CN101023968A - Coenzyme composition preparing method - Google Patents
Coenzyme composition preparing method Download PDFInfo
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- CN101023968A CN101023968A CN 200710037422 CN200710037422A CN101023968A CN 101023968 A CN101023968 A CN 101023968A CN 200710037422 CN200710037422 CN 200710037422 CN 200710037422 A CN200710037422 A CN 200710037422A CN 101023968 A CN101023968 A CN 101023968A
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Abstract
The present invention relates to a preparation method of coenzyme compound. Said invention adopts mature technical processes of temperature difference wall breakage and reduced pressure concentration, and utilizes the steps of ultrafiltering separation, column chromatography to make purification, concentration and precipitation so as to obtain the invented coenzyme compound.
Description
Technical field
The present invention relates to a kind of preparation method of coenzyme composition.
Background technology
Coenzyme composition is made up of multiple coenzyme and bioactive substance, mainly contains bioactive substances such as coenzyme A, nadide, adenosine triphosphate, reduced glutathione and nucleotide.They mostly are acetylization reactions in the human body; the coenzyme of the important enzyme of redox reaction, transmethylase reaction and energy metabolism; sugar, protein, fat and energy metabolism in the body are played an important role; at glycolysis, tricarboxylic acid cycle; aspects such as synthetic, the Tissue respiration of the synthetic and decomposition of fatty acid beta oxidation, liver glycogen, acetylcholine, energy transfer, protecting liver and detoxication, anti-radiation (radiation) effect; all closely related with it, its produce market prospect is boundless.
At present,---active carbon extraction separation---ion-exchange resin purification---ultrafiltration purification---steps such as acetone precipitation that domestic Coenzyme Complex production mainly is according to the high pressure homogenize breaking cellular wall described in the Chinese patent 200410058086.X.Its shortcoming is: 1. the linking that can't link up between each technology, need carry out just entering subsequent processing after the intermediate treatment.2. the alkaline environment that is adopted during the active carbon eluting in the processing step has serious destruction to the coenzyme activity.3. ultrafiltration is placed after the ion-exchange resin purification, the action effect of ultrafiltration is declined to a great extent.4. each process procedure is loaded down with trivial details, and operator are had relatively high expectations.
Summary of the invention
The object of the present invention is to provide a kind of preparation method of coenzyme composition.
For achieving the above object, the present invention adopts following technical scheme:
A kind of preparation method of coenzyme composition is characterized in that the step of this method is as follows:
A) raw-material processing: after yeast and mixture of ice and water mixed by 1: 1: 2～4 mass ratio, place boiling water, under 80 ℃～100 ℃ temperature, be incubated 5～10min then, drop into the trash ice cooling rapidly, through the centrifuge centrifugalize, get supernatant, centrifugal condition is: 4000rpm～6000rpm, and the time is: 15～30min;
B) ultra-filtration and separation: step a gained supernatant is placed ultrafilter, depress in 0.1～0.3MPa ultrafiltration and carry out separation and purification, get filter liquor; Described ultrafilter adopts the ultrafilter membrane of molecular weight cut off 8000～12000;
C) column chromatography purification: the filter liquor of step b gained is adsorbed with strong basic type anion-exchange resin, eluent system is a 0.2mol/L NaCl solution, and regulate pH to 1.5～2.0 with 0.1mol/L HCl, elution flow rate is 150～250mL/h, and, be collected in the part that light absorption is arranged under the 254nm wavelength with the real-time monitoring stream fluid of nucleic acid-protein detector OD value;
D) concentrate: step c gained eluent is placed Rotary Evaporators, under 55 ℃～60 ℃ temperature, be evaporated to 1/15～1/20 of original volume;
E) precipitation is separated out: the pH value of steps d gained concentrated solution is transferred to 2～3, slowly pour 10～20 times of volumes into, in-10 ℃～-20 ℃ the acetone soln, this solution is regulated pH to 2～3 with 5～8mol/L nitric acid, and standing over night is got precipitation, promptly gets coenzyme composition.
Above-mentioned yeast is beer yeast, bakery yeast or edible yeast; Described strong basic type anion-exchange resin has: 717 type resins and 711 type resins.
Compare with prior art, the inventive method adopts maturation process such as breaking cellular wall by temperature difference, concentrating under reduced pressure, when obtaining gratifying product yield, has reduced the requirement to production equipment.Secondly, made full use of existing equipment, ultrafiltration step is shifted to an earlier date, replaced the separation and Extraction of active carbon on the one hand, reduced process procedure, alkaline environment is to the active destruction of coenzyme when having avoided eluting simultaneously.On the other hand,, enter chromatographic column again, reduced separating difficulty, improved the efficient of chromatography purification and the speed of production of chromatography because ultrafiltration can be removed by impurity most of molecular weight is big, that viscosity is high.The 3rd, respectively extracting in technology of the present invention between the purification step need not various additional treatments, can directly enter next step operation, therefore, can make whole technology accomplish gapless, serialization, automation mechanized operation, improve the globality of whole process, reduced the production cycle, simplified operating procedure.It is faint yellow, loose that the products obtained therefrom color and luster is, and meets relevant national standard WS
1-XG-010-2002.
The specific embodiment
With bakery yeast and water, ice by after 1: 1: 2 the mixed, place 1 volume boiling water, 90 ℃ of insulation 5min drop into the trash ice cooling rapidly.Put centrifuge 4800rpm centrifugalize 30min, get supernatant.
2. supernatant is placed the ultrafilter that cutting 10000 molecular weight ultrafilter membranes are housed, the 0.2MPa ultrafiltration is depressed and is carried out separation and purification, gets filter liquor.
3. filter liquor is carried out the column chromatography adsorption and purification with 717 strong basic type anion-exchange resins, (0.2mol/LNaCl regulating pH with 0.1mol/L HCl is 1.7) system eluting, when eluting, adopt peristaltic pump control flow velocity at 200mL/h, the real-time monitoring stream fluid of nucleic acid-protein detector OD value is collected in the part that light absorption is arranged under the 254nm wavelength.
4. eluent is placed Rotary Evaporators, 58 ℃ are evaporated to 1/20 of original volume.
5. concentrated solution is regulated pH to 2.3 with the nitric acid of 8mol/L, slowly pour 15 times of volumes into, in-20 ℃ acid acetone (regulating pH with 5～8mol/L nitric acid the is 2.3) solution, standing over night is got precipitation, gets product.
The finished product that finally obtains is pale yellow powder, loose, and yield is about 8%.Wherein contain outside coenzyme A (CoA), nadide (CoI), the glutathione main components such as (GSH), also contain the adenosine triphosphate (ATP) of trace, flavin adenine dinucleotide (FAD) (FAD), flavin mononucleotide (FMN) (FMN), adenosine phosphate (AMP), gland two phosphorus (ADP), ademetionine (SAM).Coenzyme A purity is that 1.5～3.0U/mg, nadide purity are that 0.0015～0.003mg/mg, glutathione purity are 0.017～0.07mg/mg.
Adopt above method products obtained therefrom according to national drug standards WS
1-XG-010-2002 assay is as follows:
| Character | Differentiate | Acidity is checked | Loss on drying | GSH content | The undue toxicity | Thermal source | ||||
| Drip acetone | Drip 1,2,3-indantrione monohydrate | Paper electrophoresis | Maximum, minimum light absorbs | |||||||
| National standard | White or faint yellow | White precipitate | Show bluish violet | Identical with the adenosine triphosphate disodium salt position | The 258nm absorption maximum, the 236nm minimal absorption | pH5. 0～ 7.0 | Less than 5.0% | Must not be lower than 0.8mg in every 100U coenzyme A | Do not have | Do not have |
| Finished product | Meet | Meet | Meet | Meet | Meet | Meet | Meet | Meet | Meet | Meet |
Its result meets national standard.
Claims (2)
1. the preparation method of a coenzyme composition is characterized in that the step of this method is as follows:
A. raw-material processing: after yeast, water and ice mixture mixed by 1: 1: 2～4 mass ratio, place boiling water, under 80 ℃～100 ℃ temperature, be incubated 5～10min then, drop into the trash ice cooling rapidly, through the centrifuge centrifugalize, get supernatant, centrifugal condition is: 4000rpm～6000rpm, and the time is: 15～30min;
B. ultra-filtration and separation: step a gained supernatant is placed ultrafilter, depress in 0.1～0.3MPa ultrafiltration and carry out separation and purification, get filter liquor; Described ultrafilter adopts the ultrafilter membrane of molecular weight cut off 8000～12000;
C. column chromatography purification: the filter liquor of step b gained is adsorbed with strong basic type anion-exchange resin, eluent system is the NaCl solution of 0.2mol/L, and regulate pH to 1.5～2.0 with 0.1mol/L HCl, elution flow rate is 150～250mL/h, and, be collected in the part that light absorption is arranged under the 254nm wavelength with the real-time monitoring stream fluid of nucleic acid-protein detector OD value;
D. concentrate: step c gained eluent is placed Rotary Evaporators, under 55 ℃～60 ℃ temperature, be evaporated to 1/15～1/20 of original volume;
E. precipitation is separated out: the pH value of steps d gained concentrated solution is transferred to 2～3, slowly pour 10～20 times of volumes into, in-10 ℃～-20 ℃ the acetone soln, this acetone soln is regulated pH to 2～3 with 5～8mol/L nitric acid, standing over night is got precipitation, promptly gets coenzyme composition.
2. the preparation method of a kind of coenzyme composition according to claim 1 is characterized in that described yeast is beer yeast, bakery yeast or edible yeast; Described strong basic type anion-exchange resin has: 717 type resins or 711 type resins.
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| CN200710037422A CN101023968B (en) | 2007-02-09 | 2007-02-09 | Coenzyme composition preparing method |
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| CN200710037422A CN101023968B (en) | 2007-02-09 | 2007-02-09 | Coenzyme composition preparing method |
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| CN101023968A true CN101023968A (en) | 2007-08-29 |
| CN101023968B CN101023968B (en) | 2010-05-19 |
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| CN200710037422A Expired - Fee Related CN101023968B (en) | 2007-02-09 | 2007-02-09 | Coenzyme composition preparing method |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104876994A (en) * | 2015-05-19 | 2015-09-02 | 邦泰生物工程(深圳)有限公司 | Method for purifying oxidized beta-nicotinamide adenine dinucleotide |
| CN105131065A (en) * | 2015-07-16 | 2015-12-09 | 合肥平光制药有限公司 | Preparation method of oxidizing-type coenzyme I |
| WO2016091121A1 (en) * | 2015-05-19 | 2016-06-16 | 邦泰生物工程(深圳)有限公司 | Method for purifying reduced β-nicotinamide adenine dinucleotide |
| CN107098943A (en) * | 2017-06-07 | 2017-08-29 | 广东隆赋药业股份有限公司 | A kind of preparation method of high-purity Vidarabine Monophosphate |
| CN109836469A (en) * | 2019-02-27 | 2019-06-04 | 开封康诺药业有限公司 | A kind of decoloration process of Coenzyme I |
| CN114195835A (en) * | 2021-12-20 | 2022-03-18 | 上海蔚之星生物科技有限公司 | New process for preparing coenzyme I injection raw material medicine |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1259925C (en) * | 2004-08-13 | 2006-06-21 | 北京奥路特生物医药研发有限公司 | Process for preparing compound coemzyme medicine and its compound coemzyme medicine and clinical use |
-
2007
- 2007-02-09 CN CN200710037422A patent/CN101023968B/en not_active Expired - Fee Related
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104876994A (en) * | 2015-05-19 | 2015-09-02 | 邦泰生物工程(深圳)有限公司 | Method for purifying oxidized beta-nicotinamide adenine dinucleotide |
| WO2016091121A1 (en) * | 2015-05-19 | 2016-06-16 | 邦泰生物工程(深圳)有限公司 | Method for purifying reduced β-nicotinamide adenine dinucleotide |
| WO2016091119A1 (en) * | 2015-05-19 | 2016-06-16 | 邦泰生物工程(深圳)有限公司 | Method for purifying oxidized β-nicotinamide adenine dinucleotide |
| US10214556B2 (en) | 2015-05-19 | 2019-02-26 | Bontac Bio-Engineering (Shenzhen) Co., Ltd | Method for purifying reduced form of β-nicotinamide adenine dinucleotide |
| CN105131065A (en) * | 2015-07-16 | 2015-12-09 | 合肥平光制药有限公司 | Preparation method of oxidizing-type coenzyme I |
| CN105131065B (en) * | 2015-07-16 | 2017-02-15 | 合肥平光制药有限公司 | Preparation method of oxidizing-type coenzyme I |
| CN107098943A (en) * | 2017-06-07 | 2017-08-29 | 广东隆赋药业股份有限公司 | A kind of preparation method of high-purity Vidarabine Monophosphate |
| CN109836469A (en) * | 2019-02-27 | 2019-06-04 | 开封康诺药业有限公司 | A kind of decoloration process of Coenzyme I |
| CN114195835A (en) * | 2021-12-20 | 2022-03-18 | 上海蔚之星生物科技有限公司 | New process for preparing coenzyme I injection raw material medicine |
| CN114195835B (en) * | 2021-12-20 | 2023-09-19 | 上海蔚之星生物科技有限公司 | New process for preparing coenzyme I injection bulk drug |
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| CN101023968B (en) | 2010-05-19 |
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