CN104846039A - Process for preparing trinosin - Google Patents
Process for preparing trinosin Download PDFInfo
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- CN104846039A CN104846039A CN201510280694.3A CN201510280694A CN104846039A CN 104846039 A CN104846039 A CN 104846039A CN 201510280694 A CN201510280694 A CN 201510280694A CN 104846039 A CN104846039 A CN 104846039A
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Abstract
The invention discloses a process for preparing trinosin. According to the method, an adenosine solution is prepared through dissolving adenosine with a proper volume of room-temperature reverse-osmosis water, so that the energy consumption is lowered by over 15%, the operation is simple, and the damage to enzymes is effectively avoided. According to the method, serial-column sample loading is adopted, so that the flow velocity can be remarkably increased, and the time for sample loading is shortened by over 50%; due to serial-column washing, the possibility of product flow-through is greatly lowered, and the product yield is greatly increased and is increased by over 30%. According to the method, benzalkonium bromide is used as an activating agent, is a disinfectant and is non-toxic and harmless, and the difficulty of later-stage treatment is not increased, so that benzalkonium bromide is a reasonable substitute. According to the method, serial-column sample loading is adopted, so that the flow velocity can be remarkably increased, and the time for sample loading is shortened by over 50%; due to serial-column washing, the possibility of product flow-through is greatly lowered, and the product yield is greatly increased and is increased by over 30%.
Description
Technical field
The present invention relates to technical field of medicine, be specifically related to a kind of preparation technology of Sodium ATP.
Background technology
Sodium ATP, chemical name: adenosine-5 '-triguaiacyl phosphate disodium salt trihydrate, molecular formula: C
10h
14n
5na
2o
13p
33H
2o, molecular weight: 605.19 is nucleotide derivatives, participates in the metabolism of body fat, protein, sugar, nucleic acid and Nucleotide.When body absorption, secretion, Muscle contraction and carry out biochemical building-up reactions etc. need energy time, namely Triphosaden resolves into adenosine diphosphate (ADP) and phosphate, gives off energy simultaneously.Sodium ATP can penetrate blood cerebrospinal fluid barrier, can improve the stability of neurocyte membranous structure and reconstruction ability, promote the regrowth of nervous process.Sodium ATP is auxiliary enzyme drug, clinical for because of tissue injury, cellular enzymes vigor decline caused by various diseases.As heart failure, myocarditis, myocardial infarction, cerebral arteriosclerosis, coronary sclerosis, progressive myatrophy, apoplexy sequela, acute, chronic hepatitis, liver cirrhosis and dysaudia etc., also for treatments such as progressive myatrophy, apoplexy sequela, cardiac insufficiency, cardiomyopathy and hepatitis.
Chinese patent CN200710019776.8, disclose a kind of production method of San Scales adenosine monophosphate disodium, comprise the following steps: that (1) first adds tap water in reactor, start stirring simultaneously, 43-45 DEG C is heated to chuck, then throw in sucrose, glucose, potassium primary phosphate, dipotassium hydrogen phosphate, make it dissolve, stop heating, with circulating water cooling, when temperature is down to below 40-42 DEG C, throw in quick-frozen yeast slurry, and slowly heat up with chuck heating, (2) magnesium sulfate is dissolved, another exterior-applied liquid medicine: adenosine, first by tap water heating to 80 DEG C, dissolves by the ratio of adenosine liquid=12:1 with the tap water being twice in magnesium sulfate volume, (3) when reacting kettle temperature to 25 ± 1 DEG C, throwing in magnesium sulfate, adenosine liquid successively, and reaction feed liquid is heated to 38-40 DEG C, stop heating, insulation 2h, what now start sampling and measuring adenosine passs on rate, when reaction reaches Sodium ATP one band, reaction terminates, (4) deactivation, acid adjustment alkali, reaction terminates to bleed off hot water in chuck immediately, while logical cooling water temperature, while throw in ice in reactor, stop on the rocks when being cooled to 6-8 DEG C, pH3.0-4.0 is regulated with phosphoric acid, leave standstill after 1 hour, regulate pH6.5-7.5 again, leave standstill again and after 30 minutes, start squeezing, after reaction solution squeezing terminates, again with purified water top filter wash cake, after 30 minutes, water intaking top liquid detects, water top is stopped when Sodium ATP concentration≤0.1%, then water top liquid and pressed liquor are mixed, inspection Sodium ATP concentration 0.5-1%, transparence>=90% side is qualified, mixed solution is sent centrifugal station process, (5) resin isolation: loading: above-mentioned mixed solution is added pure water and is diluted to resin column on 0.3-0.5%, loading detects ATP concentration in effluent liquid in after 8 hours every 2 hours, when stopping loading during ATP>=0.1% in loading effluent liquid, modify-water top, loading is carried out again, until end of the sample after 2 hours, washing impurity: end of the sample, is just entering washing to effluent liquid A by purified water
257≤ 0.5, then anti-water inlet is washed till resin upper strata without muddy mechanical impurity and effluent liquid A
257≤ 0.5 terminates, impurity washed by rare salt: washing terminates, wash with the anti-influent stream of 0.05% dilute sodium chloride solution, sampling in every 2 hours, ATP concentration in flow measurement fluid, when in effluent liquid during ATP concentration>=0.1%, anti-influent stream is washed impurity and is terminated, correct into, every 2 hours sampling, ATP concentration in flow measurement fluid, if during concentration>=0.5%, start wash-out, wash-out: when liquid level to be down on resin along 20cm place, carry out wash-out with 2% dense saline solution, collect elutriant, deliver to decolouring after terminating, (6) decolour: elutriant first carries out first time ultrafiltration Deproteinization by the hollow fiber ultrafiltration membrane of 200,000 molecular weight, then adds gac, stir after more than 30 minutes and leave standstill 30 minutes, then destainer is delivered to accurate filter ultrafiltration depyrogenation, (7) ultrafiltration, crystallization: ultrafiltration: destainer is carried out second time ultrafiltration depyrogenation with receiving membrane type ultra-fine filter again, then sends to: crystallization, crystallization: ultrafiltrated first uses 3N salt acid for adjusting pH to 5.0, then 30-32 DEG C is warming up to chuck hot water, start to stir, temperature reaches 32 DEG C, starts to add 95% ethanol simultaneously, and keep temperature 30-32 DEG C, when occurring that white is mixed only in pot, stopping adding ethanol, continuing to stir half an hour, then open chuck cooling, feed liquid is cooled to room temperature, centrifugal crystal solution, wet product first uses ethanol rinse, then uses soaked in absolute ethyl alcohol, and within finally centrifugal 30 minutes, dry, wet product is put into wet product stored in room, mother liquor send Distillation recovery post to reclaim, even for wet product shakedown is entered in dish, put into baking oven, vacuum-drying temperature controls at 38-42 DEG C, take out after 16 hours and smashed to pieces, then every 5 hours stirrings 1 time, after wet product is dried to 40 hours, sample thief send analyzer room to survey quick weight loss, as quick weight loss <5%, get final product discharging, dry product is put into dry product storage chamber to preserve, obtain product.
In prior art, the medicines such as such as ATP are generally that the processing step wanting the policy paper such as Surveillance Authority or pharmacopeia to advise according to state food is produced, if drug manufacturing enterprise, to the change of product production technique taking certification, needs declaration again, manpower and materials cost can be increased.Therefore, since there is the suggestion of policy paper, pharmaceutical production producer generally arbitrarily can not change production technique.But along with the development in epoch, the progress of technology, and the requirement of environmental protection and energy saving, improvements over the prior art are also in the middle of constantly carrying out, and improving each time is all constantly perfect to prior art, is also cost-saving accordingly.
Summary of the invention
The object of the invention is, in order to solve the deficiencies in the prior art, to provide a kind of preparation technology of Sodium ATP.
The preparation technology of a kind of Sodium ATP of the present invention, comprises the steps:
1) biochemical reaction
(1) preparation of magnesium chloride solution, adenosine solution, benzalkonium bromide solution:
1. dissolve magnesium chloride with a small amount of reverse osmosis water and make magnesium chloride solution;
2. dissolve adenosine with the reverse osmosis water of appropriate room temperature and make adenosine solution;
3. dissolve Morpan BB with appropriate reverse osmosis water and be diluted to 5% benzalkonium bromide solution;
(2) quick-frozen yeast is thrown in: add reverse osmosis water in reactor, start stirring chuck hot water heating to 40-45 DEG C, throw in sucrose and potassium primary phosphate and dipotassium hydrogen phosphate, make it dissolve, after it all dissolves, stop heating, open cycle water for cooling, when temperature is down to below 40 DEG C, throws in quick-frozen yeast, and slowly heat up with chuck hot water;
(3) ATP biochemical reaction: when reacting kettle temperature and rising to 20 DEG C ± 1, throw in magnesium chloride solution, benzalkonium bromide solution, adenosine solution successively, reaction starts timing, reaction solution is continued to be heated to 36-39 DEG C, insulation, bubble to be had overflows, and starts sampling and measuring ATP transformation efficiency, when reaction reaches ATP-band, reaction terminates;
(4) deactivation, acid adjustment alkali: reaction terminates, and bleeds off chuck hot water immediately, throw in rapidly a large amount of ice in reactor, and logical cooling water temperature, when temperature is down to less than 4 DEG C, adjust pH to 2.5-3.0 with phosphoric acid, after 1 hour, use liquid adjusting PH with base to 5.5-6.0 again, within after mixing up alkali 30 minutes, start squeezing, squeeze complete, again with reverse osmosis water top filter wash cake, water top liquid merges pressed liquor, inspection content, transparence, qualifiedly send centrifugal station process;
2) resin isolation
(1) go here and there post loading: pressed liquor and water top liquid are diluted to 0.2-0.45%, upper D296 resin column, flow velocity is 4-6L/min, and timing detects effluent liquid, look into and see if there is ATP and spill; When in effluent liquid during ATP concentration >=0.03%, stop loading, just pushing up to effluent liquid ATP concentration≤0.01% with reverse osmosis water, continue loading, so repeatedly until end of the sample;
(2) wash impurity: string post end of the sample, just enter washing to effluent liquid A with reverse osmosis water
257nm≤ 0.2; Then anti-water inlet is washed till resin upper strata without muddy mechanical impurity and effluent liquid A
257nmterminate after≤0.2; Flow rate control is at 4-6L/min;
(3) impurity washed by rare salt: washing terminates, and wash with the anti-influent stream of the rare saline solution of 0.04mol/LNaCl--0.01mol/L HCl of pH2.0-2.2, flow velocity is 6-10L/min; Sampling per hour, ATP concentration in flow measurement fluid, when in effluent liquid during ATP concentration >=0.03%, instead enters to wash impurity and terminates; Correct into, positive influent stream speed is constant, sampling per hour once, the concentration of ATP in flow measurement fluid, when in effluent liquid during ATP concentration >=0.025%, per sampling half an hour is once monitored; When effluent liquid pH skips to 2-4, and when ADP substantially washes to the greatest extent or only has ATP, washing terminates, and starts wash-out;
(4) wash-out: carry out wash-out with the dense saline solution of 1mol/LnaCl-0.01mol/L HCl of pH3.5-3.8, as wash-out effluent liquid ATP concentration >0.38%, collects elutriant, send decolouring; As wash-out effluent liquid ATP concentration <0.38%, namely stop collecting;
3) refining
(1) decolour: elutriant adds 0.1-0.5% gac according to color and luster and 0.1-0.3% diatomite stirs decolouring in 1 hour, after centrifugation is filtered, destainer is through sending ultrafiltration removing protein after the assay was approved;
(2) ultrafiltration: the ultra-filtration membrane of destainer <1 ten thousand molecular weight is carried out ultrafiltration removing protein, sends to crystallization after qualified;
(3) crystallization: ultrafiltrated first adjusts pH to 2.5-3.3 with CP level HCl, ultrafiltration removing protein is carried out again with the ultra-filtration membrane of > 10,000 molecular weight, crystallization kettle is put into after qualified, start stirring, start to add 95% (v/v) ethanol, before there is white casse, alcohol adds speed control at 100L/h; When there is white casse, stop adding alcohol, stir half an hour, then continue to add alcohol to 1.5-2.0 times of material liquid volume, keep temperature to stir half an hour, then open chuck water coolant, feed liquid is chilled to room temperature, static 1 hour;
Centrifugation crystal solution, wet product first uses 95% (v/v) ethanol wash, then uses soaked in absolute ethyl alcohol, finally dries and sends to drying; Mother liquor send distillation workshop section to carry out recovered alcohol after adjusting pH to 6.0-7.0;
4) dry packing
(1) dry: wet product is put into loft drier, vacuum tightness≤-0.085Mpa, control temperature, at 34-40 DEG C, adds P
2o
5moisture absorption, drying course timing stirring, and detects moisture, can discharging when moisture≤8%;
(2) pack: qualified product is put into mixing tank mixing 15-20 minute by the batch of regulation, then by carrying out packaging censorship, qualified warehouse-in.
In the present invention, raw materials used consumption is counted by weight: adenosine 38-42 part, quick-frozen yeast 880-920 part, potassium primary phosphate 38-42 part, dipotassium hydrogen phosphate 230-235 part, magnesium chloride 18-22 part, sucrose 190-210 part, benzalkonium bromide solution 18-22 part, reverse osmosis water 1950-2050 part.
Further preferably, raw materials used consumption is counted by weight: adenosine 40 parts, 900 parts, quick-frozen yeast, potassium primary phosphate 40 parts, dipotassium hydrogen phosphate 232 parts, 20 parts, magnesium chloride, sucrose 200 parts, benzalkonium bromide solution 20 parts, reverse osmosis water 2000 parts.
Step 2) described in quick-frozen yeast, refer to and the appropriate reverse osmosis water of beer yeast slurry cleaned, and then filter with plate filter, obtain white or filbert purees, then to be obtained by quick-frozen.
Step 2) described string post loading, timing detects effluent liquid, and preferably when in effluent liquid during ATP concentration≤0.025%, every two hours sampling calibrating is once; When 0.025%<ATP concentration <0.03% in effluent liquid (caning be controlled in less than 0.05% when summer or yeast exception), sampling calibrating per hour once.
Step 2) described in ATP concentration unit be g/ml, described wash-out, elution flow rate controls at 100-300ml/min (6-18L/h).
Compared with prior art, the present invention has the following advantages:
1, in prior art, existing technique or old technique, the adenosine solution preparation of biochemical reaction makes adenosine solution with the reverse osmosis water or hot water dissolving's adenosine that to be heated to more than 85 DEG C in right amount, by reverse osmosis water or hot water steam heating to more than 85 DEG C.The water being in room temperature to be heated to more than 85 DEG C, need steam or the heat energy of at substantial on the one hand, and, technique power consumption is large, troublesome poeration and add fashionable because high temperature and easily make enzyme deactivation cause the enzyme impact reaction destroyed in reaction solution, meanwhile, heat-processed needs to expend time in, another aspect more than 85 DEG C hot water, from the angle of production safety, are also a hidden danger.And the present invention dissolves adenosine with the reverse osmosis water of appropriate room temperature to make adenosine solution, not only energy efficient more than 15%, and infringement that is simple to operate and that effectively avoid enzyme.
2, in prior art, the selection of promoting agent uses toluene, toluene is toxic substance, to the toxic effect of people, and toluene vapor can form explosive mixture with air, has requirements at the higher level to factory building, equipment, facility, increase extra input, meanwhile, toluene insoluble, in water, increases post-processed difficulty.And the present invention uses Morpan BB as promoting agent, Morpan BB is a kind of sterilizing agent, nontoxic, and does not increase difficulty to post-processed, is rational surrogate.
3, in prior art, when the loading of resin isolation operation, be adopt wall scroll resin column loading to terminating, wall scroll resin column loading has a lot of drawback, such as wall scroll resin column loading often easily stream to wear and flow velocity need control in real time, also yield is lower to extend loading time and product volume; And the present invention adopts string post loading can significantly improve flow velocity, shorten loading time more than 50%, and because be the washing of string post, the possibility that product flow is worn is also little a lot, greatly improves the yield of product, and yield improves more than 30%.
4, prior art uses yeast, it is generally yeast slurry discarded after directly using brew-house to produce beer, can come with some shortcomings like this, the sanitary condition of such as yeast slurry is bad, not resistance to stored for a long time etc., and the present invention is by cleaning beer yeast slurry with appropriate reverse osmosis water, and then filter with plate filter, obtain white or filbert purees, obtained by quick-frozen again, yeast slurry can be cleaned up on the one hand, and pass through quick-frozen, yeast slurry can be preserved for a long time, quick-frozen yeast is obtained by the present invention, aborning, the AR of biochemical reaction transforms and can not reverse, the transformation efficiency of adenosine reaches 100%.
Embodiment
With embodiment, the invention will be further described below, but the present invention is not limited to these embodiments.
Embodiment 1:
A preparation technology for Sodium ATP, comprises the steps:
1) biochemical reaction
(1) preparation of magnesium chloride solution, adenosine solution, benzalkonium bromide solution:
1. dissolve magnesium chloride with a small amount of reverse osmosis water and make magnesium chloride solution;
2. dissolve adenosine with the reverse osmosis water of appropriate room temperature and make adenosine solution;
3. dissolve Morpan BB with appropriate reverse osmosis water and be diluted to 5% benzalkonium bromide solution;
(2) quick-frozen yeast is thrown in: add reverse osmosis water in reactor, start stirring chuck hot water heating to 40-45 DEG C, throw in sucrose and potassium primary phosphate and dipotassium hydrogen phosphate, make it dissolve, after it all dissolves, stop heating, open cycle water for cooling, when temperature is down to below 40 DEG C, throws in quick-frozen yeast, and slowly heat up with chuck hot water;
(3) ATP biochemical reaction: when reacting kettle temperature and rising to 20 DEG C ± 1, throw in magnesium chloride solution, benzalkonium bromide solution, adenosine solution successively, reaction starts timing, reaction solution is continued to be heated to 36-39 DEG C, insulation, bubble to be had overflows, and starts sampling and measuring ATP transformation efficiency, when reaction reaches ATP-band, reaction terminates;
(4) deactivation, acid adjustment alkali: reaction terminates, and bleeds off chuck hot water immediately, throw in rapidly a large amount of ice in reactor, and logical cooling water temperature, when temperature is down to less than 4 DEG C, adjust pH to 2.5-3.0 with phosphoric acid, after 1 hour, use liquid adjusting PH with base to 5.5-6.0 again, within after mixing up alkali 30 minutes, start squeezing, squeeze complete, again with reverse osmosis water top filter wash cake, water top liquid merges pressed liquor, inspection content, transparence, qualifiedly send centrifugal station process;
2) resin isolation
(1) go here and there post loading: pressed liquor and water top liquid are diluted to 0.2-0.45%, upper D296 resin column, flow velocity is 4-6L/min, and timing detects effluent liquid, look into and see if there is ATP and spill; When in effluent liquid during ATP concentration >=0.03%, stop loading, just pushing up to effluent liquid ATP concentration≤0.01% with reverse osmosis water, continue loading, so repeatedly until end of the sample;
(2) wash impurity: string post end of the sample, just enter washing to effluent liquid A with reverse osmosis water
257nm≤ 0.2; Then anti-water inlet is washed till resin upper strata without muddy mechanical impurity and effluent liquid A
257nmterminate after≤0.2; Flow rate control is at 4-6L/min;
(3) impurity washed by rare salt: washing terminates, and wash with the anti-influent stream of the rare saline solution of 0.04mol/LNaCl--0.01mol/L HCl of pH2.0-2.2, flow velocity is 6-10L/min; Sampling per hour, ATP concentration in flow measurement fluid, when in effluent liquid during ATP concentration >=0.03%, instead enters to wash impurity and terminates; Correct into, positive influent stream speed is constant, sampling per hour once, the concentration of ATP in flow measurement fluid, when in effluent liquid during ATP concentration >=0.025%, per sampling half an hour is once monitored; When effluent liquid pH skips to 2-4, and when ADP substantially washes to the greatest extent or only has ATP, washing terminates, and starts wash-out;
(4) wash-out: carry out wash-out with the dense saline solution of 1mol/LnaCl-0.01mol/L HCl of pH3.5-3.8, as wash-out effluent liquid ATP concentration >0.38%, collects elutriant, send decolouring; As wash-out effluent liquid ATP concentration <0.38%, namely stop collecting;
3) refining
(1) decolour: elutriant adds 0.1-0.5% gac according to color and luster and 0.1-0.3% diatomite stirs decolouring in 1 hour, after centrifugation is filtered, destainer is through sending ultrafiltration removing protein after the assay was approved;
(2) ultrafiltration: the ultra-filtration membrane of destainer <1 ten thousand molecular weight is carried out ultrafiltration removing protein, sends to crystallization after qualified;
(3) crystallization: ultrafiltrated first adjusts pH to 2.5-3.3 with CP level HCl, ultrafiltration removing protein is carried out again with the ultra-filtration membrane of > 10,000 molecular weight, crystallization kettle is put into after qualified, start stirring, start to add 95% (v/v) ethanol, before there is white casse, alcohol adds speed control at 100L/h; When there is white casse, stop adding alcohol, stir half an hour, then continue to add alcohol to 1.5-2.0 times of material liquid volume, keep temperature to stir half an hour, then open chuck water coolant, feed liquid is chilled to room temperature, static 1 hour;
Centrifugation crystal solution, wet product first uses 95% (v/v) ethanol wash, then uses soaked in absolute ethyl alcohol, finally dries and sends to drying; Mother liquor send distillation workshop section to carry out recovered alcohol after adjusting pH to 6.0-7.0;
4) dry packing
(1) dry: wet product is put into loft drier, vacuum tightness≤-0.085Mpa, control temperature, at 34-40 DEG C, adds P
2o
5moisture absorption, drying course timing stirring, and detects moisture, can discharging when moisture≤8%;
(2) pack: qualified product is put into mixing tank mixing 15-20 minute by the batch of regulation, then by carrying out packaging censorship, qualified warehouse-in;
Raw materials used consumption is counted by weight: adenosine 38 parts, 920 parts, quick-frozen yeast, potassium primary phosphate 38 parts, dipotassium hydrogen phosphate 235 parts, 18 parts, magnesium chloride, sucrose 210 parts, benzalkonium bromide solution 22 parts, reverse osmosis water 1950 parts.
Embodiment 2:
A preparation technology for Sodium ATP, comprises the steps:
1) biochemical reaction
(1) preparation of magnesium chloride solution, adenosine solution, benzalkonium bromide solution:
1. dissolve magnesium chloride with a small amount of reverse osmosis water and make magnesium chloride solution;
2. dissolve adenosine with the reverse osmosis water of appropriate room temperature and make adenosine solution;
3. dissolve Morpan BB with appropriate reverse osmosis water and be diluted to 5% benzalkonium bromide solution;
(2) quick-frozen yeast is thrown in: add reverse osmosis water in reactor, start stirring chuck hot water heating to 40-45 DEG C, throw in sucrose and potassium primary phosphate and dipotassium hydrogen phosphate, make it dissolve, after it all dissolves, stop heating, open cycle water for cooling, when temperature is down to below 40 DEG C, throws in quick-frozen yeast, and slowly heat up with chuck hot water;
(3) ATP biochemical reaction: when reacting kettle temperature and rising to 20 DEG C ± 1, throw in magnesium chloride solution, benzalkonium bromide solution, adenosine solution successively, reaction starts timing, reaction solution is continued to be heated to 36-39 DEG C, insulation, bubble to be had overflows, and starts sampling and measuring ATP transformation efficiency, when reaction reaches ATP-band, reaction terminates;
(4) deactivation, acid adjustment alkali: reaction terminates, and bleeds off chuck hot water immediately, throw in rapidly a large amount of ice in reactor, and logical cooling water temperature, when temperature is down to less than 4 DEG C, adjust pH to 2.5-3.0 with phosphoric acid, after 1 hour, use liquid adjusting PH with base to 5.5-6.0 again, within after mixing up alkali 30 minutes, start squeezing, squeeze complete, again with reverse osmosis water top filter wash cake, water top liquid merges pressed liquor, inspection content, transparence, qualifiedly send centrifugal station process;
2) resin isolation
(1) go here and there post loading: pressed liquor and water top liquid are diluted to 0.2-0.45%, upper D296 resin column, flow velocity is 4-6L/min, and timing detects effluent liquid, look into and see if there is ATP and spill; When in effluent liquid during ATP concentration >=0.03%, stop loading, just pushing up to effluent liquid ATP concentration≤0.01% with reverse osmosis water, continue loading, so repeatedly until end of the sample;
(2) wash impurity: string post end of the sample, just enter washing to effluent liquid A with reverse osmosis water
257nm≤ 0.2; Then anti-water inlet is washed till resin upper strata without muddy mechanical impurity and effluent liquid A
257nmterminate after≤0.2; Flow rate control is at 4-6L/min;
(3) impurity washed by rare salt: washing terminates, and wash with the anti-influent stream of the rare saline solution of 0.04mol/LNaCl--0.01mol/L HCl of pH2.0-2.2, flow velocity is 6-10L/min; Sampling per hour, ATP concentration in flow measurement fluid, when in effluent liquid during ATP concentration >=0.03%, instead enters to wash impurity and terminates; Correct into, positive influent stream speed is constant, sampling per hour once, the concentration of ATP in flow measurement fluid, when in effluent liquid during ATP concentration >=0.025%, per sampling half an hour is once monitored; When effluent liquid pH skips to 2-4, and when ADP substantially washes to the greatest extent or only has ATP, washing terminates, and starts wash-out;
(4) wash-out: carry out wash-out with the dense saline solution of 1mol/LnaCl-0.01mol/L HCl of pH3.5-3.8, as wash-out effluent liquid ATP concentration >0.38%, collects elutriant, send decolouring; As wash-out effluent liquid ATP concentration <0.38%, namely stop collecting;
3) refining
(1) decolour: elutriant adds 0.1-0.5% gac according to color and luster and 0.1-0.3% diatomite stirs decolouring in 1 hour, after centrifugation is filtered, destainer is through sending ultrafiltration removing protein after the assay was approved;
(2) ultrafiltration: the ultra-filtration membrane of destainer <1 ten thousand molecular weight is carried out ultrafiltration removing protein, sends to crystallization after qualified;
(3) crystallization: ultrafiltrated first adjusts pH to 2.5-3.3 with CP level HCl, ultrafiltration removing protein is carried out again with the ultra-filtration membrane of > 10,000 molecular weight, crystallization kettle is put into after qualified, start stirring, start to add 95% (v/v) ethanol, before there is white casse, alcohol adds speed control at 100L/h; When there is white casse, stop adding alcohol, stir half an hour, then continue to add alcohol to 1.5-2.0 times of material liquid volume, keep temperature to stir half an hour, then open chuck water coolant, feed liquid is chilled to room temperature, static 1 hour;
Centrifugation crystal solution, wet product first uses 95% (v/v) ethanol wash, then uses soaked in absolute ethyl alcohol, finally dries and sends to drying; Mother liquor send distillation workshop section to carry out recovered alcohol after adjusting pH to 6.0-7.0;
4) dry packing
(1) dry: wet product is put into loft drier, vacuum tightness≤-0.085Mpa, control temperature, at 34-40 DEG C, adds P
2o
5moisture absorption, drying course timing stirring, and detects moisture, can discharging when moisture≤8%;
(2) pack: qualified product is put into mixing tank mixing 15-20 minute by the batch of regulation, then by carrying out packaging censorship, qualified warehouse-in;
Raw materials used consumption is counted by weight: adenosine 42 parts, 880 parts, quick-frozen yeast, biphosphate K42 part, dipotassium hydrogen phosphate 230 parts, 22 parts, magnesium chloride, sucrose 190 parts, benzalkonium bromide solution 18 parts, reverse osmosis water 2050 parts.
Embodiment 3:
A preparation technology for Sodium ATP, comprises the steps:
1) biochemical reaction
(1) preparation of magnesium chloride solution, adenosine solution, benzalkonium bromide solution:
1. dissolve magnesium chloride with a small amount of reverse osmosis water and make magnesium chloride solution;
2. dissolve adenosine with the reverse osmosis water of appropriate room temperature and make adenosine solution;
3. dissolve Morpan BB with appropriate reverse osmosis water and be diluted to 5% benzalkonium bromide solution;
(2) quick-frozen yeast is thrown in: add reverse osmosis water in reactor, start stirring chuck hot water heating to 40-45 DEG C, throw in sucrose and potassium primary phosphate and dipotassium hydrogen phosphate, make it dissolve, after it all dissolves, stop heating, open cycle water for cooling, when temperature is down to below 40 DEG C, throws in quick-frozen yeast, and slowly heat up with chuck hot water;
(3) ATP biochemical reaction: when reacting kettle temperature and rising to 20 DEG C ± 1, throw in magnesium chloride solution, benzalkonium bromide solution, adenosine solution successively, reaction starts timing, reaction solution is continued to be heated to 36-39 DEG C, insulation, bubble to be had overflows, and starts sampling and measuring ATP transformation efficiency, when reaction reaches ATP-band, reaction terminates;
(4) deactivation, acid adjustment alkali: reaction terminates, and bleeds off chuck hot water immediately, throw in rapidly a large amount of ice in reactor, and logical cooling water temperature, when temperature is down to less than 4 DEG C, adjust pH to 2.5-3.0 with phosphoric acid, after 1 hour, use liquid adjusting PH with base to 5.5-6.0 again, within after mixing up alkali 30 minutes, start squeezing, squeeze complete, again with reverse osmosis water top filter wash cake, water top liquid merges pressed liquor, inspection content, transparence, qualifiedly send centrifugal station process;
2) resin isolation
(1) go here and there post loading: pressed liquor and water top liquid are diluted to 0.2-0.45%, upper D296 resin column, flow velocity is 4-6L/min, and timing detects effluent liquid, look into and see if there is ATP and spill; When in effluent liquid during ATP concentration >=0.03%, stop loading, just pushing up to effluent liquid ATP concentration≤0.01% with reverse osmosis water, continue loading, so repeatedly until end of the sample;
(2) wash impurity: string post end of the sample, just enter washing to effluent liquid A with reverse osmosis water
257nm≤ 0.2; Then anti-water inlet is washed till resin upper strata without muddy mechanical impurity and effluent liquid A
257nmterminate after≤0.2; Flow rate control is at 4-6L/min;
(3) impurity washed by rare salt: washing terminates, and wash with the anti-influent stream of the rare saline solution of 0.04mol/LNaCl--0.01mol/L HCl of pH2.0-2.2, flow velocity is 6-10L/min; Sampling per hour, ATP concentration in flow measurement fluid, when in effluent liquid during ATP concentration >=0.03%, instead enters to wash impurity and terminates; Correct into, positive influent stream speed is constant, sampling per hour once, the concentration of ATP in flow measurement fluid, when in effluent liquid during ATP concentration >=0.025%, per sampling half an hour is once monitored; When effluent liquid pH skips to 2-4, and when ADP substantially washes to the greatest extent or only has ATP, washing terminates, and starts wash-out;
(4) wash-out: carry out wash-out with the dense saline solution of 1mol/LnaCl-0.01mol/L HCl of pH3.5-3.8, as wash-out effluent liquid ATP concentration >0.38%, collects elutriant, send decolouring; As wash-out effluent liquid ATP concentration <0.38%, namely stop collecting;
3) refining
(1) decolour: elutriant adds 0.1-0.5% gac according to color and luster and 0.1-0.3% diatomite stirs decolouring in 1 hour, after centrifugation is filtered, destainer is through sending ultrafiltration removing protein after the assay was approved;
(2) ultrafiltration: the ultra-filtration membrane of destainer <1 ten thousand molecular weight is carried out ultrafiltration removing protein, sends to crystallization after qualified;
(3) crystallization: ultrafiltrated first adjusts pH to 2.5-3.3 with CP level HCl, ultrafiltration removing protein is carried out again with the ultra-filtration membrane of > 10,000 molecular weight, crystallization kettle is put into after qualified, start stirring, start to add 95% (v/v) ethanol, before there is white casse, alcohol adds speed control at 100L/h; When there is white casse, stop adding alcohol, stir half an hour, then continue to add alcohol to 1.5-2.0 times of material liquid volume, keep temperature to stir half an hour, then open chuck water coolant, feed liquid is chilled to room temperature, static 1 hour;
Centrifugation crystal solution, wet product first uses 95% (v/v) ethanol wash, then uses soaked in absolute ethyl alcohol, finally dries and sends to drying; Mother liquor send distillation workshop section to carry out recovered alcohol after adjusting pH to 6.0-7.0;
4) dry packing
(1) dry: wet product is put into loft drier, vacuum tightness≤-0.085Mpa, control temperature, at 34-40 DEG C, adds P
2o
5moisture absorption, drying course timing stirring, and detects moisture, can discharging when moisture≤8%;
(2) pack: qualified product is put into mixing tank mixing 15-20 minute by the batch of regulation, then by carrying out packaging censorship, qualified warehouse-in;
Raw materials used consumption is counted by weight: adenosine 40 parts, 900 parts, quick-frozen yeast, potassium primary phosphate 40 parts, dipotassium hydrogen phosphate 232 parts, 20 parts, magnesium chloride, sucrose 200 parts, benzalkonium bromide solution 20 parts, reverse osmosis water 2000 parts.
Claims (4)
1. a preparation technology for Sodium ATP, is characterized in that, comprises the steps:
1) biochemical reaction
(1) preparation of magnesium chloride solution, adenosine solution, benzalkonium bromide solution:
1. dissolve magnesium chloride with a small amount of reverse osmosis water and make magnesium chloride solution;
2. dissolve adenosine with the reverse osmosis water of appropriate room temperature and make adenosine solution;
3. dissolve Morpan BB with appropriate reverse osmosis water and be diluted to 5% benzalkonium bromide solution;
(2) quick-frozen yeast is thrown in: add reverse osmosis water in reactor, start stirring chuck hot water heating to 40-45 DEG C, throw in sucrose and potassium primary phosphate and dipotassium hydrogen phosphate, make it dissolve, after it all dissolves, stop heating, open cycle water for cooling, when temperature is down to below 40 DEG C, throws in quick-frozen yeast, and slowly heat up with chuck hot water;
Described quick-frozen yeast, refers to and is cleaned by the appropriate reverse osmosis water of beer yeast slurry, and then filters with plate filter, obtains white or filbert purees, then is obtained by quick-frozen;
(3) ATP biochemical reaction: when reacting kettle temperature and rising to 20 DEG C ± 1, throw in magnesium chloride solution, benzalkonium bromide solution, adenosine solution successively, reaction starts timing, reaction solution is continued to be heated to 36-39 DEG C, insulation, bubble to be had overflows, and starts sampling and measuring ATP transformation efficiency, when reaction reaches ATP-band, reaction terminates;
(4) deactivation, acid adjustment alkali: reaction terminates, and bleeds off chuck hot water immediately, throw in rapidly a large amount of ice in reactor, and logical cooling water temperature, when temperature is down to less than 4 DEG C, adjust pH to 2.5-3.0 with phosphoric acid, after 1 hour, use liquid adjusting PH with base to 5.5-6.0 again, within after mixing up alkali 30 minutes, start squeezing, squeeze complete, again with reverse osmosis water top filter wash cake, water top liquid merges pressed liquor, inspection content, transparence, qualifiedly send centrifugal station process;
2) resin isolation
(1) go here and there post loading: pressed liquor and water top liquid are diluted to 0.2-0.45%, upper D296 resin column, flow velocity is 4-6L/min, and timing detects effluent liquid, look into and see if there is ATP and spill; When in effluent liquid during ATP concentration >=0.03%, stop loading, just pushing up to effluent liquid ATP concentration≤0.01% with reverse osmosis water, continue loading, so repeatedly until end of the sample;
(2) wash impurity: string post end of the sample, just enter washing to effluent liquid A with reverse osmosis water
257nm≤ 0.2; Then anti-water inlet is washed till resin upper strata without muddy mechanical impurity and effluent liquid A
257nmterminate after≤0.2; Flow rate control is at 4-6L/min;
(3) impurity washed by rare salt: washing terminates, and wash with the anti-influent stream of the rare saline solution of 0.04mol/LNaCl--0.01mol/L HCl of pH2.0-2.2, flow velocity is 6-10L/min; Sampling per hour, ATP concentration in flow measurement fluid, when in effluent liquid during ATP concentration >=0.03%, instead enters to wash impurity and terminates; Correct into, positive influent stream speed is constant, sampling per hour once, the concentration of ATP in flow measurement fluid, when in effluent liquid during ATP concentration >=0.025%, per sampling half an hour is once monitored; When effluent liquid pH skips to 2-4, and when ADP substantially washes to the greatest extent or only has ATP, washing terminates, and starts wash-out;
(4) wash-out: carry out wash-out with the dense saline solution of 1mol/LnaCl-0.01mol/L HCl of pH3.5-3.8, as wash-out effluent liquid ATP concentration >0.38%, collects elutriant, send decolouring; As wash-out effluent liquid ATP concentration <0.38%, namely stop collecting;
3) refining
(1) decolour: elutriant adds 0.1-0.5% gac according to color and luster and 0.1-0.3% diatomite stirs decolouring in 1 hour, after centrifugation is filtered, destainer is through sending ultrafiltration removing protein after the assay was approved;
(2) ultrafiltration: the ultra-filtration membrane of destainer <1 ten thousand molecular weight is carried out ultrafiltration removing protein, sends to crystallization after qualified;
(3) crystallization: ultrafiltrated first adjusts pH to 2.5-3.3 with CP level HCl, ultrafiltration removing protein is carried out again with the ultra-filtration membrane of > 10,000 molecular weight, crystallization kettle is put into after qualified, start stirring, start to add 95% (v/v) ethanol, before there is white casse, alcohol adds speed control at 100L/h; When there is white casse, stop adding alcohol, stir half an hour, then continue to add alcohol to 1.5-2.0 times of material liquid volume, keep temperature to stir half an hour, then open chuck water coolant, feed liquid is chilled to room temperature, static 1 hour;
Centrifugation crystal solution, wet product first uses 95% (v/v) ethanol wash, then uses soaked in absolute ethyl alcohol, finally dries and sends to drying; Mother liquor send distillation workshop section to carry out recovered alcohol after adjusting pH to 6.0-7.0;
4) dry packing
(1) dry: wet product is put into loft drier, vacuum tightness≤-0.085Mpa, control temperature, at 34-40 DEG C, adds P
2o
5moisture absorption, drying course timing stirring, and detects moisture, can discharging when moisture≤8%;
(2) pack: qualified product is put into mixing tank mixing 15-20 minute by the batch of regulation, then by carrying out packaging censorship, qualified warehouse-in.
2. the preparation technology of a kind of Sodium ATP according to claim 1, is characterized in that: raw materials used consumption is counted by weight: adenosine 38-42 part, quick-frozen yeast 880-920 part, potassium primary phosphate 38-42 part, dipotassium hydrogen phosphate 230-235 part, magnesium chloride 18-22 part, sucrose 190-210 part, benzalkonium bromide solution 18-22 part, reverse osmosis water 1950-2050 part.
3. the preparation technology of a kind of Sodium ATP according to claim 2, is characterized in that: raw materials used consumption is counted by weight: adenosine 40 parts, 900 parts, quick-frozen yeast, potassium primary phosphate 40 parts, dipotassium hydrogen phosphate 232 parts, 20 parts, magnesium chloride, sucrose 200 parts, benzalkonium bromide solution 20 parts, reverse osmosis water 2000 parts.
4. the preparation technology of a kind of Sodium ATP according to claim 1, is characterized in that: step 2) described string post loading, timing detects effluent liquid, and when in effluent liquid during ATP concentration≤0.025%, every two hours sampling calibrating is once; When in effluent liquid during 0.025%<ATP concentration <0.03%, sampling calibrating per hour once.
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| CN104846041A (en) * | 2015-05-28 | 2015-08-19 | 广西浦北制药厂 | Method for preparing trinosin |
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