CN116023419B - Preparation method of high-purity butanedisulfonic acid ademetionine - Google Patents
Preparation method of high-purity butanedisulfonic acid ademetionine Download PDFInfo
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- CN116023419B CN116023419B CN202310057050.2A CN202310057050A CN116023419B CN 116023419 B CN116023419 B CN 116023419B CN 202310057050 A CN202310057050 A CN 202310057050A CN 116023419 B CN116023419 B CN 116023419B
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- MEFKEPWMEQBLKI-AIRLBKTGSA-N S-adenosyl-L-methioninate Chemical compound O[C@@H]1[C@H](O)[C@@H](C[S+](CC[C@H](N)C([O-])=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 MEFKEPWMEQBLKI-AIRLBKTGSA-N 0.000 title claims abstract description 132
- 229960001570 ademetionine Drugs 0.000 title claims abstract description 40
- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- FUDAIDRKVVTJFF-UHFFFAOYSA-N butane-1,1-disulfonic acid Chemical compound CCCC(S(O)(=O)=O)S(O)(=O)=O FUDAIDRKVVTJFF-UHFFFAOYSA-N 0.000 title claims description 15
- 239000012535 impurity Substances 0.000 claims abstract description 42
- 238000001728 nano-filtration Methods 0.000 claims abstract description 40
- 239000012528 membrane Substances 0.000 claims abstract description 36
- 239000002808 molecular sieve Substances 0.000 claims abstract description 35
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 claims abstract description 35
- QDHFHIQKOVNCNC-UHFFFAOYSA-N butane-1-sulfonic acid Chemical compound CCCCS(O)(=O)=O QDHFHIQKOVNCNC-UHFFFAOYSA-N 0.000 claims abstract description 33
- 239000007788 liquid Substances 0.000 claims abstract description 29
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 26
- 239000011347 resin Substances 0.000 claims abstract description 21
- 229920005989 resin Polymers 0.000 claims abstract description 21
- ULUAUXLGCMPNKK-UHFFFAOYSA-N Sulfobutanedioic acid Chemical compound OC(=O)CC(C(O)=O)S(O)(=O)=O ULUAUXLGCMPNKK-UHFFFAOYSA-N 0.000 claims abstract description 19
- 239000007787 solid Substances 0.000 claims abstract description 12
- QDHFHIQKOVNCNC-UHFFFAOYSA-M butane-1-sulfonate Chemical compound CCCCS([O-])(=O)=O QDHFHIQKOVNCNC-UHFFFAOYSA-M 0.000 claims abstract description 11
- 238000000926 separation method Methods 0.000 claims abstract description 7
- VERAMNDAEAQRGS-UHFFFAOYSA-N butane-1,4-disulfonic acid Chemical compound OS(=O)(=O)CCCCS(O)(=O)=O VERAMNDAEAQRGS-UHFFFAOYSA-N 0.000 claims abstract description 4
- 239000000047 product Substances 0.000 claims description 29
- XQCHMGAOAWZUPI-UHFFFAOYSA-M sodium;butane-1-sulfonate Chemical compound [Na+].CCCCS([O-])(=O)=O XQCHMGAOAWZUPI-UHFFFAOYSA-M 0.000 claims description 24
- 238000001914 filtration Methods 0.000 claims description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 14
- 239000008213 purified water Substances 0.000 claims description 11
- 241001052560 Thallis Species 0.000 claims description 10
- 238000000855 fermentation Methods 0.000 claims description 9
- 230000004151 fermentation Effects 0.000 claims description 9
- 150000003839 salts Chemical class 0.000 claims description 9
- 238000003756 stirring Methods 0.000 claims description 9
- 239000002244 precipitate Substances 0.000 claims description 7
- 239000000843 powder Substances 0.000 claims description 6
- 239000000725 suspension Substances 0.000 claims description 6
- 238000009777 vacuum freeze-drying Methods 0.000 claims description 6
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 4
- 239000000706 filtrate Substances 0.000 claims description 4
- 241000235342 Saccharomycetes Species 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- 238000000034 method Methods 0.000 abstract description 22
- 238000005185 salting out Methods 0.000 abstract description 14
- 238000011282 treatment Methods 0.000 abstract description 13
- 229930024421 Adenine Natural products 0.000 abstract description 12
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 abstract description 12
- 229960000643 adenine Drugs 0.000 abstract description 12
- 238000004128 high performance liquid chromatography Methods 0.000 abstract description 11
- 150000001768 cations Chemical class 0.000 abstract description 10
- 238000001179 sorption measurement Methods 0.000 abstract description 9
- 238000001514 detection method Methods 0.000 abstract description 7
- 238000010828 elution Methods 0.000 abstract description 5
- 238000001035 drying Methods 0.000 abstract description 4
- 230000007547 defect Effects 0.000 abstract description 3
- 238000010612 desalination reaction Methods 0.000 abstract description 2
- 238000004090 dissolution Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 55
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 12
- 239000000126 substance Substances 0.000 description 11
- 230000000052 comparative effect Effects 0.000 description 8
- 238000004519 manufacturing process Methods 0.000 description 7
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 6
- 229960005305 adenosine Drugs 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 229910021645 metal ion Inorganic materials 0.000 description 5
- WUUGFSXJNOTRMR-IOSLPCCCSA-N 5'-S-methyl-5'-thioadenosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CSC)O[C@H]1N1C2=NC=NC(N)=C2N=C1 WUUGFSXJNOTRMR-IOSLPCCCSA-N 0.000 description 4
- ZJUKTBDSGOFHSH-WFMPWKQPSA-N S-Adenosylhomocysteine Chemical compound O[C@@H]1[C@H](O)[C@@H](CSCC[C@H](N)C(O)=O)O[C@H]1N1C2=NC=NC(N)=C2N=C1 ZJUKTBDSGOFHSH-WFMPWKQPSA-N 0.000 description 4
- 239000012467 final product Substances 0.000 description 4
- 239000011148 porous material Substances 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 239000002253 acid Substances 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003480 eluent Substances 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 238000011031 large-scale manufacturing process Methods 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 238000005374 membrane filtration Methods 0.000 description 2
- 231100000614 poison Toxicity 0.000 description 2
- 238000007873 sieving Methods 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- 239000003440 toxic substance Substances 0.000 description 2
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- ZKHQWZAMYRWXGA-KQYNXXCUSA-N Adenosine triphosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-N 0.000 description 1
- DATIDMJSWOVVRS-UHFFFAOYSA-N C(CCC)S(=O)(=O)O.C(CCC)S(=O)(=O)O Chemical compound C(CCC)S(=O)(=O)O.C(CCC)S(=O)(=O)O DATIDMJSWOVVRS-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- DMNNAGLOODQTOQ-HXTWIPGBSA-N [C@@H]1([C@H](O)[C@H](O)[C@@H](CN[C@@H](CCSC)C(=O)O)O1)N1C=NC=2C(N)=NC=NC12.C(CCCS(=O)(=O)O)S(=O)(=O)O Chemical compound [C@@H]1([C@H](O)[C@H](O)[C@@H](CN[C@@H](CCSC)C(=O)O)O1)N1C=NC=2C(N)=NC=NC12.C(CCCS(=O)(=O)O)S(=O)(=O)O DMNNAGLOODQTOQ-HXTWIPGBSA-N 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- -1 aminopropyl Chemical group 0.000 description 1
- 239000000729 antidote Substances 0.000 description 1
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- 210000000941 bile Anatomy 0.000 description 1
- 238000005842 biochemical reaction Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000003729 cation exchange resin Substances 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 238000011284 combination treatment Methods 0.000 description 1
- 230000002301 combined effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000008451 emotion Effects 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
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- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 210000005229 liver cell Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 239000002858 neurotransmitter agent Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 238000001694 spray drying Methods 0.000 description 1
- 125000000446 sulfanediyl group Chemical group *S* 0.000 description 1
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- Saccharide Compounds (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention belongs to the field of biological pharmacy, and particularly relates to a preparation method of high-purity butanesulfonic acid ademetionine. The invention processes the prepared adenosylmethionine separating liquid through a molecular sieve and an ultrafiltration membrane, and then sequentially carries out nanofiltration concentration, salting out, re-dissolution, resin decoloration, nanofiltration desalination and drying to finally obtain the adenosylmethionine sulfosuccinate solid. The invention is characterized in that after the molecular sieve and the ultrafiltration membrane are combined, the purity of the adenosyl methionine of the butanedisulfonate can be synergistically improved, and the ash content of a finished product can be reduced; the combination of molecular sieve, ultrafiltration membrane treatment, nanofiltration concentration and salting-out overcomes the defect that impurities such as adenine and the like still cannot be effectively removed after cation resin adsorption elution is adopted for the adenosylmethionine separation liquid in the prior art, and obviously reduces the impurities in the finished product; through HPLC detection, the purity of the adenosyl methionine butanesulfonate prepared by the method is high, the total content can reach 100.4%, and the ash content of a finished product can reach 0.03%.
Description
Technical Field
The invention relates to the field of biological pharmacy, in particular to a preparation method of high-purity butanedisulfonic acid ademetionine.
Background
Adenosylmethionine (S-adenosyl methionine), which is widely present in organisms as Adenosylmethionine, SAM, SAMe, which is important next to ATP, is an important physiologically active substance having the actions of transferring methyl, transferring thio, transferring aminopropyl, etc.; takes part in many biochemical reactions in the body, such as by sodium, potassium, ATP pumps; the butanesulfonic acid ademetionine can be used for treating liver and gall diseases, has multiple action mechanisms and pharmacological actions, can improve the mobility of liver cell membranes, can enhance the activity of corresponding enzymes, can promote secretion and operation of bile, and can also participate in synthesis of neurotransmitters, so that the emotion of a patient can be improved to a certain extent; in addition, the medicine is taken as an endogenous antidote, can play a role in detoxifying some toxic substances, can reduce the damage of the toxic substances to the liver, and has high practical value.
The current production method of the adenosylmethionine mainly comprises a microbial fermentation method, a chemical synthesis method and an enzymatic conversion method. Wherein, the preparation of the chemical synthesis method and the enzymatic conversion method have the defect of difficult separation and purification; at present, the method is mainly suitable for mass production, mainly comprises a microbial fermentation method, and is used for producing the adenosylmethionine through microbial fermentation and extracting the adenosylmethionine from the adenosylmethionine. When used as an injection, the residual metal ions (ash) and other impurities in the ademetionine are very high in requirement. The prior art ademetionine has no mention of metal ion (ash) residues in the finished product, although the ademetionine has a concern for liquid phase impurities; and the purity of the adenosylmethionine prepared at present still needs to be further improved.
Along with the increasing of life quality, SAM demands are continuously expanded, and the adenosylmethionine drugs are gradually brought into the medical insurance category, so that the quality requirements on the adenosylmethionine drugs are higher and higher.
Disclosure of Invention
In order to solve the problems, the invention provides a preparation method of high-purity butanedisulfonic acid ademetionine suitable for industrial stable production, and the ash content and other impurities in the finished product of the butanedisulfonic acid ademetionine prepared by the specific method are low, so that the impurities such as adenosine, adenine, S-adenosine-L-homocysteine and the like are effectively removed, and the quality of the finished product is greatly improved. The total content of the adenosyl methionine butanesulfonate is about 100 percent, the ash content is less than or equal to 0.05 percent, and the method is suitable for large-scale production.
The specific technical scheme of the invention is as follows:
a process for preparing high-purity adenosylmethionine sulfosuccinate includes such steps as treating the separated adenosylmethionine liquid by molecular sieve and ultrafiltration membrane, nano-filtering, concentrating, salting out, re-dissolving, resin decoloring, nano-filtering, desalting and drying.
The adenosylmethionine separating liquid is obtained by crushing cells of a fermentation liquid separating thalli containing adenosylmethionine, and the fermentation liquid is yeast fermentation liquid.
Molecular sieve, ultrafiltration membrane treatments are in the art relatively conventional adsorption and filtration techniques, respectively; however, the method for preparing the adenosylmethionine by salting out after combining the two has not been carried out; in the experimental process, the invention discovers that the purity of the adenosylmethionine can be obviously improved by salting out treatment after the molecular sieve is combined with the ultrafiltration membrane; before, the person skilled in the art generally uses the cationic resin to adsorb and then elute the filtered adenosylmethionine to achieve the aim of purifying and removing impurities, and also plays a good role; in the invention, the purity of the adenomethionine prepared by adopting molecular sieve, ultrafiltration membrane treatment, nanofiltration concentration and salting-out treatment is higher than that of the adenomethionine prepared by adopting cation resin treatment, so that impurities such as adenine which cannot be removed effectively after adsorption and elution by adopting the cation resin can be removed effectively, the total impurity content is also greatly reduced, and the effect is remarkable; in addition, from the viewpoint of production cost, although the process is increased, the time length and cost are little changed.
The invention provides a preparation method of high-purity butanedisulfonic acid ademetionine, which comprises the following specific steps:
(1) Adsorbing the adenomethionine separating liquid by a molecular sieve, and filtering by an ultrafiltration membrane to obtain an ultrafiltrate;
(2) Nanofiltration and concentration are carried out on the ultrafiltrate to obtain concentrated solution of the adenosylmethionine;
(3) Adding sodium butanesulfonate into the concentrated solution for salting out to obtain an adenomethionine salting-out substance;
(4) Separating the salt out and then redissolving to obtain an adenomethionine compound solution;
(5) Decolorizing the ademetionine complex solution by macroporous resin to obtain ademetionine decolorized solution;
(6) Carrying out nanofiltration desalination on the decolorized solution to remove redundant butanesulfonic acid, thereby obtaining concentrated solution containing adenosylmethionine;
(7) Adding butanedisulfonic acid into the concentrated solution obtained in the step (6) in proportion to form a butanedisulfonic acid adenosyl methionine solution;
(8) And (3) drying the adenosylmethionine sulfosuccinate solution obtained in the step (7) to obtain a solid powder product of adenosylmethionine sulfosuccinate.
Molecular sieves are a material containing precise and single minute pores that can be used to adsorb gases or liquids. Sufficiently small molecules can be adsorbed through the pore channels, while larger molecules cannot. Unlike a conventional screen, it operates at the molecular level. Small molecule impurities smaller than the molecules of the adenosylmethionine in the adenosylmethionine separating liquid, particularly metal ions in the form of salts, can be removed by selecting a suitable molecular sieve.
Ultrafiltration is a membrane filtration process, also known as ultrafiltration, used to retain colloid-sized particles in water, while water and low molecular weight solutes are allowed to permeate the membrane. The mechanism of ultrafiltration refers to the combined effect of mechanical sieving by the membrane surface, membrane Kong Zuzhi and membrane surface and membrane pore adsorption, and is mainly sieving. The selection of an appropriate ultrafiltration membrane removes macromolecular impurities larger than the ademetionine molecules.
The nano-filtration mechanism is basically the same as that of ultrafiltration, the molecular weight cut-off is lower, small impurities of the adenosylmethionine molecules can be removed, and the concentration effect is realized.
After the SAM membrane filtrate is treated by a molecular sieve, an ultrafiltration membrane and a nanofiltration membrane, part of impurities with larger difference between the metal ions and the SAM can be removed. After nanofiltration concentration, sodium butanesulfonate with higher concentration is added, so that the adenomethionine is separated out from the water phase in the form of butanesulfonate salt, and further purification is obtained.
Preferably, the molecular sieve in step (1) has a pore size of from 4 to 20 angstroms.
Preferably, the ultrafiltration membrane in step (1) has a molecular weight cut-off of 3000-20000Da.
Preferably, the nanofiltration membrane as described in step (2) has a molecular weight cut-off of 300-500Da.
Preferably, the SAM content in the concentrate in step (2) is 100 to 300g/kg.
Preferably, the final concentration of the sodium butanesulfonate added in the step (3) is 2-5mol/kg.
Preferably, 1, 4-butanesulfonic acid (butanesulfonic acid) is added in the step (7) according to the proportion, and the butanedisulfonic acid ademetionine SAM is formed after the additional process: the mol ratio of the butanedisulfonic acid is 1:1.65-1.75.
Preferably, the drying mode in the step (8) is freeze drying or spray drying.
In summary, the invention has the following beneficial effects:
1. the molecular sieve and the ultrafiltration membrane are combined and then are subjected to salting-out treatment, so that the purity of the adenomethionine is obviously improved; the invention discovers that the purity of the adenomethionine prepared by adopting molecular sieve, ultrafiltration membrane treatment, nanofiltration concentration and salting-out treatment is higher than that of the adenomethionine prepared by adopting cation resin treatment generally adopted by the person skilled in the art, so that impurities such as adenine which cannot be removed effectively after adsorption elution by adopting the cation resin can be removed effectively, the total impurity content is also greatly reduced, and the effect is obvious; in addition, from the viewpoint of production cost, although the process is increased, the time length and the cost are little changed;
2. the invention uses membrane separation and salting-out purification to replace the existing positive resin adsorption elution, and is simpler and more convenient and has higher controllability and reproducibility;
3. according to the invention, metal ions and other small molecular impurities in the adenosyl methionine separating liquid are adsorbed by the molecular sieve, then the macromolecular impurities are removed by ultrafiltration membrane filtration, and the obtained finished product of the adenosyl methionine sulfosuccinate is high in purity, low in ash content, about 100% in total content of the adenosyl methionine sulfosuccinate, less than or equal to 0.05% in ash content, and suitable for large-scale production.
Drawings
FIG. 1 is a HPLC chart of the final product of example 1;
FIG. 2 is a HPLC chart of the final product of example 2;
FIG. 3 is a HPLC chart of the final product of comparative example 1;
FIG. 4 is a HPLC chart of comparative example 2;
FIG. 5 is a HPLC chart of the final product of comparative example 3.
Detailed Description
The present invention will be described in detail with reference to examples, which are illustrative only and are not limiting the scope of application of the present invention.
The present invention will be described in detail with reference to examples, which are illustrative only and are not limiting the scope of application of the present invention.
Example 1
Preparation of S1 adenomethionine separating liquid
Taking a yeast fermentation broth containing adenosylmethionine, separating thalli, and washing thalli with purified water; then adding purified water until the concentration of thalli is 51%, thus obtaining a heavy suspension containing saccharomycetes; heating the heavy suspension to 70 ℃, maintaining for 8 minutes, then cooling to below 25 ℃ to obtain a cell disruption solution, filtering, and collecting filtrate to obtain an adenomethionine separation solution;
s2 high-purity sodium butanesulfonate ademetionine preparation
(1) Taking 5kg of adenomethionine separating liquid, adding 200g of 4 angstrom molecular sieve, stirring for half an hour, and then filtering to separate out the molecular sieve to obtain a molecular sieve treated feed liquid;
filtering the feed liquid treated by the molecular sieve by using an ultrafiltration membrane of 3000Da to obtain an adenosylmethionine ultrafiltrate;
(2) Concentrating the adenomethionine ultrafiltrate by nanofiltration, wherein the molecular weight cut-off of the nanofiltration membrane is 300Da, obtaining nanofiltration concentrated solution, and detecting the SAM content to be 185.3g/kg;
(3) Adding solid sodium butanesulfonate into the nanofiltration concentrated solution under stirring to ensure that the final concentration of the sodium butanesulfonate is 3.5mol/kg, obtaining a light yellow precipitate of the adenosyl methionine of the butanesulfonate, and filtering and collecting the salt-out;
(4) Re-dissolving the salt with purified water to obtain ademetionine re-solution;
(5) Decolorizing with macroporous resin to remove impurities and obtain colorless transparent butanedisulfonic acid ademetionine decolorized solution;
(6) Removing redundant sodium butanesulfonate from the decolorized solution by nanofiltration to obtain an adenomethionine concentrated solution containing SAM115.8 g/kg;
(7) Detecting the content of butanesulfonate in the concentrated solution, and according to SAM: 1, 4-butanesulfonic acid (butanesulfonic acid, the same applies hereinafter) is added to the butanesulfonic acid in a ratio of (mol ratio) =1:1.68, so that butanesulfonic acid ademetionine solution is obtained;
(8) And (3) performing vacuum freeze drying on the adenosylmethionine sulfosuccinate solution obtained in the step (7) to obtain a high-purity adenosylmethionine sulfosuccinate solid powder product.
The obtained product was subjected to HPLC detection for substances, wherein the known impurities of adenosine, adenine and S-adenosyl-L-homocysteine were not detected, and the total content of the other unknown impurities was 0.032% and 0.253%. The content of the cation of the adenosylmethionine is 52.8%, the content of the butanesulfonic acid is 47.6%, and the total content of the adenosylmethionine of the butanesulfonic acid is 100.4%. The ash content of the finished product is 0.03%.
The product obtained by the invention has various indexes such as related substances, content and the like which are superior to or equal to the existing standard. In particular, the indexes of related substances and ash are obviously superior to those of the commercial products.
Example 2
Preparation of S1 adenomethionine separating liquid
Taking a yeast fermentation broth containing adenosylmethionine, separating thalli, and washing thalli with purified water; then adding purified water until the concentration of thalli is 52.5%, thus obtaining heavy suspension containing saccharomycetes; heating the heavy suspension to 68 ℃ for 9 minutes, then cooling to below 25 ℃ to obtain a cell disruption solution, filtering and collecting filtrate to obtain an adenomethionine separation solution;
s2 high-purity sodium butanesulfonate ademetionine preparation
(1) Taking 5kg of adenomethionine separating liquid, adding 200g of 10 angstrom molecular sieve, stirring for half an hour, and then filtering to separate out the molecular sieve to obtain a molecular sieve treated feed liquid;
filtering the feed liquid treated by the molecular sieve by using a 5000Da ultrafiltration membrane to obtain an adenosylmethionine ultrafiltrate;
(2) Concentrating the adenomethionine ultrafiltrate by nanofiltration, wherein the molecular weight cut-off of the nanofiltration membrane is 300Da, thus obtaining nanofiltration concentrated solution, and detecting the SAM content as 216.7g/kg;
(3) Adding solid sodium butanesulfonate into the nanofiltration concentrated solution under stirring to ensure that the final concentration of the sodium butanesulfonate is 4mol/kg, obtaining a light yellow precipitate of the adenosyl methionine of the butanesulfonate, and filtering and collecting the salt precipitate;
(4) Re-dissolving the salt with purified water to obtain ademetionine re-solution;
(5) Decolorizing with macroporous resin to remove impurities and obtain colorless transparent butanedisulfonic acid ademetionine decolorized solution;
(6) Removing redundant sodium butanesulfonate from the decolorized solution by nanofiltration to obtain an adenosylmethionine concentrated solution containing SAM109.6 g/kg;
(7) Detecting the content of butanesulfonate in the concentrated solution, and according to SAM: 1, 4-butanesulfonic acid is added to the butanesulfonic acid in a ratio of (mol ratio) =1:1.7, so that a butanesulfonic acid ademetionine solution is obtained;
(8) And (3) performing vacuum freeze drying on the adenosylmethionine sulfosuccinate solution obtained in the step (7) to obtain a high-purity adenosylmethionine sulfosuccinate solid powder product.
The obtained product was subjected to HPLC detection for substances, wherein adenosine, adenine, S-adenosyl-L-homocysteine, which are known impurities, were not detected, methylthioadenosine was 0.275%, the total of other unknown impurities was 0.163%, and the total of impurities was 0.438%. The content of the cation of the adenosylmethionine is 52.5%, the content of the butanesulfonic acid is 47.2%, and the total content of the adenosylmethionine of the butanesulfonic acid is 99.7%. The ash content of the finished product is 0.05%.
The product obtained by the invention has various indexes such as related substances, content and the like which are superior to or equal to the existing standard. In particular, the indexes of related substances and ash are obviously superior to those of the commercial products.
Comparative example 1
S1 is as in example 1;
s2 sodium butanesulfonate ademetionine preparation
(1) Taking 5kg of adenomethionine separating liquid for resin adsorption, wherein the adsorption resin is weak acid type cation exchange resin;
(2) Desorbing the resin by using 0.2 mol/L1, 4-butanedisulfonic acid to obtain an eluent containing SAM;
(3) Decolorizing and removing impurities from the eluent by macroporous resin to obtain a decolorized solution containing SAM;
(4) Nanofiltration and concentration are carried out on the decolorized solution to obtain concentrated solution containing SAM 112.6 g/kg;
(5) Ding Erhuang acid radical in the concentrated solution is detected, and the method is carried out according to SAM: 1, 4-butanesulfonic acid is added to the butanesulfonic acid in a ratio of (mol ratio) =1:1.7, so that a butanesulfonic acid ademetionine solution is obtained;
(6) And (3) carrying out vacuum freeze drying on the solution to obtain a butanedisulfonic acid ademetionine product.
The obtained product was subjected to HPLC detection for substances, wherein the known impurities of adenosine were 0.09%, adenine was 1.058%, S-adenosyl-L-homocysteine was 0.037%, methylthioadenosine was 0.731%, the total of other unknown impurities was 0.68%, and the total of impurities was 2.596%. The content of the cation of the adenosylmethionine is 51.3%, the content of the butanesulfonic acid is 46.8%, and the total content of the adenosylmethionine of the butanesulfonic acid is 98.1%. The finished ash content was 0.52%.
Comparative example 2
S1 is as in example 1;
s2 sodium butanesulfonate ademetionine preparation
(1) Filtering 5kg of the adenomethionine separating liquid with a 3000Da ultrafiltration membrane to obtain an adenomethionine ultrafiltrate;
(2) Concentrating the adenomethionine ultrafiltrate by nanofiltration, wherein the molecular weight cut-off of the nanofiltration membrane is 300Da, thus obtaining nanofiltration concentrated solution, and detecting SAM content to be 196.6g/kg;
(3) Adding solid sodium butanesulfonate into the nanofiltration concentrated solution under stirring to ensure that the final concentration of the sodium butanesulfonate is 3.5mol/kg, obtaining a light yellow precipitate of the adenosyl methionine of the butanesulfonate, and filtering and collecting the salt-out;
(4) Re-dissolving the salt with purified water to obtain ademetionine re-solution;
(5) Decolorizing with macroporous resin to remove impurities and obtain colorless transparent butanedisulfonic acid ademetionine decolorized solution;
(6) Removing redundant sodium butanesulfonate from the decolorized solution by nanofiltration to obtain a concentrated solution containing SAM104.9 g/kg;
(7) Detecting the content of butanesulfonate in the concentrated solution, and according to SAM: 1, 4-butanesulfonic acid is added to the butanesulfonic acid in a ratio of (mol ratio) =1:1.68, so that a butanesulfonic acid ademetionine solution is obtained;
(8) And (3) performing vacuum freeze drying on the adenosylmethionine sulfosuccinate solution obtained in the step (7) to obtain a high-purity adenosylmethionine sulfosuccinate solid powder product.
The obtained finished product is subjected to HPLC detection on related substances, wherein the known impurities of adenosine 0.069%, adenine 0.883%, S-adenosine-L-homocysteine 0.065%, methylthioadenosine 0.485%, and the total of other unknown impurities is 0.472%, and the total impurity is 1.974%. The content of the cation of the adenosylmethionine is 51.6%, the content of the butanesulfonic acid is 46.9%, and the total content of the adenosylmethionine of the butanesulfonic acid is 98.5%. The finished ash content was 0.46%.
Comparative example 3
S1 is as in example 1;
s2 sodium butanesulfonate ademetionine preparation
(1) Taking 5kg of adenomethionine separating liquid, adding 200g of 4 angstrom molecular sieve, stirring for half an hour, and then filtering to separate out the molecular sieve to obtain a molecular sieve treated feed liquid;
(2) Concentrating the feed liquid treated by the molecular sieve through nanofiltration, wherein the molecular weight cut-off of the nanofiltration membrane is 300Da, thus obtaining nanofiltration concentrate, and detecting the SAM content to be 182.2g/kg;
(3) Adding solid sodium butanesulfonate into the nanofiltration concentrated solution under stirring to ensure that the final concentration of the sodium butanesulfonate is 4mol/kg, obtaining a light yellow precipitate of the adenosyl methionine of the butanesulfonate, and filtering and collecting the salt precipitate;
(4) Re-dissolving the salt with purified water to obtain ademetionine re-solution;
(5) Decolorizing with macroporous resin to remove impurities and obtain colorless transparent butanedisulfonic acid ademetionine decolorized solution;
(6) Removing redundant sodium butanesulfonate from the decolorized solution by nanofiltration to obtain a concentrated solution containing SAM113.5 g/kg;
(7) Detecting the content of butanesulfonate in the concentrated solution, and according to SAM: 1, 4-butanesulfonic acid is added to the butanesulfonic acid in a ratio of (mol ratio) =1:1.7, so that a butanesulfonic acid ademetionine solution is obtained;
(8) And (3) performing vacuum freeze drying on the adenosylmethionine sulfosuccinate solution obtained in the step (7) to obtain a high-purity adenosylmethionine sulfosuccinate solid powder product.
The obtained product was subjected to HPLC detection for substances, wherein the known impurities of adenosine 0.066%, adenine 0.335%, S-adenosyl-L-homocysteine 0.363%, methylthioadenosine 0.401%, and the total of other unknown impurities was 0.556%, and the total impurity was 1.721%. The content of the cation of the adenosylmethionine is 51.9%, the content of the butanesulfonic acid is 47.0%, and the total content of the adenosylmethionine of the butanesulfonic acid is 98.9%. The ash content of the finished product is 0.05%.
TABLE 1 detection of finished product of ademetionine butandisulfonate
According to the table and the attached figures 1-5, compared with the comparative example 1, the embodiment of the invention adopts the combination treatment of molecular sieve, ultrafiltration membrane treatment, nanofiltration concentration and salting-out, overcomes the defect that the impurity such as adenine and the like still cannot be effectively removed after the separation liquid of the adenosylmethionine is adsorbed and eluted by adopting the positive resin in the prior art, and obviously reduces the impurity and ash content in the finished product; the embodiment of the method has high purity of the adenosylmethionine sulfosuccinate, the total content of the adenosylmethionine sulfosuccinate can reach 100.4%, and the ash content of a finished product can reach 0.03%.
The steps of molecular sieve, ultrafiltration membrane, nanofiltration, salting out and the like have the functions of separating and purifying substances in the field; however, as can be seen from comparative examples 2 and 3, when the molecular sieve or ultrafiltration membrane is singly adopted for treatment, the subsequent steps of nanofiltration, salting out and the like are matched, the purity of the obtained finished product of the ademetionine butanedisulfonate is improved compared with that of the positive resin adsorption elution method, but the level of single impurities such as adenine and the like and the total impurities is still very high at the moment; when the molecular sieve and the ultrafiltration membrane are combined and then are matched with the subsequent purification step, the purity of the obtained finished product is greatly improved, impurities such as adenine and the like are effectively removed, and the ash content of the finished product is remarkably reduced. In fact, in the art of adenosylmethionine production, it is very difficult to remove ash further on the basis of the effects of the prior art; it is generally difficult for those skilled in the art to apply several treatment processes having partially identical effects to the organic combination mentioned in the present invention to prepare high purity and low ash-content adenosylmethionine sulfosuccinate.
Claims (1)
1. A preparation method of adenosylmethionine butanedisulfonate is characterized by comprising the following specific steps:
preparation of S1 adenomethionine separating liquid
Taking a yeast fermentation broth containing adenosylmethionine, separating thalli, and washing thalli with purified water; then adding purified water until the concentration of thalli is 51%, thus obtaining a heavy suspension containing saccharomycetes; heating the heavy suspension to 70 ℃, maintaining for 8 minutes, then cooling to below 25 ℃ to obtain a cell disruption solution, filtering, and collecting filtrate to obtain an adenomethionine separation solution;
preparation of S2 sodium butanesulfonate ademetionine
(1) Adding 200g of 4 angstrom molecular sieve into 5kg adenomethionine separating liquid, stirring for half an hour, and then filtering to separate out the molecular sieve to obtain a molecular sieve treated feed liquid;
filtering the feed liquid treated by the molecular sieve by using an ultrafiltration membrane of 3000Da to obtain an adenosylmethionine ultrafiltrate;
(2) Concentrating the adenosylmethionine ultrafiltrate by nanofiltration, wherein the molecular weight cut-off of the nanofiltration membrane is 300 and Da, so as to obtain nanofiltration concentrated solution, and detecting that the content of the adenosylmethionine is 185.3g/kg;
(3) Adding solid sodium butanesulfonate into the nanofiltration concentrated solution under stirring to ensure that the final concentration of the sodium butanesulfonate is 3.5mol/kg, obtaining a light yellow precipitate of the adenosyl methionine of the butanesulfonate, and filtering and collecting the salt-out;
(4) Re-dissolving the salt with purified water to obtain ademetionine re-solution;
(5) Decolorizing with macroporous resin to remove impurities and obtain colorless transparent butanedisulfonic acid ademetionine decolorized solution;
(6) Removing redundant sodium butanesulfonate from the decolorized solution by nanofiltration to obtain an adenomethionine concentrated solution containing 115.8/g/kg of adenomethionine;
(7) Detecting the content of butanedisulfonate in the concentrated solution according to the adenosylmethionine: 1, 4-butanesulfonic acid is supplemented in a molar ratio of butanesulfonic acid=1:1.68, so that a butanesulfonic acid adenomethionine solution is obtained;
(8) And (3) carrying out vacuum freeze drying on the adenosylmethionine sulfosuccinate solution obtained in the step (7) to obtain a solid powder product of the adenosylmethionine sulfosuccinate.
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