CN1586502A - Process for preparing compound coemzyme medicine and its compound coemzyme medicine and clinical use - Google Patents

Process for preparing compound coemzyme medicine and its compound coemzyme medicine and clinical use Download PDF

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CN1586502A
CN1586502A CN 200410058086 CN200410058086A CN1586502A CN 1586502 A CN1586502 A CN 1586502A CN 200410058086 CN200410058086 CN 200410058086 CN 200410058086 A CN200410058086 A CN 200410058086A CN 1586502 A CN1586502 A CN 1586502A
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coenzyme
medicine
yeast
atp
adp
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CN1259925C (en
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郑昌学
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HAINAN KANGYONG PHARMACEUTICALS CO Ltd
Aolute Biomedicines Research & Development Co Ltd Beijing
Academy of Military Medical Sciences AMMS of PLA
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HAINAN KANGYONG PHARMACEUTICALS CO Ltd
Aolute Biomedicines Research & Development Co Ltd Beijing
Academy of Military Medical Sciences AMMS of PLA
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Abstract

The present invention belongs to the field of medicine technology. The compound coenzyme medicine contains CoA, NAD, GSH and ATP; and features that it contains also FMN, FAD, ADP, AMP and SAM. The present invention has improved compound coenzyme medicine preparing process. The compound coenzyme medicine can protect liver cell, myocardial cell and other cells, maintain or restore their normal metabolism, and may be used in heart and liver surgical operation, restoring of damaged liver, restoring damaged cell caused by radiotherapy and chemotherapy.

Description

A kind of preparation method of Coenzyme Complex medicine and Coenzyme Complex medicine thereof and clinical purposes
Technical field
The invention belongs to medical technical field, relate to a kind of preparation method and a kind of NEW TYPE OF COMPOSITE coenzyme medicine and clinical new purposes of new Coenzyme Complex medicine especially.
Background technology
Develop a kind of injection Coenzyme Complex lyophilized injectable powder the seventies, it is a kind of extraction from yeast (beer yeast, edible yeast and geotrichum candidum), contains coenzyme A (CoA), nadide (NAD), the natural coenzyme compound formulation of ATP and glutathion.Be used for the treatment of acute, chronic hepatitis, idiopathic thrombocytopenic purpura, thrombocytopenia for a long time, coronary atherosclerosis, chronic arterits, myocardial infarction, the weak oliguria that causes of renal function, uremia etc. be can be used as auxiliary therapeutic agent.
Yeast is a unicellular eukaryote, contains the necessary various coenzyme of biological substance metabolism such as sugar, fat, protein, nucleic acid (synthetic and decomposition) in its cell.But because original production process has been used the heating breaking cellular wall, vacuum decompression concentrates, and dialysis waits personated old man's production. art, causes multiple coenzyme inactivation and degraded to destroy, thereby has influenced the scope of its coenzyme kind and clinical practice.
Summary of the invention
The objective of the invention is for overcoming the weak point of prior art, a kind of preparation method and Coenzyme Complex medicine and clinical purposes of new Coenzyme Complex medicine are proposed, by changing the production technology of existing Coenzyme Complex, make that the coenzyme material in the former Coenzyme Complex drug products is increased to nine kinds from original four kinds; This Coenzyme Complex can protect various kinds of cell to avoid anoxia-induced apoptosis, keeps or recovers its homergy, makes it can be used for heart, operation on liver, and hepatic injury recovers, clinical treatments such as the cell injury recovery that ray or chemotherapy cause.
A kind of Coenzyme Complex medicament preparation that the present invention proposes may further comprise the steps:
1) fresh yeast being added 5-8 times of pH value is that PH3.0~5.0,0.01M~0.03M hac buffer are made the yeast cells suspension;
2) with the cell wall of high pressure homogenizer at 70~150Mpa pressure breaking yeast cells, temperature maintenance is at 0 ℃~20 ℃ when broken;
3) with the yeast cells suspension frozen centrifugation after the fragmentation, the frozen centrifugation condition is 0 ℃~5 ℃, and speed is 20000~35000CPM, 30~60 minutes time, obtains clear liquid;
4) use the activated carbon adsorption filter liquor, concentrated usefulness contains 40% alcoholic solution eluting of 2.0~3.5% ammonia;
5) adopt ion-exchange chromatography: desorbed solution is transferred PH7.5-9.0, and last anion-exchange column is with PH8.0~9.0,0.01~0.03M phosphate buffer eluant solution;
6) eluent is the 5000-10000 ultrafiltration through the molecular weight that dams;
7) ultrafiltrate removes H through nanofiltration or reverse osmosis 2O is concentrated into original volume 1/5-1/10, transfers PH2.5~3.5, adds 5~10 times of volumes ,-10 ℃~-20 ℃ cold acetones, collecting precipitation;
9) the degerming embedding of the water-soluble back of precipitation, lyophilizing.
Adopt the Coenzyme Complex medicine of above-mentioned preparation method, its coenzyme composition contains coenzyme A (CoA), nadide (NAD), glutathione (GSH), adenosine triphosphate (ATP) is characterized in that, also contains flavin mononucleotide (FMN) (FMN), flavin adenine dinucleotide (FAD) (FAD), gland two phosphorus (ADP), adenosine phosphate (AMP), ademetionine (SAM).
Above-mentioned Coenzyme Complex not only is used for acute, chronic hepatitis, idiopathic thrombocytopenic purpura, thrombocytopenia; as coronary atherosclerosis, chronic arterits, myocardial infarction, the weak oliguria that causes of renal function, uremic auxiliary therapeutic agent, also be used for the medicine of operation on heart, myocardial ischemia, cerebral anoxia, pesticide intoxication, atomic disease and the protection medicine of various histiocytic anoxia-induced apoptosis, physics and chemical damage.
Characteristics of the present invention and effect:
Preparation method of the present invention has used the high pressure homogenizer smudge cells to replace original heating breaking cellular wall; Use hyperfiltration technique to replace original dialysis; Use anti-dialysis and nanofiltration to replace original vacuum decompression to concentrate; Make the coenzyme material in the former Coenzyme Complex drug products be increased to nine kinds from original four kinds; and show that through zoopery new Coenzyme Complex can protect various kinds of cell to avoid anoxia, radical damage; keep or recover its homergy; make it can be used for heart, operation on liver; hepatic injury recovers, clinical treatments such as the cell injury recovery that ray, chemotherapy and operation cause.
The specific embodiment
Embodiment 1:
The soft tommy yeast adds 5 times of PH4.0 0.01M hac buffers and makes the yeast cells suspension; At 100Mpa pressure breaking yeast cells, keep cell suspension below 20 ℃ with high pressure homogenizer; With the yeast cells suspension frozen centrifugation after the fragmentation, speed is that 20000CPM got clear liquid in centrifugal 60 minutes under 5 ℃ of conditions; Activated carbon adsorption, with the 40% cholamine solution stripping that contains 2% ammonia, stripping liquid is transferred PH8.0, and last Ambrite IRC50 ion exchange column is used PH8.5, and the 0.02M phosphoric acid liquid is towards the liquid eluting; Eluent concentrates through nanofiltration and removes H with the ultrafilter ultrafiltration of the molecular weight 8000 that dams, filter liquor 2O is concentrated into original volume 1/10, transfers PH3.5, adds 5 times of volumes ,-10 ℃ of cold acetone precipitations, and collecting precipitation is dissolved in the apirogen water, degerming embedding, lyophilizing.
Measure the concentration of each organic principle, degerming is freeze-up to be done.
The product analysis result:
?CoA ?120U/ml
?NAD ?0.12mg/ml
?ATP ?2.1mg/ml
?ADP ?0.3mg/ml
?AMP ?0.2mg/ml
?FMN ?8ug/ml
?FAD ?6ug/ml
?GSH ?4.1mg/ml
?SAM ?10.0ug/ml
Embodiment 2:
The young beer yeast adds 6 times of PH3.0,0.03M acetate buffer solution, with high pressure homogenizer smudge cells under 70Mpa pressure, keeps cell breakage liquid below 10 ℃, and 30000 CPM centrifugal 45 minutes under 3 ℃ of conditions, gets clear liquid; It with the molecular weight that dams 8000 ultrafilter membrane ultrafiltration; Activated carbon adsorption is resolved with 40% cholamine that contains 3.0% ammonia; Desorbed solution is transferred Ambrite IRC 50 ion exchange column on the PH to 7.5, uses PH9.0,0.01M phosphate buffer eluting; Eluent is 5000 ultrafilter membrane ultrafiltration through the molecular weight that dams; Ultrafiltrate removes H with reverse osmosis 2O is concentrated into original volume 1/5, transfers PH2.5, adds the cold acetone of 10 times of volumes-15 ℃, and 0 ℃ is spent the night, next day collecting precipitation.Precipitation is dissolved in apirogen water, measures the concentration of each composition, degerming embedding, lyophilizing.
The product analysis result:
?CoA ?180U/ml
?NAD ?0.22mg/ml
?ATP ?2.3mg/ml
?ADP ?0.4mg/ml
?AMP ?0.5mg/ml
?FMN ?2ug/ml
?FAD ?4ug/ml
?GSH ?3.3mg/ml
?SAM ?18.0ug/ml
Embodiment 3:
With the fresh food yeast, add 8 times of PH4.5, the 0.01M hac buffer; Use high pressure homogenizer, under 150Mpa pressure, broken yeast cell is kept broken liquid below 5 ℃; Centrifugal 35000cpm under 0 ℃ of condition obtained clear liquid in 30 minutes; Collect filter liquor; The activated carbon adsorption filter liquor is resolved with 40% ethanol that contains 3.5% ammonia; Desorbed solution is transferred PH to 9.0, last Ambrite IRC 50 ion exchange column, with PH8.0,0.03M phosphate buffer eluting, eluent is 9000 ultrafilter membrane ultrafiltration through the molecular weight that dams, ultrafiltrate through reverse osmosis concentration to original volume 1/8, transfer PH3.0, add the cold acetone of 8 times of volumes-20 ℃, next day collecting precipitation.Precipitation is dissolved in apirogen water, measures the concentration of each composition, degerming embedding, lyophilizing.
The product analysis result:
The coenzyme title Concentration
????CoA ????120U/ml
????NAD + ????0.32mg/ml
????FMN ????8ug/ml
????FAD ????6ug/ml
????ATP ????1.0mg/ml
????ADP ????0.3mg/ml
????AMP ????0.5mg/ml
????GSH ????2.1mg/ml
????SAM ????5.0ug/ml
Embodiment 4: fresh geotrichum candidum is added 5 times of PH3.0,0.01M acetate buffer solution; Under high pressure homogenizer 150Mpa pressure, smudge cells, broken liquid are kept below 15 ℃; Centrifugal 35 minutes of 4 ℃ of 30000cpm, clear liquid; Activated carbon adsorption, with 40% alcohol desorption that contains 2.0% ammonia, stripping liquid is transferred PH8.5, last Ambrite IRC 50 ion exchange column, use PH8.5,0.02M the phosphate buffer eluting, eluent is 5000 ultrafilter membrane ultrafiltration through the molecular weight that dams, and filtrate is condensed into 1/8 of original volume through nanofiltration, transfer PH3.5, add 10 times of-15 ℃ of cold acetones, put 0 ℃ and spend the night collecting precipitation.Be dissolved in a certain amount of apirogen water, measure each composition and through degerming embedding, lyophilizing.
The product analysis result:
The coenzyme title Concentration
????CoA ????250U/ml
????NAD ????0.41mg/ml
????ATP ????0.5mg/ml
????ADP ????0.23mg/ml
????AMP ????0.22mg/ml
????FAD ????7ug/ml
????FMN ????10ug/ml
????GSH ????1.5mg/ml
????SAM ????6ug/ml
Use Coenzyme Complex of the present invention, the protective effect to cardiac resuccitation (CPR) back rat thyroid is described as follows:
Use suffocate (succinylcholine) merge 0.5M ice chlorination potassium arrest liquid and cause the rat cardiac arrest, begin cardio-pulmonary resuscitation after 5 minutes.Experiment divides three groups: 1. matched group be sham operated rats (without suffocate, the rat of cardiac arrest and cardio-pulmonary resuscitation process); 2. conventional recovery group is 24 hours groups after the cardio-pulmonary resuscitation; 3. Coenzyme Complex experimental group.The conventional medicine recovery gives Coenzyme Complex simultaneously and (by one/10Kg) normal saline dilution pneumoretroperitoneum injection, recovers and gave Isodose in back 12 hours again.Every group of 8 rats.
(1) test rat blood serum thyroxine T 3, T 4The variation of thyrotropin is as shown in Table I:
Table 1 serum T 3, T4, TSH change (X ± S)
Grouping T3 (ng/ml) T4 (Hg/dl) TSH (μ IU/ml)
Matched group 0.56 ± 0.06 5.11 ± 0.39 1.61 ± 0.40 conventional recovery group 0.23 ± 0.05 *??1.55±0.35 *1.44 ± 0.35 Coenzyme Complex group 0.29 ± 0.4 *#??2.45±0.58 *#?1.52±0.24
With matched group ratio and matched group ratio *P<0.05, with routine recovery group than #P<0.05.
The result shows: CPR can cause serum T 3, T 4, TSH concentration obviously descends, but conventional recovery group is starkly lower than the Coenzyme Complex group, two groups of T 3, T 4There was a significant difference.
(2) MDA (malonaldehyde) resultant in the parathyroid tissue, superoxide dismutase (SOD) vigor and Na +-K +The variation of-ATPase vigor is as shown in table 2:
MDA content in each group tissue of table 2, SOD vigor, Na+-K+-ATPase vigor (X ± S)
MDA content SOD vigor Na+-K+-ATPase vigor grouping (nmol/mgprot) is (μ mol/mgprot/hour) (U/mgprot)
Matched group 0.49 ± 0.09 11.14 ± 0.98 1.07 ± 0.17 conventional recovery group 1.52 ± 0.21 *???7.82±1.14 *0.45 ± 0.17 Coenzyme Complex group 1.78 ± 0.20 *#??9.64±1.04 *#??0.58±0.10 *#
With matched group ratio and matched group ratio *P<0.05, with routine recovery group than #P<0.05.
The result shows: conventional recovery group parathyroid tissue MDA content is apparently higher than matched group, and apparently higher than the Coenzyme Complex group, and SOD and Na +-K +-Atpase vigor is lower than matched group, and significantly is lower than the Coenzyme Complex group.
(3) experimental mouse original position terminal transferase labelling (Tunel labelling) changes as shown in table 3:
Table 3 is respectively organized TUNEL. positive cell number average gray value (X ± S)
Grouping n positive cell number (individual) average gray value (%)
Matched group 7 4.71 ± 1.39 71.31 ± 6.65 conventional recoveries organize 8 35.88 ± 3.72 *?????34.86±3.95 *Coenzyme Complex group 8 25.88 ± 4.17 *#????44.66±4.18 *#
With contrast ratio *P<0.05, with routine recovery group than #P<0.05.
The result shows: conventional recovery group Tunel positive cell is apparently higher than matched group, and is significantly higher than the Coenzyme Complex group, and its average gray value is starkly lower than matched group and is lower than the Coenzyme Complex group, and the difference between conventional group and Coenzyme Complex group has statistical significance.
(4) research of test rat Fas differential expression is as shown in table 4:
Grouping n positive cell number (individual) positive cell number average gray value (average gray value (%) of X ± S)
Matched group 7 7.00 ± 1.31 65.32 ± 5.72 conventional recoveries organize 8 37.00 ± 4.03 *????26.41±3.82 *Bei Keneng organizes 8 27.25 ± 4.21 *#???36.69±4.62 *#
With contrast ratio *P<0.05, with routine recovery group than #P<0.05.
The result shows: Coenzyme Complex group Fas gene expression positive cell number and average gray and conventional recovery group relatively have significant difference.
More than four groups of tests show:
1. Coenzyme Complex has improved CPR rat SOD, Na +-K +-ATPase vigor improves the damage of histiocyte energy metabolism to antioxidant radical, and the resultant of lipid peroxidation catabolite MDA is lower than conventional recovery group in the exhibit tissue.
2. Coenzyme Complex has been reduced and has been transferred the expression of related gene Fas of dying, and shows as and transfer the obvious minimizing of cell (Tunel positive cell) of dying, thereby has alleviated the histiocyte damage that anoxia, perfusion cause, and rat behind the CPR is had protective effect.
3. Coenzyme Complex has alleviated the degree that recovery back rat blood serum thyroxin changes, and is of value to the prognosis that improves heart beating time-out and CPR.

Claims (6)

1, a kind of Coenzyme Complex medicament preparation may further comprise the steps:
1) fresh yeast being added 5-8 times of pH value is that PH3.0~5.0,0.01M~0.03M hac buffer are made the yeast cells suspension;
2) with the cell wall of high pressure homogenizer at 70~150Mpa pressure breaking yeast cells, temperature maintenance is at 0 ℃~20 ℃ when broken;
3) with the yeast cells suspension frozen centrifugation after the fragmentation, the frozen centrifugation condition is 0 ℃~5 ℃, and speed is 20000~35000CPM, 30~60 minutes time, obtains clear liquid;
4) use the activated carbon adsorption filter liquor, with the 40% alcoholic solution eluting that contains 2.0~3.5% ammonia;
5) adopt ion-exchange chromatography: desorbed solution is transferred PH7.5-9.0, and last anion-exchange column is with PH8.0~9.0,0.01~0.03M phosphate buffer eluant solution;
6) eluent is the 5000-10000 ultrafiltration through the molecular weight that dams;
7) ultrafiltrate removes H through nanofiltration or reverse osmosis 2O is concentrated into original volume 1/5-1/10, transfers PH2.5~3.5, adds 5~10 times of volumes ,-10 ℃~-20 ℃ cold acetones, collecting precipitation;
9) the degerming embedding of the water-soluble back of precipitation, lyophilizing.
2, preparation method as claimed in claim 1 is characterized in that, described fresh yeast bacterium is a kind of of fresh beer yeast, bakery yeast, edible yeast or geotrichum candidum.
3, adopt as requiring the Coenzyme Complex medicine of preparation method as described in 1, its coenzyme composition contains coenzyme A, nadide, glutathione, adenosine triphosphate, it is characterized in that, also contain flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), gland two phosphorus, adenosine phosphate, ademetionine.
4, Coenzyme Complex as claimed in claim 3 is characterized in that, the concentration of described coenzyme composition is as follows:
FAD?????????????????2~20ug/ml
FMN?????????????????2~20ug/ml
ADP?????????????????0.1~0.5mg/ml
AMP?????????????????0.1~0.5mg/ml
SAM?????????????????1~20ug/ml
CoA?????????????????100~300U/ml、
NAD?????????????????0.1~0.5mg/ml、
ATP?????????????????0.1~2.5mg/ml
GSH?????????????????1~5mg/ml
5, Coenzyme Complex as claimed in claim 3, the ratio that it is characterized in that ATP, ADP, AMP in the described coenzyme is E:
E = ATP + 1 / 2 ADP ATP + ADP + AMP = 0.30 ~ 0.95 Between
6, a kind of Coenzyme Complex as claimed in claim 3 not only is used for acute, chronic hepatitis, idiopathic thrombocytopenic purpura, thrombocytopenia; as coronary atherosclerosis, chronic arterits, myocardial infarction, the weak oliguria that causes of renal function, uremic auxiliary therapeutic agent; the medicine that also is used for operation on heart, myocardial ischemia, cerebral anoxia, pesticide intoxication, atomic disease, and the protection medicine of the anoxia-induced apoptosis of myocardial cell, liver cell and neurocyte, physics and chemotherapy damage.
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Cited By (11)

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CN101023968B (en) * 2007-02-09 2010-05-19 上海大学 Coenzyme composition preparing method
CN101984978A (en) * 2010-09-30 2011-03-16 海南康永药品有限公司 Method for preparing compound coenzyme medicine
CN102174512A (en) * 2011-02-23 2011-09-07 武汉工业学院 Process method for producing yeast polysaccharide and yeast nucleotide from yeast
CN103191145A (en) * 2013-03-27 2013-07-10 南京理工大学 Application of adenosine and derivatives thereof in prevention and treatment of medicament-induced liver injury
CN103341155A (en) * 2013-07-17 2013-10-09 北京双鹭药业股份有限公司 Stable compound coenzyme preparation and preparation method as well as application thereof
CN104623626A (en) * 2013-07-17 2015-05-20 北京双鹭药业股份有限公司 Stable compound coenzyme preparation as well as preparation method and applications thereof
CN104844487A (en) * 2015-04-05 2015-08-19 杭州蓝然环境技术有限公司 Method for decoloring methionine crystallization mother liquor by adopting membrane integration technology
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
CN111202745A (en) * 2020-03-06 2020-05-29 广东药科大学 Application of FAD in preparation of medicine for inhibiting or treating cardiovascular system diseases
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101023968B (en) * 2007-02-09 2010-05-19 上海大学 Coenzyme composition preparing method
CN101984978A (en) * 2010-09-30 2011-03-16 海南康永药品有限公司 Method for preparing compound coenzyme medicine
CN101984978B (en) * 2010-09-30 2012-06-27 海南康永药品有限公司 Method for preparing compound coenzyme medicine
CN102174512A (en) * 2011-02-23 2011-09-07 武汉工业学院 Process method for producing yeast polysaccharide and yeast nucleotide from yeast
CN103191145A (en) * 2013-03-27 2013-07-10 南京理工大学 Application of adenosine and derivatives thereof in prevention and treatment of medicament-induced liver injury
CN103341155A (en) * 2013-07-17 2013-10-09 北京双鹭药业股份有限公司 Stable compound coenzyme preparation and preparation method as well as application thereof
CN103341155B (en) * 2013-07-17 2015-03-11 北京双鹭药业股份有限公司 Stable compound coenzyme preparation and preparation method as well as application thereof
CN104623626A (en) * 2013-07-17 2015-05-20 北京双鹭药业股份有限公司 Stable compound coenzyme preparation as well as preparation method and applications thereof
CN104844487A (en) * 2015-04-05 2015-08-19 杭州蓝然环境技术有限公司 Method for decoloring methionine crystallization mother liquor by adopting membrane integration technology
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
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
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
US10214556B2 (en) 2015-05-19 2019-02-26 Bontac Bio-Engineering (Shenzhen) Co., Ltd Method for purifying reduced form of β-nicotinamide adenine dinucleotide
CN111202745A (en) * 2020-03-06 2020-05-29 广东药科大学 Application of FAD in preparation of medicine for inhibiting or treating cardiovascular system diseases
CN111202745B (en) * 2020-03-06 2021-09-10 广东药科大学 Application of FAD in preparation of medicine for inhibiting or treating cardiovascular system diseases
WO2022082487A1 (en) * 2020-10-21 2022-04-28 朱上翔 Anti-aging composite nutrient containing coenzymes and bioactive hydrogen, preparation method therefor and application thereof

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