotatae, utilize the method for activated carbon column chromatography method effective site total iridoid glycoside of separating and purifying high-purity from Radix Lamiophlomidis Rotatae.
Background technology
Radix Lamiophlomidis Rotatae
lamiophlomis rotata(Benth.) Kudo is Labiatae acaulescence herbaceos perennial, has the effects such as blood circulation promoting and blood stasis dispelling, pain easing and hemostasis, functions of detumescence, relieving inflammation.Modern pharmacological research shows, in Radix Lamiophlomidis Rotatae aerial parts and root, main chemical compositions has flavonoid, iridoid glycosides and phenethyl alcohol glycosides etc., and wherein total iridoid glycoside is to stop blooding in Radix Lamiophlomidis Rotatae, analgesia, the main active site of antiinflammatory.In existing Radix Lamiophlomidis Rotatae, the separation method of iridoid methods of glycosides is for adopting polyamide de-etiolation ketone, again through enriching and purifying macroporous resin total iridoid methods of glycosides, this method is complex steps not only, and purity is not high, therefore, due to its special pharmacological effect, how from the raw medicinal herbs crude extract, separate fast and efficiently and purification to obtain the total iridoid methods of glycosides be the problem that everybody relatively is concerned about.
To the separation and purification of Radix Lamiophlomidis Rotatae total iridoid glycosides, existing method comprises: polyamide is in conjunction with Flavonoids by Macroporous Adsorption Resin, the medium pressure column chromatography method.
Polyamide is in conjunction with macroporous absorption resin chromatography: this method is first utilized flavones ingredient in polyamide column absorption Radix Lamiophlomidis Rotatae, effluent is through macroporous adsorptive resins, with the ethanol elution of 50%-70%, in products obtained therefrom, the content of total iridoid glycoside is 57.91%, and the response rate is 78.22%.(Chinese experimental pharmacology of Chinese medical formulae magazine, 2011,17(13): 32-35) the method can be used in the enrichment of Radix Lamiophlomidis Rotatae total iridoid glycosides, but also comprise the part flavone in product, phenethyl alcohol glycoside and principal component not, color is darker, and complex steps.
The medium pressure column chromatography method can obtain highly purified total iridoid glycoside, monomer component even, but yield is too low, and the amount once obtained very little, can not realize suitability for industrialized production.
At present, active carbon is used to decolouring more, but active carbon is nonpolar absorption, and absorbability is the strongest, can be used to the separating water-soluble material, especially separating compositions such as glycoside, saccharide and aminoacid.
Summary of the invention
For the problem existed in existing technique, the present invention aims to provide a kind of method of active carbon chromatographic column one step separation and purification total iridoid methods of glycosides of utilizing to obtain easy, as to separate fast and efficiently a Radix Lamiophlomidis Rotatae total iridoid glycosides constituents method.
The object of the present invention is achieved like this:
A kind of method of utilizing total iridoid methods of glycosides in active carbon chromatographic column one step separation and purification Radix Lamiophlomidis Rotatae, the Radix Lamiophlomidis Rotatae aerial parts is shredded, cross the 40-50 mesh sieve, with the 60-70% ethanol of 10 times of amounts at 60 ℃-100 ℃ by 40 order Radix Lamiophlomidis Rotatae reflux, extract, three times, each 2 h, merge the gained medicinal liquid, filter, decompression and solvent recovery evaporate to dryness obtain extractum.Get the active carbon water and mix thoroughly, wet method dress post, column internal diameter used is 2.1cm, column length 25cm; Get extractum, dissolve rear normal pressure loading fully with distilled water, treat that loading is complete, take active carbon as chromatographic column fixed phase, under the flow velocity of 4BV/h, wash 5BV with water and be colourless to remove impurity to effluent, then be that mobile phase eluting 4BV obtains the Radix Lamiophlomidis Rotatae total iridoid glycosides product that content reaches 80-90% with ethanol.
The activated carbon particle size of above-mentioned use is at 0.42-0.5 mm.
The 8-12 that in above-mentioned chromatographic column, the weight of active carbon is extractum weight doubly.
Above-mentioned organic solvent is ethanol, and eluting concentration is at 60%-90%; The active carbon eluting is 90% by the optium concentration of ethanol, and collecting amount is 5 times of amount column volumes.
Radix Lamiophlomidis Rotatae total iridoid glycosides physicochemical property: white crystals body, bitter in the mouth.Soluble in water, methanol; Be dissolved in ethanol, n-butyl alcohol equal solvent; Be insoluble in the lipophilic solvents such as chloroform, ether, benzene.
In separation and purification of the present invention:
According to the physicochemical property of iridoid glycoside, it dissolves in ethanol, acetone and n-butyl alcohol, is insoluble in the lipotropy organic solvents such as chloroform, ether, benzene, easily is acid hydrolysis.The present invention, in adopting the activated carbon column chromatography process, had once attempted having compared and has used n-butyl alcohol, acetone, ethanol as elution system, and the yield size is ethanol > acetone > n-butyl alcohol.The rate of transform size of utilizing 8-0-acetyl Shanzhiside methyl ester in high-efficient liquid phase chromatogram technique analysis gained sample is ethanol > acetone > n-butyl alcohol.Comprehensive the above results, select ethanol as eluting solvent.
Utilize ethanol as eluant, under the prerequisite that guarantees purity and the response rate, investigated the concentration of applied sample amount and eluant.Under constant prerequisite, applied sample amount has been investigated to 1:24 in other conditions (temperature, flow velocity, loading volume); 1:12; 1:8; 1:6; 1:4.8(extractum amount/amounts of activated carbon; G/g) five conditions, the adsorption rate of each group is respectively 97.04%, 96.78%, 93.5%, 76.79%, 41.62%, therefore, applied sample amount (extractum amount/amounts of activated carbon; G/g) preferably between 1:12-1:8.Select 30%, 50%, 70%, 90% concentration of alcohol to carry out preferably, ultraviolet 235nm place investigates the absorption peak result and shows eluting rate 90% > 70% > 50% > 10%; The 8-0 acetylshanzhiside methyl ester result that adopts the HPLC method to measure wherein obtains the rate of transform: 90% ethanol elution 82%, 70% ethanol elution: 70%, 50% ethanol elution: 43%, 30% ethanol elution: 12%.The purity of calculating total iridoid glycoside with areas of peak normalization method obtains: 90% ethanol elution: 88.69%, 70% ethanol elution: 79.04%.Optimize above-mentioned condition, in the separation and purification Radix Lamiophlomidis Rotatae, the preferred plan of total iridoid glycoside is: activated carbon particle size 0.42-0.5mm, and applied sample amount is in 1:12-1:8(extractum amount/amounts of activated carbon; G/g) between, the eluting concentration of alcohol is 90%, and elution volume is 5 times of amount column volumes.
The preferred wet method dress of active carbon chromatographic column post, solvent for use is distilled water, the 8-12 that in chromatographic column, the weight of active carbon is extractum weight is doubly.
Advantage of the present invention:
Mainly comprise flavones ingredient, iridoid glycoside constituents and phenylethanoid glycoside in Radix Lamiophlomidis Rotatae.Flavones ingredient has maximum absorption band at 250nm and 344nm place, and the iridoid glycoside constituents has maximum absorption band at the 235nm place, and phenylethanoid glycoside has maximum absorption band at the 332nm place.By Fig. 1 result, can be found out, by document (Chinese experimental pharmacology of Chinese medical formulae magazine, 2011,17(13): 32-35) Radix Lamiophlomidis Rotatae total iridoid glycosides of method gained (1) is at 235nm, 285nm and 332nm all have maximum absorption band, illustrate and wherein not only comprise the iridoid glycoside constituents, also contain phenylethanoid glycoside and flavones ingredient.It is immobile phase that the present invention adopts active carbon, adopts the Radix Lamiophlomidis Rotatae total iridoid glycosides of 90% and 70% ethanol elution gained, and it has maximum absorption band at the 235nm place, and at 250nm, 332nm, the 344nm place there is no absworption peak or interference, shows that it does not contain or seldom contain flavonoid and phenylethanoid glycoside.
Material therefor economy of the present invention, method therefor are simple, solvent for use also easily reclaims, avoided the iridoid glycoside constituents to decompose rotten, in addition, applying this invention only needs a step separation and purification to obtain content and the high total iridoid glycoside of yield, the product loss of having avoided the loaded down with trivial details of multi-step and having brought.The present invention is efficient, quick, technique is simple, can utilize on the basis of simple equipment and economic parting material and reach significant separating effect, have that separation efficiency is high, the content of total iridoid methods of glycosides reaches 80-90% in product, and the response rate can reach 70-82%.
The accompanying drawing explanation
Fig. 1 is the uv-spectrogram that the present invention utilizes total iridoid methods of glycosides in active carbon chromatographic column one step separation and purification Radix Lamiophlomidis Rotatae, in figure:
1. the uv-spectrogram of macroporous resin enrichment Radix Lamiophlomidis Rotatae total iridoid glycosides;
2. employing activated-charcoal column, the uv-spectrogram of 90% ethanol elution gained Radix Lamiophlomidis Rotatae total iridoid glycosides;
3. employing activated-charcoal column, the uv-spectrogram of 70% ethanol elution gained Radix Lamiophlomidis Rotatae total iridoid glycosides.
the specific embodiment
Embodiment 1
Get 500g Radix Lamiophlomidis Rotatae aerial parts chopping, cross 50 mesh sieves, with 70% ethanol of 10 times of amounts at 60 ℃ by 50 order Radix Lamiophlomidis Rotatae reflux, extract, three times, each 2 h, merge the gained medicinal liquid, filtration, decompression and solvent recovery evaporate to dryness obtain 15g extractum.Get 5g, 40 order active carbons are mixed thoroughly by 10 times of water yields, wet method dress post, column internal diameter used is 2.1cm, column length 25cm; Get 1g extractum, with 5 times of volume distilled water, dissolve rear normal pressure loading fully, treat that loading is complete.Take active carbon as chromatographic column fixed phase, under the flow velocity of 4BV/h, wash 5BV with water and be colourless to remove impurity to effluent, then be that mobile phase eluting 4BV obtains content and reaches 84% Radix Lamiophlomidis Rotatae total iridoid glycosides product with 70% ethanol, with the 8-O acetylshanzhiside methyl ester, calculate, the response rate reaches 71.4%.
Embodiment 2
Get 500g Radix Lamiophlomidis Rotatae aerial parts chopping, cross 50 mesh sieves, with 60% ethanol of 10 times of amounts at 100 ℃ by 50 order Radix Lamiophlomidis Rotatae reflux, extract, three times, each 2 h, merge the gained medicinal liquid, filtration, decompression and solvent recovery evaporate to dryness obtain 12.5g extractum.Get 5g, 40 order active carbons are mixed thoroughly by 10 times of water yields, wet method dress post, column internal diameter used is 2.1cm, column length 25cm; Get 1g extractum, with 5 times of volume distilled water, dissolve rear normal pressure loading fully, treat that loading is complete.Take active carbon as chromatographic column fixed phase, under the flow velocity of 4BV/h, wash 5BV with water and be colourless to remove large polar substances to effluent, 90% ethanol is that mobile phase eluting 4BV obtains content and reaches 89.4% Radix Lamiophlomidis Rotatae total iridoid glycosides product again, with the 8-O acetylshanzhiside methyl ester, calculate, its response rate reaches 85.3%.
Due to the adsorption of active carbon, the strongest in aqueous solution, in organic solvent a little less than, therefore attached by the organic solvent desorption.While with alcohol-water, carrying out eluting, with concentration of alcohol increase progressively and eluting power increase.While therefore adopting 90% ethanol elution, the response rate is higher than 70% ethanol elution.
Claims (4)
1. a method of utilizing total iridoid methods of glycosides in active carbon chromatographic column one step separation and purification Radix Lamiophlomidis Rotatae, the Radix Lamiophlomidis Rotatae aerial parts is shredded, cross the 40-50 mesh sieve, with the 60-70% ethanol of 10 times of amounts, 50 order Radix Lamiophlomidis Rotataes are extracted three times at 60 ℃ of-100 ℃ of reduced-pressure backflows, each 2 h, merge the gained medicinal liquid, filter, decompression and solvent recovery evaporate to dryness obtain extractum; Get the active carbon water and mix thoroughly, wet method dress post, column internal diameter used is 2.1cm, column length 25cm; It is characterized in that: get extractum, dissolve rear normal pressure loading fully with distilled water, treat that loading is complete, take active carbon as chromatographic column fixed phase, under the flow velocity of 4BV/h, wash 5BV with water and be colourless to remove large polar substances to effluent, then be that mobile phase eluting 4BV obtains the Radix Lamiophlomidis Rotatae total iridoid glycosides product that content reaches with ethanol.
2. a kind of method of utilizing total iridoid methods of glycosides in active carbon chromatographic column one step separation and purification Radix Lamiophlomidis Rotatae as claimed in claim 1, it is characterized in that: described activated carbon particle size is 0.42-0.5 mm.
3. a kind of method of utilizing total iridoid methods of glycosides in active carbon chromatographic column one step separation and purification Radix Lamiophlomidis Rotatae as claimed in claim 1, it is characterized in that: the 8-12 that in described chromatographic column, the weight of active carbon is extractum weight doubly.
4. a kind of method of utilizing total iridoid methods of glycosides in active carbon chromatographic column one step separation and purification Radix Lamiophlomidis Rotatae as claimed in claim 1, it is characterized in that: described organic solvent is that ethanol, eluting concentration are 60%-90%; The active carbon eluting is 90% by the optium concentration of ethanol, and collecting amount is 5 times of amount column volumes.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1660163A (en) * | 2004-12-29 | 2005-08-31 | 贾正平 | Method for extracting general iridoid glycoside from 'Duyiwei' of Tibet medicine and application |
| CN101744873A (en) * | 2008-12-02 | 2010-06-23 | 甘肃独一味生物制药股份有限公司 | Externally used liquid preparation containing only iridoid glycoside and application thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1660163A (en) * | 2004-12-29 | 2005-08-31 | 贾正平 | Method for extracting general iridoid glycoside from 'Duyiwei' of Tibet medicine and application |
| CN101744873A (en) * | 2008-12-02 | 2010-06-23 | 甘肃独一味生物制药股份有限公司 | Externally used liquid preparation containing only iridoid glycoside and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| 董娟娥等: "植物中环烯醚萜类化合物研究进展", 《西北林学院学报》 * |
| 贾正平等: "独一味抗肿瘤活性成分的体外筛选", 《西北国防医学杂志》 * |
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Application publication date: 20131211 |