CN101774992A - Method for separating and preparing polymethoxyflavone monomer from orange skin - Google Patents
Method for separating and preparing polymethoxyflavone monomer from orange skin Download PDFInfo
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- CN101774992A CN101774992A CN201010104126A CN201010104126A CN101774992A CN 101774992 A CN101774992 A CN 101774992A CN 201010104126 A CN201010104126 A CN 201010104126A CN 201010104126 A CN201010104126 A CN 201010104126A CN 101774992 A CN101774992 A CN 101774992A
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- polymethoxyflavone
- orange peel
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Abstract
The invention relates to a method for separating and preparing a polymethoxyflavone monomer from orange skin, which comprises the following steps of: leaching polymethoxyflavone from orange skin by using ethanol; and separating the polymethoxyflavone monomer from a crude polymethoxyflavone extract from the orange skin by using a countercurrent chromatography technology. The solvent system comprises sherwood oil which is a liquid under normal temperature and pressure, ethyl acetate, methanol and water in the volume ratio of 1:(0.5-2):1:(1-3). The invention effectively reduces the preparation cost of the polymethoxyflavone monomer by using a high-speed countercurrent chromatograph (HSCCC) and a slow-rate countercurrent chromatograph (SRCCC) as separation devices.
Description
Technical field
The invention belongs to the light industry technical field, relate to the deep process technology of Pon orange peel, be specially and from the Pon orange peel, separate preparation high purity polymethoxyflavone monomer methods.
Technical background
The Pon orange peel contains rich nutrient substances and polymethoxyflavone, existing studies show that natural polymethoxyflavone class material has good anticancer, anti-inflammatory, anti-oxidant, antimutagenic and cardiovascular effect, can be developed to is of value to healthy medicine of people or healthcare products.
Have now and mainly adopt column chromatography and preparative high performance liquid chromatography about the separation of polymethoxyflavone, its limitation is that inferior separating effect or preparation amount are few.Secondly, column chromatography and the relative difficulty of preparative high performance liquid chromatography regeneration use, it is higher that separation obtains polymethoxyflavone monomer cost, is difficult to develop into the big isolation technique of preparation amount.
Summary of the invention
The purpose of this invention is to provide a kind of preparation cost and be lower than prior art, can from the Pon orange peel, separate the method for preparing the monomeric economical and efficient of high purity polymethoxyflavone in enormous quantities.
For achieving the above object, the present invention by the following technical solutions: adopt the polymethoxyflavone in the ethanol lixiviate Pon orange peel, obtain Pon orange peel polymethoxyflavone crude extract; With the counter current chromatograph is that separating device separates the polymethoxyflavone monomer in the crude extract, and its solvent systems is formed by being in liquid sherwood oil, ethyl acetate, first alcohol and water under the normal temperature and pressure, and volume ratio is 1: 0.5～2: 1: 1～3.On the phase that fixes mutually, do moving phase down mutually.
The present invention program further narrates with following step:
1. the polymethoxyflavone in the employing ethanol lixiviate raw material Pon orange peel is 3 hours, will contain the vat liquor concentrating under reduced pressure of polymethoxyflavone, is removed until ethanol, obtains extracting concentrated solution;
2. will extract concentrated solution with isopyknic dichloromethane extraction twice, extraction liquid concentrating under reduced pressure, vacuum-drying, the polymethoxyflavone crude extract;
3. adopt the adverse current chromatogram method to separate the polymethoxyflavone crude extract;
4. collect the adverse current chromatogram target components separated and carry out concentrating under reduced pressure, reach more than 40%, adopt lyophilize again, obtain Powdered polymethoxyflavone monomer until the solid quality mark.
The described ethanol of step (1) is massfraction 60-100% aqueous ethanolic solution (water-ethanol).
The vacuum tightness of concentrating under reduced pressure process is lower than 0.09Mpa in step (1), the step (3), and temperature is 50～60 ℃.
Its solvent systems of adverse current chromatogram method that step (3) adopts is formed by being in liquid sherwood oil, ethyl acetate, first alcohol and water under the normal temperature and pressure.
The employed counter current chromatograph of step (3) is high-speed counter-current chromatograph (HSCCC) or low speed counter current chromatograph (SRCCC).The detailed process of separating Pon orange peel polymethoxyflavone crude extract is: the preparation solvent systems, leave standstill after fully shaking up, treat layering after, will separate mutually up and down; The chromatographic column of counter current chromatograph will be injected mutually with pump on the solvent systems; The following phased soln Pon orange peel polymethoxyflavone crude extract of getting solvent systems prepares adverse current chromatogram sample separation solution; Open counter current chromatograph, inject adverse current chromatogram sample separation solution, inject moving phase then, detect the adverse current chromatogram effluent liquid, collect target components according to the color atlas of gained with the ultraviolet-visible detector.
The vacuum tightness of concentrating under reduced pressure process is lower than 0.02Mpa in the step (4), and temperature is 30～40 ℃, in cryodesiccated vacuum tightness be lower than 50Pa, temperature is lower than-35 ℃.
Advantage of the present invention is: good separating effect, efficient height, the purity of polymethoxyflavone monomer product reaches more than 95%, polymethoxyflavone monomer preparation cost is far below prior art, is a kind of economy, separates preparation high purity polymethoxyflavone monomer methods efficiently from the Pon orange peel.
Description of drawings
Fig. 1 is a process flow sheet of the present invention.
Fig. 2 is the HSCCC-D400 separating spectrum of embodiment 1.Among the figure: I, II, IV: the unknown; III: tetramethoxy flavones monomer; V: pentamethoxyl flavones monomer; VI: hexa methoxy flavones monomer
Fig. 3 is the HSCCC-D1000 separating spectrum of embodiment 2.
Among the figure: III: tetramethoxy flavones monomer; V: pentamethoxyl flavones monomer; VI: hexa methoxy flavones monomer
Fig. 4 is the SRCCC-D3500 separating spectrum of embodiment 3.
Among the figure: II: tetramethoxy flavones monomer; IV: pentamethoxyl flavones monomer; V: hexa methoxy flavones monomer
Fig. 5 is the SRCCC-D32L separating spectrum of embodiment 4.
Among the figure: II: tetramethoxy flavones monomer; IV: pentamethoxyl flavones monomer; V: hexa methoxy flavones monomer
Embodiment
With reference to the accompanying drawings 1:
1, the polymethoxyflavone in the employing 60%-100% ethanol lixiviate raw material Pon orange peel is 3 hours, the vat liquor that will contain polymethoxyflavone is lower than 0.09Mpa in vacuum tightness, temperature is 50～60 ℃ of concentrating under reduced pressure, substantially removed until ethanol, obtained extracting concentrated solution.
2, will extract concentrated solution with isopyknic dichloromethane extraction twice, extraction liquid is lower than 0.09Mpa in vacuum tightness, and temperature is 30～40 ℃ of concentrating under reduced pressure concentrating under reduced pressure, vacuum-drying, the polymethoxyflavone crude extract.
3, adopt the adverse current chromatogram method to separate the polymethoxyflavone crude extract.
A) preparation solvent systems, leave standstill after fully shaking up, treat layering after, will be separately standby mutually up and down.
B) will inject the chromatographic column of counter current chromatograph on the above-mentioned solvent systems mutually with pump, and get phased soln Pon orange peel polymethoxyflavone extract under the part of above-mentioned solvent systems, preparation adverse current chromatogram sample separation solution.
C) open counter current chromatograph, inject the sample solution of above-mentioned dissolved Pon orange peel polymethoxyflavone extract and moving phase (the injection order can be to inject sample solution earlier to inject moving phase then, also can inject the sample solution that reinjects behind the moving phase pre-equilibration earlier).
D) the ultraviolet-visible detector detects the adverse current chromatogram effluent liquid, collects target components according to the color atlas of gained.
4, the target components of Shou Jiing carries out being lower than 0.09Mpa in vacuum tightness, temperature is 50～60 ℃ of concentrating under reduced pressure, reaches more than 40% until solid concentration, adopts lyophilize again, obtains the polymethoxyflavone monomer powders.Purity is 95～99.
For further describing the counter current chromatograph purge process of step 4 of the present invention, as follows with 4 routine divisions:
Embodiment 1
The solvent systems of present embodiment adopts sherwood oil by volume: ethyl acetate: methyl alcohol: water=1: 1.5: 1: 2, counter current chromatograph is the HSCCC-D400 high-speed counter-current chromatograph, and polymethoxyflavone content accounts for 36% in the Pon orange peel polymethoxyflavone crude extract.
Measure sherwood oil 250ml, ethyl acetate 475ml, methyl alcohol 250ml and water 500ml, place the 1500ml separating funnel, fully shake up, behind the standing demix, two-phase is respectively charged into reagent bottle up and down.With the last chromatographic column of injecting high speed adverse current chromatogram with the flow velocity of 20ml/min.Open counter current chromatograph to 800 commentaries on classics/min, pump into sample solution (being dissolved with phase solution under the 15ml of 1.0g Pon orange peel polymethoxyflavone crude extract) with the flow velocity of 1.0ml/min then, after treating that sample introduction finishes, again with the polymethoxyflavone component in the flow velocity injection moving phase wash-out post of 2.0ml/min, under 330nm, detect elutant with the UV, visible light detector, collect the polymethoxyflavone component according to the collection of illustrative plates that detector is gathered.Separate and obtain tetramethoxy flavones monomer; V: tetramethoxy flavones monomer (II) 45.5mg, pentamethoxyl flavones monomer (V) 135.1mg, hexa methoxy flavones monomer (VI) 106.3mg.Purity is all more than 97%.
Embodiment 2
The solvent systems of present embodiment adopts sherwood oil by volume: ethyl acetate: methyl alcohol: water=1: 1: 1: 1.5, counter current chromatograph is the HSCCC-D1200 high-speed counter-current chromatograph, and polymethoxyflavone content accounts for 36% in the Pon orange peel polymethoxyflavone crude extract.
Measure sherwood oil 750ml, ethyl acetate 750ml, methyl alcohol 750ml and water 1125ml, place the 3000ml separating funnel, fully shake up, behind the standing demix, two-phase is respectively charged into reagent bottle up and down.With the last chromatographic column of injecting high speed adverse current chromatogram with the flow velocity of 20ml/min.Open counter current chromatograph to 800 commentaries on classics/min, pump into sample solution (being dissolved with phase solution under the 50ml of 5.0g Pon orange peel polymethoxyflavone crude extract) with the flow velocity of 2.0ml/min then, after treating that into bar finishes, again with the polymethoxyflavone component in the flow velocity injection moving phase wash-out post of 5.0ml/min, under 330nm, detect elutant with the UV, visible light detector, collect the polymethoxyflavone component according to the collection of illustrative plates that detector is gathered.Separate and obtain tetramethoxy flavones monomer; V: tetramethoxy flavones monomer (II) 231mg, pentamethoxyl flavones monomer (V) 534mg, hexa methoxy flavones monomer (VI) 413mg.Purity is all more than 96%.
Embodiment 3
The solvent systems of present embodiment adopts sherwood oil by volume: ethyl acetate: methyl alcohol: water=1: 1: 1: 2, and counter current chromatograph is a SRCCC-D3500 low speed counter current chromatograph, polymethoxyflavone content accounts for 36% in the Pon orange peel polymethoxyflavone crude extract.
Measure sherwood oil 2000ml, ethyl acetate 2000ml, methyl alcohol 2000ml and water 4000ml, place the 10000ml separating funnel, fully shake up, behind the standing demix, two-phase is respectively charged into reagent bottle up and down.With the last chromatographic column of injecting high speed adverse current chromatogram with the flow velocity of 100ml/min.Open counter current chromatograph to 80 commentaries on classics/min, pump into sample solution (being dissolved with phase solution under the 500ml of 50g Pon orange peel polymethoxyflavone crude extract) with the flow velocity of 10.0ml/min then, after treating that sample introduction finishes, again with the polymethoxyflavone component in the flow velocity injection moving phase wash-out post of 20.0ml/min, under 330nm, detect elutant with the UV, visible light detector, collect the polymethoxyflavone component according to the collection of illustrative plates that detector is gathered.Separate and obtain tetramethoxy flavones monomer; V: tetramethoxy flavones monomer (II) 2.3g, pentamethoxyl flavones monomer (V) 5.4g, hexa methoxy flavones monomer (Vl) 4.3g.Purity is all more than 96%.
Embodiment 4
The solvent systems of present embodiment adopts sherwood oil by volume: ethyl acetate: methyl alcohol: water=1: 0.8: 1: 3, counter current chromatograph is a SRCCC-D32L low speed counter current chromatograph, and polymethoxyflavone content accounts for 36% in the Pon orange peel polymethoxyflavone crude extract.
Measure sherwood oil 20L, ethyl acetate 16L, methyl alcohol 20L and water 60L, place the 160L solvent barrel, fully shake up, behind the standing demix, two-phase is respectively charged into the reagent bucket up and down.With the last chromatographic column of injecting high speed adverse current chromatogram with the flow velocity of 1L/min.Open counter current chromatograph to 80 commentaries on classics/min, pump into sample solution (being dissolved with phase solution under the 5L of 600g Pon orange peel polymethoxyflavone crude extract) with the flow velocity of 20.0ml/min then, after treating that sample introduction finishes, again with the polymethoxyflavone component in the flow velocity injection moving phase wash-out post of 50ml/min, under 330nm, detect elutant with the UV, visible light detector, collect the polymethoxyflavone component according to the collection of illustrative plates that detector is gathered.Separate and obtain tetramethoxy flavones monomer; V: tetramethoxy flavones monomer (II) 28g, pentamethoxyl flavones monomer (V) 63g, hexa methoxy flavones monomer (VI) 48g.Purity is all more than 95%.
Claims (7)
1. from the Pon orange peel, separate preparation polymethoxyflavone monomer methods, it is characterized in that following steps:
(1) polymethoxyflavone in the ethanol lixiviate raw material Pon orange peel of employing massfraction 60～100% is 3 hours, will contain the vat liquor concentrating under reduced pressure of polymethoxyflavone, is removed substantially until ethanol, obtains concentrated extracting solution;
(2) with concentrated extracting solution with isopyknic dichloromethane extraction twice, extraction liquid concentrating under reduced pressure, vacuum-drying, the polymethoxyflavone crude extract;
(3) adopt the adverse current chromatogram method to separate the polymethoxyflavone crude extract;
(4) collect the adverse current chromatogram target components separated and carry out concentrating under reduced pressure, reach more than 40%, adopt lyophilize again, obtain Powdered polymethoxyflavone monomer until the solid quality mark.
2. according to the described preparation polymethoxyflavone monomer methods of from the Pon orange peel, separating of claim 1, it is characterized in that step (1) employing massfraction is 75% extraction using alcohol.
3. according to the described preparation polymethoxyflavone monomer methods of from the Pon orange peel, separating of claim 1, the vacuum tightness that it is characterized in that concentrating under reduced pressure process in step (1), the step (3) is lower than 0.09Mpa, temperature is 50～60 ℃, the vacuum tightness of concentrating under reduced pressure process is lower than 0.02Mpa in the step (4), and temperature is 30～40 ℃.
4. according to the described preparation polymethoxyflavone monomer methods of separating of claim 1, it is characterized in that cryodesiccated vacuum tightness is lower than 50Pa in the step (4) from the Pon orange peel, temperature is lower than-35 ℃.
5. according to the described preparation polymethoxyflavone monomer methods of from the Pon orange peel, separating of claim 1, it is characterized in that its solvent systems of adverse current chromatogram method of step (3) employing is formed by being in liquid sherwood oil, ethyl acetate, first alcohol and water under the normal temperature and pressure.
6. according to the described preparation polymethoxyflavone monomer methods of from the Pon orange peel, separating of claim 1, it is characterized in that the employed counter current chromatograph of step (3) is high-speed counter-current chromatograph HSCCC or low speed counter current chromatograph SRCCC.
7. according to the described preparation polymethoxyflavone monomer methods of from the Pon orange peel, separating of claim 1, it is characterized in that the detailed process that step (3) adopts counter current chromatograph to separate Pon orange peel polymethoxyflavone crude extract is: the preparation solvent systems, leave standstill after fully shaking up, after treating layering, will separate mutually up and down; The chromatographic column of counter current chromatograph will be injected mutually with pump on the solvent systems; The following phased soln Pon orange peel polymethoxyflavone crude extract of getting solvent systems prepares adverse current chromatogram sample separation solution; Open counter current chromatograph, inject adverse current chromatogram sample separation solution, inject moving phase then, detect the adverse current chromatogram effluent liquid, collect target components according to the color atlas of gained with the ultraviolet-visible detector.
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| CN201010104126A CN101774992A (en) | 2010-01-26 | 2010-01-26 | Method for separating and preparing polymethoxyflavone monomer from orange skin |
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| CN201010104126A CN101774992A (en) | 2010-01-26 | 2010-01-26 | Method for separating and preparing polymethoxyflavone monomer from orange skin |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102603697A (en) * | 2012-02-06 | 2012-07-25 | 四川亿乙显生物科技有限公司 | Process for extracting polymethoxylated flavones from lemon |
| CN107011308A (en) * | 2017-04-17 | 2017-08-04 | 浙江大学 | The method that polymethoxyflavone class compound is isolated and purified from bowl mandarin orange fruit |
-
2010
- 2010-01-26 CN CN201010104126A patent/CN101774992A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102603697A (en) * | 2012-02-06 | 2012-07-25 | 四川亿乙显生物科技有限公司 | Process for extracting polymethoxylated flavones from lemon |
| CN107011308A (en) * | 2017-04-17 | 2017-08-04 | 浙江大学 | The method that polymethoxyflavone class compound is isolated and purified from bowl mandarin orange fruit |
| CN107011308B (en) * | 2017-04-17 | 2019-09-13 | 浙江大学 | The method of polymethoxyflavone class compound is isolated and purified from bowl mandarin orange fruit |
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Open date: 20100714 |