CN101987815B - Purification process for preparing high-purity coenzyme Q10 - Google Patents
Purification process for preparing high-purity coenzyme Q10 Download PDFInfo
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- CN101987815B CN101987815B CN 201010293858 CN201010293858A CN101987815B CN 101987815 B CN101987815 B CN 101987815B CN 201010293858 CN201010293858 CN 201010293858 CN 201010293858 A CN201010293858 A CN 201010293858A CN 101987815 B CN101987815 B CN 101987815B
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Abstract
The invention relates to a purification process for preparing high-purity coenzyme Q10 from crude coenzyme Q10 extracts obtained from thallus fermentation, which belongs to the technical field of compound separation purification. The purification process is characterized by sequentially comprising the following steps of: carrying out adsorbent resin adsorption, elution, concentration, crystallization and recrystallization on the crude coenzyme Q10 extracts; then, carrying out chromatography on a silicagel column; and purifying and refining by using a petroleum ether -aether or normal hexane-ethyl acetate mixed solvents as eluant. The invention has the following advantages that by adopting an adsorbent resin, good adsorption performance can be exerted on the coenzyme Q10, the desorption operation is simple, the stability is good, and the adsorbent resin can be reused many times; and by combining the adsorbent resin with the silica gel chromatography method, the silica gel utilization rate is high, the required silica gel is low quantity and can maintain good purification effect after being reused more than 10 times, the waste quantity of the silica gel is small, and the solvents can be recycled so that the purpose of environment protection is achieved. The production operation is simple and convenient, and the purity of the obtained coenzyme Q10 is higher than or equal to 98 percent.
Description
Technical field
The invention belongs to compound separation purification technique field, relate to the purifying process of biomedical product Coenzyme Q10 99.0, relate in particular to the technique of separation and purification Coenzyme Q10 99.0 from a kind of Coenzyme Q10 99.0 crude extract that fermentation obtains from thalline.
Background technology
Coenzyme Q10 99.0 (coenzyme Q10) claim again ubiquinone, is a kind of fat-soluble quinones, and structure and vitamin K are close.It is widely distributed at occurring in nature, mainly is present in the heart of yeast, plant leaf, seed and animal, hepatic and/or renal cell.
American scholar Frederick nineteen fifty-seven separates from bovine cardiac first and obtains Coenzyme Q10 99.0, and it combines with mitochondrial inner membrane, participation cellular oxidation phosphorylation and ATP generative process.Nearest research discovery, Coenzyme Q10 99.0 also has preventive and therapeutic action to various diseases such as cancer, cardiovascular disorder, Parkinson's disease and acquired immune deficiency syndrome (AIDS), and has the effects such as enhancing and adjusting immunity of human body.Coenzyme Q10 99.0 is one of important member of mitochondrial respiratory chain.
The existing production method of Coenzyme Q10 99.0 is divided into four kinds: animal vegetable tissue extraction method, chemical synthesis, Salanesol semi-synthesis method and microbe fermentation method.Wherein microbial fermentation because its production cost is lower, is acknowledged as the production method of tool potentiality and advantage.And existing production technology adopts microbial fermentation processes mostly.An important factor of restriction Coenzyme Q10 99.0 cost is the downstream extraction purifying.As how cheaply method obtain highly purified Coenzyme Q10 99.0, become purifies and separates worker's research emphasis.
Existing from fermented liquid the method for purifying high-purity Coenzyme Q10 99.0 be divided into for two steps:
1. Coenzyme Q10 99.0 is slightly carried: with organic solvent extraction method (Unexamined Patent 11-178595 etc.), and the thick formulation of cell fragmentation (JP 56-072694 etc.), the crude extract product purity that obtains is low, and it is lower that the organic solvent consumption is put forward greatly and slightly rate.
2. crude extract purification refine.Process for purification has: silica gel or activated alumina column chromatography (JP 56-164791, JP 55-111793 etc.).But existing method has the following disadvantages: higher its industrial applications cost that makes of activated alumina cost rises; Silica gel recycling number of times is low; Thereby cause the industrial operation environment abominable, easily cause environmental pollution, the after-cost is increased.
Summary of the invention
The present invention seeks to, overcome the shortcoming that prior art exists, for fermented liquid Coenzyme Q10 99.0 crude extract, provide a kind of simple to operate, with low cost, silica gel and resin repeating utilization factor high Coenzyme Q10 99.0 purifying process.
For achieving the above object, the technical scheme of employing is: a kind of purifying process for preparing the high purity Coenzyme Q10 99.0 comprises the following steps successively:
(1) with organic solvent A dissolving Coenzyme Q10 99.0 crude extract, and with 2～3 times of column volumes/hour flow velocity this solution is pumped in the glass column that is filled with polymeric adsorbent, reach leak wear a little after, stop sample introduction;
(2) with the resin column in organic solvent B washing step (1), after 5 times of column volumes of wash-out, use organic solvent C wash-out resin column instead, obtain the Coenzyme Q10 99.0 elutriant;
(3) the Coenzyme Q10 99.0 elutriant of step (2) is concentrated, be statically placed in 0 ℃ of lower crystallization 12h, recrystallization 2 times with organic solvent D dissolving;
(4) crystal that further step (3) is obtained carries out purification refine, and take final product quality: the chromatographic silica gel mass ratio was as 1: 25 loading, and organic solvent E collects the Coenzyme Q10 99.0 elutriant as eluent.
Described in step (1), organic solvent A refers to ethanol, and perhaps the volume ratio of acetone and water is (8～20): 1 acetone and water mixture.
Described in step (2), organic solvent B refers to ethanol, and perhaps acetone and water volume ratio are (6～7): 1 acetone and water mixture.
Described in step (2), organic solvent C refers to acetone or ethyl acetate.
Described in step (3), organic solvent D refers to ethanol, acetone or ethyl acetate.
Described in step (4), organic solvent E is sherwood oil: the ether volume ratio is (15～25): 1 or normal hexane: the ethyl acetate volume ratio is (20～30): 1 mixed organic solvents.
Polymeric adsorbent described in step (1) refers to HZ series 816,818,820,830 nonpolar adsorption resins such as grade.
Compared with the prior art, the present invention has the following advantages:
(1) select polymeric adsorbent, Coenzyme Q10 99.0 is had good absorption property, desorb is simple to operate, and good stability and recycling number of times are high, thereby make suitability for industrialized production easy and simple to handle, alleviate environmental stress, and solvent all can be more easy to be recycled.
(2) select polymeric adsorbent to be combined with the silica gel column chromatography method, greatly reduce silica gel purification pressure, compare with existing single dependence silica gel chromatography, in the present invention, the silica gel utilization ratio is high, not only required silica gel amount is little, and reuse the purification effect (prior art silica gel recycling number of times is only 3～4 times) that still can keep good more than 10 times, thereby can reduce the discarded amount of silica gel.That the present invention has is simple to operate, with low cost, silica gel and resin repeating utilization factor advantages of higher, gained Coenzyme Q10 99.0 purity 〉=98%, total recovery 〉=75%.
Embodiment
The invention will be further described below by embodiment.
A kind of purifying process for preparing the high purity Coenzyme Q10 99.0, step is as follows:
(1) with organic solvent A dissolving Coenzyme Q10 99.0 crude extract, and with 2～3 times of column volumes/hour flow velocity this solution is pumped in the glass column that is filled with polymeric adsorbent, reach leak wear point (effluent liquid Coenzyme Q10 99.0 concentration/initial soln Coenzyme Q10 99.0 concentration=0.05) after, stop sample introduction;
(2) with the resin column in organic solvent B washing step (1), after 5 times of column volumes of wash-out, use organic solvent C wash-out resin column instead, obtain the Coenzyme Q10 99.0 elutriant;
(3) the Coenzyme Q10 99.0 elutriant of step (2) is concentrated, be statically placed in 0 ℃ of lower crystallization 12h, recrystallization 2 times with organic solvent D dissolving;
(4) crystal that further step (3) is obtained carries out purification refine, and with finished product: 1: 25 loading of chromatographic silica gel mass ratio, organic solvent E collects the Coenzyme Q10 99.0 elutriant as eluent, concentrated gained elutriant drying under reduced pressure.
Described in step (1), organic solvent A refers to ethanol, perhaps the mixture of acetone and water.In the mixture of described acetone and water, the volume ratio of acetone and water is (8～20): 1.
Described in step (2), organic solvent B refers to ethanol, and perhaps acetone and water volume ratio are (6～7): 1 acetone and water mixture.
Described in step (2), organic solvent C refers to acetone or ethyl acetate.
Described in step (3), organic solvent D refers to ethanol, acetone or ethyl acetate.
Described in step (4), the E organic solvent refers to sherwood oil: the ether volume ratio is (15～25): 1 or normal hexane: the ethyl acetate volume ratio is (20～30): 1 mixed organic solvents.
Polymeric adsorbent described in step (1) refers to commercially available HZ series 816,818,820,830 non-polar resins such as grade.
Embodiment 1
Take Coenzyme Q10 99.0 crude extract 1g and be dissolved in 300ml ethanol, with HZ816 resin filling separator column, with 2 times of column volumes/hour loading, with ethanolic soln wash-out 5 times of column volumes, use 2 times of column volumes of acetone soln wash-out instead after removing most of impurity after end of the sample.With gained elutriant concentrating under reduced pressure, will obtain enriched material dissolving with 100ml ethanol, under 0 ℃ standing 12 hours, recrystallization can get 93.4% Coenzyme Q10 99.0 crystal for 2 times.With gained Coenzyme Q10 99.0 crystal: chromatographic silica gel=1: 25 loading, with normal hexane: ethyl acetate=20: 1 is collected Coenzyme Q10 99.0 as eluent, and gained Coenzyme Q10 99.0 solution decompression concentrate drying can get purity 〉=98% crystal.
Embodiment 2
Take Coenzyme Q10 99.0 crude extract 1g and be dissolved in 300ml acetone: water=in 8: 1.With HZ820 resin filling separator column, with 3 times of column volumes/hour loading, use acetone after end of the sample: water=5 times of volumes of 6: 1 eluant solutions, use 2 times of column volumes of ethyl acetate solution wash-out instead after removing most of impurity.With gained elutriant concentrating under reduced pressure, will obtain enriched material dissolving with the 50ml acetone soln, under 0 ℃ standing 12 hours, recrystallization can get 92.5% Coenzyme Q10 99.0 crystal for 2 times.With gained Coenzyme Q10 99.0 crystal: 1: 25 loading of chromatographic silica gel mass ratio, with normal hexane: the ethyl acetate volume ratio as eluent is collected Coenzyme Q10 99.0 at 30: 1, and gained Coenzyme Q10 99.0 solution decompression concentrate drying can get purity 〉=98% crystal.
Embodiment 3
Take Coenzyme Q10 99.0 crude extract 1g and be dissolved in 300ml acetone: water volume ratio is in the mixing solutions of 20: 1.With HZ830 resin filling separator column, with 3 times of column volumes/hour loading, use acetone after end of the sample: water volume ratio is 5 times of volumes of 7: 1 eluant solutions, uses 2 times of column volumes of acetone soln wash-out instead after removing most of impurity.With gained elutriant concentrating under reduced pressure, will obtain enriched material dissolving with the 30ml ethyl acetate, under 0 ℃ standing 12 hours, recrystallization can get 91.4% Coenzyme Q10 99.0 crystal for 2 times.With gained Coenzyme Q10 99.0 crystal: 1: 25 loading of chromatographic silica gel mass ratio, with sherwood oil: the ether volume ratio as eluent is collected Coenzyme Q10 99.0 at 25: 1, and gained Coenzyme Q10 99.0 solution decompression concentrate drying can get purity 〉=98% crystal.
Make respectively the HPLC collection of illustrative plates of Coenzyme Q10 99.0 after Coenzyme Q10 99.0 crude extract, standard model and silica gel column chromatography.HPLC figure by Coenzyme Q10 99.0 after Coenzyme Q10 99.0 crude extract HPLC figure and silica gel column chromatography relatively can obviously find out, has removed impurity through the Coenzyme Q10 99.0 after purifying.After contrast Coenzyme Q10 99.0 standard model figure and silica gel column chromatography, the HPLC of Coenzyme Q10 99.0 figure as can be known, Coenzyme Q10 99.0 finished product after this technique purifying and purity consistent with the standard substance collection of illustrative plates is to 〉=98%, illustrate that this technique can reach the purpose of purification refine Coenzyme Q10 99.0, and the advantage such as this purifying process has that purity is high, yield is high, cost is low, environmental friendliness, operational safety are convenient.
Claims (2)
1. purifying process for preparing the high purity Coenzyme Q10 99.0 is characterized in that comprising the following steps successively:
(1) with solvent orange 2 A dissolving Coenzyme Q10 99.0 crude extract, and with 2～3 times of column volumes/hour flow velocity this solution is pumped in the glass resin post that is filled with polymeric adsorbent, reach leak wear a little after, stop sample introduction;
(2) with the resin column in solvent B washing step (1), after 5 times of column volumes of wash-out, use organic solvent C2 times of column volume wash-out resin column instead, obtain the Coenzyme Q10 99.0 elutriant;
(3) the Coenzyme Q10 99.0 elutriant of step (2) is concentrated, be statically placed in 0 ℃ of lower crystallization 12h, recrystallization 2 times with organic solvent D dissolving;
(4) crystal that further step (3) is obtained carries out purification refine, and take finished product and chromatographic silica gel mass ratio as 1: 25 loading, organic solvent E collects the Coenzyme Q10 99.0 elutriant as eluent;
Described in step (1), solvent orange 2 A refers to ethanol, and perhaps acetone and water volume ratio are (8～20): the mixture of 1 acetone and water;
Described in step (2), solvent B refers to ethanol, and perhaps acetone and water volume ratio are (6～7): 1 acetone and water mixture; Described organic solvent C refers to acetone or ethyl acetate;
Described in step (3), organic solvent D refers to ethanol, acetone or ethyl acetate;
Described in step (4), organic solvent E is sherwood oil: the ether volume ratio is (15～25): 1 or normal hexane: the ethyl acetate volume ratio is (20～30): 1 mixed organic solvents.
2. a kind of purifying process for preparing the high purity Coenzyme Q10 99.0 as claimed in claim 1, it is characterized in that: polymeric adsorbent described in step (1) refers to commercially available HZ series 816,818,820,830 nonpolar adsorption resins.
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Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102351698A (en) * | 2011-08-15 | 2012-02-15 | 上海华震科技有限公司 | Adsorption chromatograph based method for recovering pleuromutilin from crystallization mother liquor |
| CN102391092B (en) * | 2011-11-22 | 2013-05-01 | 杭州华东医药集团康润制药有限公司 | Method for preparing high-purity coenzyme Q10 in large scale |
| CN108017530B (en) * | 2017-12-12 | 2020-10-13 | 浙江大学 | Method for continuously separating coenzyme Q10 from mushroom dregs |
| CN108084007B (en) * | 2017-12-12 | 2020-05-08 | 浙江大学 | Method for separating coenzyme Q10 and coenzyme Q11 by simulated moving bed chromatography |
| CN108047014B (en) * | 2017-12-12 | 2020-07-07 | 浙江大学 | Method for extracting and separating coenzyme Q10 by using ionic liquid |
| CN110944970B (en) * | 2018-08-06 | 2023-08-01 | 内蒙古金达威药业有限公司 | For the production of coenzyme Q 10 Systems and methods of (1) |
| CN110002985A (en) * | 2019-05-15 | 2019-07-12 | 丽珠集团(宁夏)制药有限公司 | One kind is from ubiquinone10Ubiquinone is isolated and purified in mother liquor10Method and ubiquinone10Crude product |
| CN112920035A (en) * | 2019-12-06 | 2021-06-08 | 中国科学院大连化学物理研究所 | Method for removing Q11 impurity in coenzyme Q10 by using preparation chromatography |
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| CN101233095A (en) * | 2005-06-10 | 2008-07-30 | 协和发酵工业株式会社 | Method of purifying ubiquinone-10 |
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| CN101445435A (en) * | 2008-11-13 | 2009-06-03 | 三明华健生物工程有限公司 | Coenzyme Q10 cleaning and purifying process |
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| JP4275621B2 (en) * | 2002-07-25 | 2009-06-10 | 協和発酵バイオ株式会社 | Method for producing ubiquinone-10-containing solution |
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| CN101233095A (en) * | 2005-06-10 | 2008-07-30 | 协和发酵工业株式会社 | Method of purifying ubiquinone-10 |
| CN101429108A (en) * | 2007-11-09 | 2009-05-13 | 浙江医药股份有限公司新昌制药厂 | Purification method for separation of cozymase Q10 |
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